Eco-Friendly Synthesis and Characterization of Crystalline Selenium Nanoparticles via Bacillus cereus

Eco-Friendly Synthesis and Characterization of Crystalline Selenium Nanoparticles via Bacillus cereus | 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 Eco-Friendly Synthesis and Characterization of Crystalline Selenium Nanoparticles via Bacillus cereus Saibal Ghosh, Shouvik Mahanty, Sristi Das, Shreeya Purkait, Gopala Krishna Darbha, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7492605/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Among all the micronutrients, selenium (Se) is highly desirable as a dietary supplement, with the potential to improve germination and seedling development in crops, as well as exhibiting anticancer and antimicrobial properties. Organic and elemental nanoforms of Se demonstrate greater reactivity, higher bioavailability, and lower toxicity compared to inorganic forms. This study proposes the biosynthesis of selenium nanoparticles (SeNPs) using a soil-borne bacterium ( Bacillus cereus ). The synthesis of SeNPs through rhizospheric bacteria isolated from mica-rich agricultural soil is more environmentally friendly and cost-effective than conventional chemical synthesis methods. The synthesized nanoparticles were purified, dried, and initially characterized by UV-VIS spectroscopy, which showed a prominent peak at 282 nm, a characteristic feature of SeNPs. The crystalline phases were further confirmed by matching the XRD results with the JCPDS reference code 06–0362. Surface characterization was carried out using FTIR and XPS analyses, and the size and morphology of the particles were finally confirmed by FE-SEM and TEM imaging. The environmentally sustainable biosynthesis of SeNPs by Bacillus cereus from mica-rich rhizospheric soil produces extremely stable, bioavailable, and low-toxicity nanoparticles with potential applications in agriculture. Selenium nanoparticles Soil-borne bacteria Bio-synthesis Mica mine Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1 Introduction In the recent era, agricultural production increasingly necessitates additional inputs aimed at enhancing crop yield, sustaining plant quality and growth, prolonging the post-harvest longevity of products, and augmenting the market value of plant foods by improving their nutritional attributes and sensory qualities [ 1 ]. To meet agricultural demands, synthetic fertilizers and toxic pesticides have been widely used, but their overapplication has led to health risks and environmental pollution. As a sustainable alternative, nanotechnology-based solutions, such as nanobiostimulants, nano-fertilizers, and nano-pesticides, are emerging. These innovations offer reduced toxicity compared to conventional products while enhancing crop productivity, disease management, and post-harvest preservation [ 2 ]. In this scenario, gold (Au) and silver (Ag) are often used in nanoparticle (NP) production; however, their usage may be limited by toxicity at certain quantities. Hence, biologically generated Au and AgNPs have enhanced stability and less phytotoxicity, making them advantageous for agricultural applications [ 1 ]. Aside from these few metalloid and metal oxide NPs, such as SiO 2 , ZnO, and CuO, which have been shown to enhance plant nutrition and bolster disease resistance [ 3 ]. Selenium (Se) is a crucial trace element present as over 25 types of selenoenzymes and selenoproteins in the human body, playing a vital role in sustaining normal physiological activities. Excessive intake or deficiency of Se, whether in organic or inorganic forms, can result in significant harm due to its inherent toxicity. Selenium nanoparticles (SeNPs), the nano-form of Se, exhibit reduced toxicity and enhanced biocompatibility relative to their counterparts [ 4 ]. Selenium is a constituent of several essential enzymes, including thioredoxin reductase and glutathione reductase, and is crucial for sustaining normal human metabolism. Although not essential for plants, selenium is advantageous for crops. At appropriate concentrations, selenium may promote plant development, augment antioxidant capacity, and mitigate damage inflicted by environmental stressors in plants [ 3 , 5 ]. According to Garcia Marquez et al.[ 6 ] plant foods are the principal source of Se for humans, with a recommended daily intake of 60–70 µg/day for adults; nevertheless, excessive consumption of Se (> 400 µg/day) may result in detrimental health consequences. Consequently, Se is used as a fertilizer to modulate crop development and yield Se-enriched food. The SeNPs have enhanced bioavailability and reduced toxicity compared to selenite and selenate, suggesting they are viable alternatives to conventional inorganic Se species. Numerous studies indicate that SeNPs function as bio-stimulators and antioxidants, enhancing food quality and providing resistance against phytopathogen assaults [ 7 , 8 ]. The synthesis of SeNPs is primarily accomplished through chemical and physical methods. Nonetheless, these methods are expensive, complex, and consistently produce toxic by-products, significantly limiting the broader application of SeNPs. Consequently, it is imperative to investigate environmentally friendly methods for the synthesis of SeNPs. Recently, the biosynthesis methods for the preparation of SeNPs have garnered significant attention [ 9 ]. Under the action of selenite reductase and selenate reductase, Selenate (SeO 4 ² − ) is converted to its red allotrope, selenium (Se 0 ). This typically results in the accumulation of SeNPs on the cell surface or in the culture medium. The biogenic synthesis of SeNPs may be facilitated by microorganisms, including bacteria, fungi, and plant parts (root, shoot, and leaf), offering a straightforward, safe, and eco-friendly method for their preparation [ 4 ]. The size and shape of SeNPs can be effectively regulated by adjusting the pH, incubation temperature, metal ion concentration, reaction time, and the quantity of organic matter in the culture medium [ 10 ]. Presently, many bacterial isolates have been recognized for their ability to synthesize SeNPs, owing to their ease of handling, rapid growth rate, and low-cost maintenance, making them ideal organisms for the green synthesis of nanoparticles [ 11 ]. Several studies have indicated that certain bacterial isolates, including Lactobacillus acidophilus [ 12 ], Bacillus cereus [ 13 ], Bacillus licheniformis [ 14 ], and Pseudomonas alcaliphila [ 15 ], are capable of producing Bio-SeNPs, with applications in antimicrobial, anticancer, bioremediation, and enhancement of plant quality. Agricultural soil associated with mica mines creates a forest that serves as a detritus-based niche, functioning as a hub for bacterial diversity. The genera Candidatus solibacter , Candidatus koribacter , Sphingomonas , Gemmatimonas , Bacillus , Sorangium, Serratia , Pseudomonas , and Azoarcus constituted a significantly diverse bacterial community observed [ 16 ]. Numerous mining locations are recognized for containing selenium, a trace metal inherently found in the Earth's crust [ 17 ]. The content fluctuates according to rock type, averaging around 0.09 mg/kg in the top continental crust. Shales generally possess high concentrations (about 0.3 mg/kg), whereas granites and limestones have around 0.025 mg/kg, and sandstones have even lower amounts, around 0.01 mg/kg. Coal often exhibits elevated selenium concentrations, sometimes reaching several mg/kg [ 18 ]. The current work successfully synthesized Bio-SeNPs using rhizospheric bacteria isolated from mica-rich agricultural soil in Giridih, Jharkhand, India. The study presents a comprehensive analysis of the characterization of Bio-SeNPs using diverse tools. 2 Materials and Methods 2.1. Moist soil sample collection Soil samples from the rhizosphere of rice were obtained from three blocks (Deori, Gawan, and Tisri) in the Giridih district of Jharkhand, India Fig. S1 . The predominant operating mica mines in Giridih are located within these three blocks of the district. Samples were obtained from rice fields next to mica mines, where leachate and surface runoff from the mines accumulate naturally owing to the lower elevation. Three soil samples were randomly taken from each mine, and all samples were homogenized to form a single soil sample for isolation. Samples were maintained in sterile plastic bags, appropriately labeled, transported in an ice box, and promptly transferred to the laboratory for further processing. 2.2. Isolation of Bacterial Strain Transforming Selenium (Se 0 ) Serial dilution and nutrient agar plating were used for the isolation of bacterial strains that convert selenium. Commercial Aleksandrow broth medium (CAB- HiMedia Laboratories, M-1997) was used for screening of bacterial isolation, which is mica-rich selective medium. The enrichment technique, followed by a serial dilution technique (in 0.87% normal sterile saline solution) was used for the isolation. In the enrichment process, 5 g of soil was inoculated into sterile CAB broth media and incubated at 28 ± 2°C under shaking conditions for seven days. After incubation, serially diluted (10 − 6 ) enriched bacterial isolates were plated on CAB media with agar-agar (3%) followed by incubation at 28 ± 2°C for seven days [ 19 ]. The pure culture of the colonies forming a clear halo zone was further established in Aleksandrow broth media. 21 Screened bacterial isolates were stained (gram-stain) for approximate detection of purity of each of the isolates, and pure colonies (n = 30) were transferred to sterile slants on nutrient agar medium (HiMedia) and sterile glycerol stock medium for long-term preservation at -20 ° C. The morphological parameters of chosen isolates, including pigmentation, Gram reaction, margin, colony height, optical density, and slime generation, were previously examined by our study [ 19 ]. 2.3. Molecular Characterization of Selected Isolate The molecular identification of chosen bacterial isolates was conducted as previously described by Ghosh et al. [ 19 ]. Genomic DNA was extracted from a single colony of bacterial isolates for confirmation of molecular identification using a DNA isolation kit (NucleoSpin Microbial DNA, MACHEREY-NAGEL). Genomic DNA was preserved at -20°C for further use. The amplification of 16S ribosomal DNA was performed with universal bacterial primer sets, namely forward primer 27f (5´-AGAGTTTGATCCTGGCTCAG-3´) and reverse primer 1492r (5´-TACGGTTACCTTGTTACGACTT-3´). The PCR amplification procedure consisted of a 25 µl mixture containing 45 ng of genomic DNA, 5 U/µl of Taq polymerase (Takara Bio Inc.), 2.5 µl of 10 × buffer, 2.5 mM dNTP mixture, 1.5 mM MgCl 2 , and 10 pmol/µl of each primer. The PCR reaction was conducted under the following parameters: initial denaturation at 94°C for 4 minutes, followed by 30 cycles comprising denaturation at 94°C for 30 seconds, annealing at 54°C for 1 minute, extension at 72°C for 1 minute, and a final extension at 72°C for 7 minutes. To verify amplification, 5 µl of the PCR result was subjected to 1% agarose gel electrophoresis and then analyzed using a gel documentation system (Bio-Rad). A 100 bp plus ladder (GeNetBio Corp, Korea) was used to ascertain the product size. Upon confirmation, the amplified PCR products were purified using gel electrophoresis with a PCR purification kit, following the manufacturer's instructions (Macherey-Nagel), and stored at -20°C for further analysis. 2.4. Synthesis of Bio-SeNPs Nutrient broth medium was made and autoclaved for 20 minutes at 121°C. Subsequently, a sterilized sodium selenate (Na 2 SeO 4 , HIMEDIA-GRM7516-100G) solution was added from a 10,000 mg/L stock solution to achieve a concentration of 300 mg/L. The flasks were inoculated with a 10% inoculum of a 24-hour-old culture of a previously identified strain ( Bacillus cereus ), followed by incubation for 48 hours in an orbital shaker at 200 rpm and 30°C. Upon completion of the incubation period, the culture medium's hue changed to red, signifying the production of nano-selenium. The fermentation broth underwent centrifugation for 20 minutes at 5000 rpm, after which the supernatant was discarded. The residual pellets were subjected to three consecutive washes with 0.9% NaCl solution, thereafter treated with 37% HCl, maintained at room temperature for two days, and then centrifuged and washed several times with autoclaved distilled water to eliminate HCl. The acquired red precipitate was desiccated at 70°C and used for further analysis [ 20 ]. Figure 1 illustrates the flow chart for the production of Bio-SeNPs. 2.5 Characterization of the Bio-Synthesis SeNPs 2.5.1. UV-Visible Absorption Spectroscopic Analysis The nanoparticle was characterized using a UV-Vis spectrophotometer (Thermo Scientific, GENESYS 180 UV-VIS spectrophotometer) throughout a wavelength range of 200 to 800 nm at 1-nm intervals, employing a 1cm thick quartz cuvette. The reaction's progress was observed by the visible alteration in the color of the mixture. 2.5.2. X-Ray Diffraction (XRD) Analysis The crystalline phase of the Bio-SeNPs was determined using XRD measurements conducted using a Philips X-ray diffractometer (SmartLab, RIGAKU). The diffraction pattern of a thin layer of synthesized NPs on a glass substrate was recorded from 10° to 80° (2θ) with a step size of 0.02°, using Cu Kα radiation (k = 1.542 Å) at 40 kV and 30 mA. The crystal phase was ascertained by juxtaposing the computed interplanar spacing values and the associated diffraction peak intensities with the theoretical values from the Joint Committee on Powder Diffraction Standards-International Centre for Diffraction Data (JCPDS-ICCD) database [ 21 ]. 2.5.3. Fourier Transform Infrared (FTIR) Spectroscopic Analysis FTIR spectroscopy to used identify and characterize biologically synthesized Se nanoparticles measuring a sample absorbed infrared (IR) radiation. Using the attenuated total reflection (ATR) technique, spectra were obtained with a Thermo Scientific Nicolet iS5, scanning the range of 3500 to 400 cm⁻¹ at a resolution of 4 cm⁻¹ [ 22 ]. 2.5.4. X-ray photoelectron spectroscopy (XPS) analysis X-ray photoelectron spectroscopy (XPS) measurements for surface chemistry investigations were conducted using a K-Alpha instrument (PHI 5000 VersaProbe III) with Al Kα monochromatic X-ray (1486.6 eV) radiation and a spot size of 300 × 300 µm². The spectrometer has a flood cannon for charge compensation, with any charge-induced energy shift rectified by anchoring the C 1s line at 284.4 eV. The peak-fitting approach included subtracting a Shirley-type background from the spectra and fitting the peaks using symmetric Gaussian functions [ 23 ]. 2.5.5. Scanning Electron Microscope (SEM) Analysis A high-resolution scanning electron microscope SUPRA 55 VP- 4132 CARL ZEISS With attachment – ED was used to ascertain the dimensions and morphology of the produced nano-selenium particles. Two distinct imaging modalities were employed—bright field (with an electron accelerating voltage of 200 kV, using LaB6 as the electron source) and diffraction pattern imaging (DPI). A 4 k x 4 k resolution eagle CCD camera was used to capture transmitted electron pictures. The TEM Imaging & Analysis program was used to evaluate the EDX peak spectrum [ 20 ]. 2.5.6. Transmission Electron Microscopy (TEM) Analysis For TEM, 5µl of the sample solution was deposited on carbon-coated copper grids and stored in desiccators. Upon drying, the grids were examined using a JEOL JEM 2100 HR. The size distribution for the polydispersity index (PDI) of SeNPs was assessed using the following Eq. ( 1 ) [ 22 , 24 ]. $$\:PDI=\:{\left(\sigma\:/\text{D}\right)}^{2}$$ 1 where D represents the mean diameter of the Bio-SeNPs and σ denotes the standard deviation (SD). 3 Results and Discussion 3.1. General features and molecular identification of the Potential Bacterial Isolates The bacterial strain was isolated from mica-contaminated rice rhizospheric agricultural soil. A previous study by Ghosh et al.[ 19 ] reported the morphological characterization of the isolate as follows: pigmentation – white, Gram reaction – positive, margin – smooth, opacity – translucent to opaque, and slime production – high. Molecular identification of the bacterial isolate was carried out using the 16S ribosomal DNA region. The isolate was identified as Bacillus (strain: Bacillus cereus strain YB1806). It was previously reported that this bacterial isolate can solubilize potassium and phosphorus from non-exchangeable forms of these elements and also possesses nitrogen-fixing ability. The selected strain can also promote plant growth by producing indole-3-acetic acid (IAA), gibberellic acid (GA₃), hydrogen cyanide (HCN), ammonia (NH₃), and siderophores. Based on all the previous characteristics, the Bacillus cereus isolate showed the potential to reduce Se⁴⁺ to Se⁰ through bacterial enzyme- and metabolite-mediated mechanisms, and the results were positive. The visual identification of changes in colour of the broth Fig. S (a & b) after 48 hr of incubation at room temperature (30 ± 2°C) was considered as a positive result for the formation of Bio-SeNPs. The process of biosynthesizing selenium nanoparticles (SeNPs) through bacterial isolates entails a bioreduction mechanism, wherein bacteria convert soluble selenium salts, such as sodium selenite (SeO₃²⁻) or sodium selenate (SeO₄²⁻), into elemental selenium (Se⁰). This method is environmentally sustainable, economically viable, and safe, in contrast to traditional chemical and physical approaches[ 25 ]. A UV-VIS spectrophotometer was used to further monitor the Bio-SeNPs that were produced. Both the negative control (Na₂SeO₄ solution) and the positive control (just nutrient broth) showed no discernible color change throughout the process. 3.2. Characterization of Bio-SeNPs 3.2.1. UV-Visible Spectra Analysis of the SeNPs Suspension UV-VIS spectroscopy was used to assess the optical characteristics of the bio-synthesized SeNP suspensions Fig. 2 (a & c). The UV–VIS spectrum of the SeNP suspensions showed an increase in absorbance intensity in the shorter UV range. The bio-synthesized SeNPs produced by Bacillus cereus exhibited a maximum absorbance at 282 nm, which might be due to a plasmon resonance spike at approximately 2 nm, a characteristic feature of selenium nanoparticles Fig. 2 (c). Additionally, no prominent peaks were observed for the bacterial broth Fig. 2 (a) and selenium salt within the studied wavelength range Fig. 2 (b). The findings align with earlier studies, including the research by Dhanjal and Cameotra [ 26 ], which illustrated the aerobic biogenesis of selenium nanospheres by Bacillus cereus and analyzed their optical properties through UV–VIS spectroscopy. Their research established that the appearance of red coloration and a broad UV–VIS peak serve as a reliable indicator of SeNP synthesis. 3.2.4. XRD Analysis XRD analysis was conducted to elucidate the crystalline characteristics of SeNPs (Fig. 3 .). The XRD patterns confirmed the crystalline structure of synthesized SeNPs exhibit a prominent diffraction peak at 2θ = 23.51°, 29.70°, 41.30°, 43.64°, 45.3°, 48.10°, 51.72° and 56.14° corresponding to the (100), (101), (110), (102), (111), (200), (201) and (112) planes respectively. The result further confirmed the hexagonal structure of SeNP according to the JCPDS reference code 06–0362. This diffraction pattern is characteristic of hexagonal SeNPs, recognized for their thermodynamic stability and distinctive physicochemical features [ 27 ]. The results align with prior research indicating that SeNPs synthesized by several green and chemical methods exhibited comparable peak positions and phase identities [ 28 , 29 ]. 3.2.3. FTIR Characterisation of Bio-SeNPs FTIR spectroscopy analysis (Fig. 4 ) was conducted to examine the presence of biomolecules in synthesized SeNPs. The FTIR spectra of SeNP span from 4000 to 400 cm -1 . The spectra reveal absorption peaks at 3400 cm -1 and 2920 cm -1 , corresponding to the stretching vibrations of O-H and aliphatic C-H, respectively. The prominent peak at 1642 cm -1 is attributed to the CO–NH stretching vibration associated with amide-I, a defining feature of proteins, while the peak at 1546 cm -1 relates to the N–H bending vibration linked to amide-II, another characteristic section of proteins. The absorption peaks at 1380 cm -1 , 1224 cm -1 , and 1064 cm -1 are indicative of symmetrical C–H stretching, C–O stretching vibrations of the carboxyl group, and C–O–C bending vibrations, respectively, within the typical area of polysaccharides. The absorption spectra of SeNPs exhibit significant peaks at 689 cm⁻¹ and 480 cm⁻¹, indicating Se–Se/Se–O bond vibrations that affirm the biogenesis of SeNPs [ 28 ]. 3.2.5. XPS analysis XPS technique has been used to analyse the surface functional group of SeNP. The XPS survey spectrum, recorded between 0 and 1100 eV (Fig. 5 ), represents the core binding energies of C, N, O, Cl and Se in SeNP (Table S1 ). The high-resolution spectra of C, N, O, Cl and Se have also been recorded to analyse the surface electronic property. The high resolution of the C 1s spectrum has been carefully deconvoluted, which consists of three peaks at 284.8, 285.7 and 288.6 eV corresponding to C–C or C–H in amino acid chains, C–O or C–H in alcohol, amine, ether or amide and C = O or O–C–C in carbonyl, amide or hemiacetal bindings, respectively Fig. S3(b) [ 30 ]. The Gaussian fitted O 1s peak is deconvolved into two peaks at 532.2 and 533.3 eV. These two peaks are assigned as C = O and C–O binding, respectively Fig. S3 (c) [ 31 ]. The presence of C = O and C–O bonds in O 1s and C 1s confirms the C and O bonding, which plays a pivotal role in the formation of SeNP. The deconvoluted N 1s and Cl 2p scans are fitted with a Gaussian profile, amine group detected at 399.3 eV, protonated amine group detected at 400.5 eV and Cl 2p 1/2 detected at 199.9 eV, respectively Fig. S3(d) [ 32 ]. In case of Se 3d XPS spectrum has been deconvoluted with care and shows two deconvoluted peaks at 55.3 and 56.5 eV and assigned as Se 3d 3/2 and Se 3d 5/2 spin-orbit coupling, respectively (Fig. 5 ) [ 33 ]. From the literature survey, it is noted that 3d 5/2 of metallic selenium Se 0 is expected at 55.1 eV, whereas Se − II , Se II , Se IV , Se IV are expected approximately at binding energies < 55, 57.7, 59.4, and 61 eV, respectively [ 34 ]. The binding energy at 55.3 eV in the NP confirmed the presence of Se 0 . Also, it has been noted that a slightly higher binding energy is formed due to polarised leading of Se 0 . It has been observed selenium signal's initial intensity is substantially lower as a result of the data being noisy. From here, it has been concluded that in our SeNP, selenium is a combination of polarized Se 0 and Se II . 3.2.4 SEM and TEM analysis The biosynthesized selenium nanoparticles (SeNPs) were thoroughly characterized to assess their shape, structure, and composition using Field Emission Scanning Electron Microscopy (FESEM), High-Resolution Transmission Electron Microscopy (HRTEM), and Energy Dispersive X-ray Spectroscopy (EDX) (Fig. 6). FESEM images showed that the SeNPs primarily had a spherical shape, with some appearing quasi-spherical and elliptical (Fig. 6a). This diversity is common in biologically synthesized nanoparticles, where factors like biomolecular capping agents influence particle growth [ 35 ]. These agents, which include fungal polysaccharides, proteins, or enzymatic cofactors, play an important role in controlling nucleation and growth during synthesis. Overall, the nanoparticles were well-dispersed, though some areas showed moderate aggregation, likely due to biomolecular bridging or incomplete electrostatic stabilization. The particle sizes, measured from the FESEM images using ImageJ software (n = 50), ranged from 48 to 115 nm, with an average size of 82.4 ± 15.6 nm. This size range is ideal for medical and environmental uses because it balances the surface area-to-volume ratio with colloidal stability [ 36 ]. The particles appeared smooth and uniformly electron-dense, suggesting a consistent selenium core without significant pores or surface defects. The brightness seen in the FESEM images is consistent with selenium’s high atomic number (Z = 34), which improves backscattered electron yield and provides excellent imaging contrast, reinforcing the elemental identity of the particles [ 37 , 38 ]. Energy Dispersive X-ray Spectroscopy (EDX) analysis was performed to examine the elemental makeup of the SeNPs and confirm the purity and uniformity of the selenium incorporation. The EDX spectrum (Fig. 6b) had a dominant sharp peak at about 11.22 keV, matching the Kα emission line of selenium and confirming it as the main element in the nanoparticle core. The peak had a narrow full-width at half maximum (FWHM), indicating high purity and no significant overlap or interference from secondary phases. Quantitative analysis through peak integration showed that selenium content was around 91.6 wt%, confirming the strong presence of elemental Se in the material. The high signal-to-noise ratio and the sharp Gaussian profile of the peak reflect efficient selenium incorporation and a strong detector response, typical of well-prepared crystalline selenium. In addition to selenium, the spectrum showed minor peaks related to oxygen (~ 0.52 keV) and carbon (~ 0.27 keV), likely from organic matter in the synthesis medium or the carbon support grid used during sample preparation [ 39 ]. Oxygen might be part of a thin layer on the nanoparticle surface or linked to capping molecules that contain hydroxyl or carboxyl groups. These results align with earlier studies on biosynthesized selenium, where organic functional groups from fungal extracts slightly oxidize the nanoparticle surface but do not alter the core elemental structure. Trace amounts of sulfur (~ 2.31 keV) and phosphorus (~ 2.01 keV) were also found, likely from leftover biomolecules that contain thiol or phosphate groups (Hashem et al. 2022)[ 40 ]. Importantly, no signals were detected for heavy metals or transition elements like Cu, Zn, or Fe, confirming the chemical purity of the synthesis process and ruling out co-precipitation or contamination [ 41 ]. To confirm and improve the structural insights from FESEM, HRTEM analysis was conducted. The high-resolution TEM images revealed individual SeNPs with sharp contrast and well-defined edges, confirming the particulate nature of the synthesized material. At low magnification, the particles maintained a nearly spherical shape with minimal aggregation, which may relate to grid preparation and drop-casting of well-dispersed colloidal samples. The average diameter from the TEM images was about 76.1 ± 10.3 nm (Fig. 6ci-ciii), which aligns with the FESEM measurements, reinforcing the accuracy and consistency of the particle sizing. At higher magnification, the SeNPs showed a strong contrast between the selenium-rich core and the surrounding organic material, indicating a surface-bound organic layer. This organic coating, likely made of fungal proteins, amino acids, and other metabolites, acts as a stabilizing shell, preventing particles from merging and improving colloidal stability in water [ 42 ]. Although the shell is thin (~ 1.5–2.0 nm), it is vital for modulating surface interactions, especially in medical applications where surface properties influence biocompatibility and targeting. The nanoparticles appeared solid and crystalline with no signs of hollowness or core-shell separation. No voids, cracks, or amorphous boundaries were seen in the SeNP cores (Fig. 6ci-ciii), supporting the idea that nucleation was driven by a uniform and quick reduction of selenium oxyanions (e.g., SeO₃²⁻ or SeO₄²⁻) to elemental selenium (Se⁰) with the help of fungal reductants [ 42 , 43 ]. The particle size distribution, while not perfectly uniform, showed a relatively narrow range, suggesting a diffusion-limited growth process instead of burst nucleation or Ostwald ripening, which are more common in chemically synthesized systems [ 44 ]. This level of control, achieved without toxic reducing agents or surfactants, highlights the effectiveness of the biological synthesis process in creating nanomaterials ready for use [ 45 ]. The compositional purity is particularly critical in assessing the potential uses of the SeNPs in sensitive biological fields, such as drug delivery or antimicrobial coatings, where impurities can seriously affect toxicity or biological function [ 46 ]. Additionally, the consistent elemental makeup across various sampling points suggests a high level of uniformity in the synthesized batch [ 47 ]. Although complete elemental mapping wasn’t conducted, the strong selenium peak across multiple EDX spots supports the idea of even Se distribution. The combined structural and compositional data obtained through FESEM, TEM, and EDX strongly confirms the successful synthesis of selenium nanoparticles through a green, biologically mediated approach. Their structural integrity, near-spherical shape, nanoscale size, and elemental purity indicate that the fungal synthesis process not only facilitated the reduction of selenium oxyanions but also directed the growth and stabilization of selenium nuclei into uniform nanostructures. The measured particle size range (45–115 nm) is beneficial, providing a high surface area while avoiding excessive aggregation or rapid settling. The organic layer seen in the TEM plays a dual role: it stabilizes the nanoparticles colloidally and offers a functional surface that can be used for further attachment to targeting ligands or biomolecules in therapeutic uses. Furthermore, the stability and composition of the SeNPs suggest excellent suitability for redox-sensitive applications, such as oxidative stress management, reactive oxygen species (ROS) removal, or targeted cancer treatment, where selenium’s redox-active nature offers therapeutic benefits. The thin organic shell also hints at possible bio-interaction behaviors, such as improved cellular uptake or enzyme-like functions. In environmental cleanup, these particles can act as effective electron donors or surface agents for immobilizing heavy metals or breaking down stubborn pollutants. Overall, the characterization confirms the success of the biological synthesis method in producing high-quality, compositionally pure, and structurally stable selenium nanoparticles suitable for various biomedical and environmental applications. The studies analyzed for the comparison of various synthesis processes are enumerated in Table 1 . This comparative study examined the physical, chemical, and green synthesis of SeNPs, focusing on the size and shape of the resulting nanoparticles. Various approaches, including microbial, fungal, green, and algal biosynthesis, have been examined within biosynthetic processes. Previous investigations, as illustrated in Table 1 , indicate that hydrothermal methods Shar et al. [ 48 ] produced hexagonal nanoparticles measuring 169 nm, whereas electrochemical methods Zhang et al. [ 49 ] yielded tubular nanoparticles of 200 nm. Photocatalysis Triantis et al. [ 50 ] exhibited no discernible shape, with nanoparticle sizes ranging from 4 to 110 nm. Among different biosynthesis processes, microbial [ 51 , 52 ], green synthesis [ 53 ], fungal [ 54 ], and algal [ 55 ] methods have demonstrated the capability to synthesize selenium nanoparticles with sizes ranging from 40 to 400 nm. Considering the above-mentioned points, the present study highlights the biosynthesis of SeNPs by bacterial isolates ( Bacillus cereus ) from mica-contaminated rhizospheric agricultural soil of Giridih district, India. In this study, Bacillus cereus produced nanoparticles measuring 282 nm, which falls within the reported range of 40–400 nm, confirming that the synthesized nanoparticles are SeNPs. Table 1 Comparative comparison of the synthesis of Bio-SeNPs by various methodologies. Synthesis method Process Shape Size (nm) reference Physical/Chemical Hydrothermal hexagonal 169.11 [ 48 ] Electrochemical tube 200 [ 49 ] Photocatalysis 40–110 [ 50 ] Biosynthesis Microbial spherical 80–220 [ 51 ] Fungal various ~ 137nm [ 54 ] Green synthesis spherical 40–100 [ 53 ] Algal synthesis spherical 45–80 [ 55 ] Microbial spherical 400 [ 52 ] 4 Conclusion This study demonstrates a green, scalable, and sustainable method for making selenium nanoparticles (SeNPs) using a strain of Bacillus cereus, which we isolated from selenium-contaminated rice field soil. The biosynthetic method in this work relies on the natural ability of the bacterial strain to reduce selenite (Se⁴⁺) ions to elemental selenium (Se⁰) nanoparticles at room temperature. This process does not require harmful chemicals or extreme conditions. Microbial synthesis reduces environmental impact and improves the biocompatibility and functionality of the resulting nanostructures. The reddish color of the reaction mixture indicated the formation of elemental selenium, which was confirmed by a distinct UV-Visible absorption peak at 282 nm, a common marker for selenium nanostructures, showing successful synthesis. Detailed analyses using FESEM and TEM showed that the biosynthesized SeNPs were mostly spherical and evenly sized, with smooth surfaces. This indicates effective nucleation and controlled growth in biological conditions. FESEM images displayed uniform particle formation with little agglomeration, while TEM provided exact size measurements, estimating the average particle size at about 76.1 ± 10.3 nm. These nanoscale dimensions are ideal for many biomedical applications since they balance cellular uptake efficiency and lower toxicity. Additionally, a faint organic layer around the SeNPs, seen in high-resolution TEM images, suggests the presence of biomolecular capping agents, likely made up of extracellular proteins, enzymes, or metabolites from the bacteria. We evaluated elemental composition and purity using energy-dispersive X-ray spectroscopy (EDX), which confirmed selenium as the principal component with a strong peak at approximately 11.22 keV. Quantitative analysis indicated that selenium content reached up to 91.6 wt%, with small amounts of carbon and oxygen, coming from organic capping agents or the carbon-coated sample grid. No signs of toxic heavy metals or synthetic impurities were found, confirming the high purity and environmentally friendly nature of the biosynthesized nanomaterial. The EDX results support the successful reduction of selenite to Se⁰ and exclude the possibility of external elemental contamination during synthesis. Fourier-transform infrared spectroscopy (FTIR) further examined the surface chemistry, revealing various functional groups linked to protein and carbohydrate biomolecules. We observed key absorption bands at 3400 cm⁻¹ (O–H and N–H stretching), 1642 cm⁻¹ (amide I), and 1546 cm⁻¹ (amide II), suggesting that bacterial proteins acted as both reducing and stabilizing agents. Additional peaks corresponding to C–H, C = O, and C–N vibrations imply the presence of polysaccharides and amino acids on the nanoparticle surface. This organic capping is vital for preventing aggregation and improving the biocompatibility and functionality of SeNPs in biological contexts. X-ray diffraction (XRD) analysis provided insights into crystallinity and phase composition. The diffractogram showed characteristic peaks for the (100), (101), (110), and (201) planes of crystalline selenium, matching the standard JCPDS card No. 06-0362. The sharpness and intensity of these peaks confirm the high crystallinity of the nanoparticles, which is important for their physical properties, redox activity, and electronic structure. The XRD pattern did not show amorphous selenium or any secondary phases, reinforcing the effectiveness of the biosynthetic method in producing phase-pure SeNPs. To gain more information on elemental oxidation states and surface bonding, we performed X-ray photoelectron spectroscopy (XPS). The high-resolution Se 3d spectrum displayed a doublet at 55.3 eV and 56.5 eV, indicating the Se⁰ oxidation state. The lack of peaks at higher binding energies suggests there were no residual selenite or selenate species, confirming complete reduction. Additional spectra for carbon (C 1s) and oxygen (O 1s) showed peaks consistent with C–C, C = O, and O–H groups, reflecting the complex organic layer around the particles. The organic functionality on the surface offers opportunities for further biofunctionalization, such as ligand conjugation or drug loading. By integrating these analytical techniques, we gain a comprehensive understanding of the biosynthesized selenium nanoparticles: they are uniform, crystalline, highly pure, and surface-functionalized nanostructures produced through a low-cost and eco-friendly microbial process. This synthesis method marks a significant shift from traditional physical and chemical approaches, which are often energy-intensive, toxic, and hard to scale. In contrast, the microbial method does not require external reducing agents or capping chemicals, operates under mild conditions, and yields nanoparticles with excellent biocompatibility and a minimal environmental impact. Beyond synthesis and characterization, the properties of these SeNPs make them promising for various applications. Their nanoscale size and organic coating boost their potential for targeted drug delivery, antioxidant therapy, antimicrobial coatings, and cancer treatment. Furthermore, their redox activity and stability make them suitable for catalytic and sensing applications, especially in biomedical and environmental fields. From an ecological standpoint, the green synthesis method aligns with sustainable nanotechnology principles and offers a practical strategy for utilizing microbial diversity in bioremediation and producing valuable nanomaterials. In summary, this study confirms the feasibility of microbial biosynthesis for high-quality selenium nanoparticles and highlights the importance of detailed physicochemical characterization in assessing their application potential. The Bacillus cereus -mediated approach described here presents a reliable, reproducible, and environmentally friendly method for producing selenium nanostructures, linking green chemistry, nanotechnology, and biotechnology. Future research can further explore optimizing these SeNPs for uses in therapeutics, diagnostics, and environmental detoxification. Declarations Acknowledgment: The authors acknowledge the financial support from SERB, Govt. of India (SERB Sanction Order No: PDF/2023/000288) and Tokclai Tea Research Institute, India for providing all the laboratory facilities. Author contribution: SG: Conceptualization, methodology, data analysis, original draft preparation, writing, investigation, reviewing and editing; SM: Data analysis, original draft preparation, writing, investigation, reviewing and editing; SD: Laboratory analysis, methodology, reviewing and editing; SP: Data analysis, writing and reviewing; GKD: Resources and reviewing; RP: Supervision, resources, reviewing and editing; SM (Majumder): Supervision, conceptualization, reviewing and editing. Funding: The authors would like to thank the Anusandhan National Research Foundation (SERB), Govt. of India, under the project (SERB Sanction Order No: PDF/2023/000288) for the necessary funding. Data availability: No datasets were generated or analysed during the current study. Declarations Ethics Approval: Not applicable. Research Involving Humans and Animals Statement: None. Informed Consent: None Conflict of interest: The authors declare no competing interests. References Garza-García, J. J. O., Hernández-Díaz, J. A., León-Morales, J. M., Velázquez-Juárez, G., Zamudio-Ojeda, A., Arratia-Quijada, J., & García-Morales, S. (2023). Selenium nanoparticles based on Amphipterygium glaucum extract with antibacterial, antioxidant, and plant biostimulant properties. Journal of nanobiotechnology , 21 (1), 252. Hofmann, T., Lowry, G. V., Ghoshal, S., Tufenkji, N., Brambilla, D., Dutcher, J. R., & Landry, M. P. (2020). Technology readiness and overcoming barriers to sustainably implement nanotechnology-enabled plant agriculture. Nature Food , 1 (7), 416–425. Song, J., Yu, S., Yang, R., Xiao, J., & Liu, J. (2023). Opportunities for the use of selenium nanoparticles in agriculture. NanoImpact , 31 , 100478. Sun, Y., Shi, Y., Jia, H., Ding, H., Yue, T., & Yuan, Y. (2021). Biosynthesis of selenium nanoparticles of Monascus purpureus and their inhibition to Alicyclobacillus acidoterrestris. Food Control , 130 , 108366. Ramakrishnan, M., Arivalagan, J., Satish, L., Mohan, M., Christyraj, J. R. S. S., Chandran, S. A., & Kalishwaralal, K. (2022). Selenium: a potent regulator of ferroptosis and biomass production. Chemosphere , 306 , 135531. García Márquez, V., Moreno, M., Mendoza, Á. B., A., & Macías, M., J (2020). Ionic selenium and nanoselenium as biofortifiers and stimulators of plant metabolism. Agronomy , 10 (9), 1399. Kumar, A., & Prasad, K. S. (2021). Role of nano-selenium in health and environment. Journal of Biotechnology , 325 , 152–163. Yang, R., Li, Q., Zhou, W., Yu, S., & Liu, J. (2022). Speciation analysis of selenium nanoparticles and inorganic selenium species by dual-cloud point extraction and ICP-MS determination. Analytical Chemistry , 94 (47), 16328–16336. Hussain, A., Lakhan, M. N., Hanan, A., Soomro, I. A., Ahmed, M., Bibi, F., & Zehra, I. (2023). Recent progress on green synthesis of selenium nanoparticles–a review. Materials Today Sustainability , 23 , 100420. Sarkar, J., Mridha, D., Davoodbasha, M. A., Banerjee, J., Chanda, S., Ray, K., & Sarkar, J. (2023). A state-of-the-art systemic review on selenium nanoparticles: Mechanisms and factors influencing biogenesis and its potential applications. Biological Trace Element Research , 201 (10), 5000–5036. Medina-Cruz, D., Truong, L. B., Sotelo, E., Martínez, L., González, M. U., Huttel, Y., & Cholula-Díaz, J. L. (2023). Bacterial-mediated selenium nanoparticles as highly selective antimicrobial agents with anticancer properties. RSC Sustainability , 1 (6), 1436–1448. Alam, H., Khatoon, N., Khan, M. A., Husain, S. A., Saravanan, M., & Sardar, M. (2020). Synthesis of selenium nanoparticles using probiotic bacteria Lactobacillus acidophilus and their enhanced antimicrobial activity against resistant bacteria. Journal of Cluster Science , 31 , 1003–1011. Kora, A. J. (2018). Bacillus cereus, selenite-reducing bacterium from contaminated lake of an industrial area: A renewable nanofactory for the synthesis of selenium nanoparticles. Bioresources and Bioprocessing , 5 (1), 1–12. Ali, E. N., El-Sonbaty, S. M., & Salem, F. M. (2013). Evaluation of selenium nanoparticles as a potential chemopreventive agent against lung carcinoma. Int J Pharm Biol Sci , 2 (4), 38–46. Zhang, W., Chen, Z., Liu, H., Zhang, L., Gao, P., & Li, D. (2011). Biosynthesis and structural characteristics of selenium nanoparticles by Pseudomonas alcaliphila. Colloids and Surfaces B: Biointerfaces , 88 (1), 196–201. Chakraborty, S., Ghosh, S., Banerjee, S., Kumar, S., & Bhattacharyya, P. (2024). Elucidating the synergistic effect of acidity and metalloid poisoning on the microbiome through metagenomics and machine learning approaches. Environmental Research , 243 , 117885. Etteieb, S., Magdouli, S., Zolfaghari, M., & Brar, S. (2020). Monitoring and analysis of selenium as an emerging contaminant in mining industry: A critical review. Science of the Total Environment , 698 , 134339. Lopes, G., Ávila, F. W., & Guilherme, L. R. G. (2017). Selenium behavior in the soil environment and its implication for human health. Ciência e Agrotecnologia , 41 , 605–615. Ghosh, S., Mondal, S., Banerjee, S., Mukherjee, A., & Bhattacharyya, P. (2023). Temporal Dynamics of Potassium Release from Waste Mica as Influenced by Potassium Mobilizing Bacteria. Journal of Pure and Applied Microbiology . https://doi.org/10.22207/JPAM.17.1.17 Ghazi, A. A., El-Nahrawy, S., El-Ramady, H., & Ling, W. (2022). Biosynthesis of nano-selenium and its impact on germination of wheat under salt stress for sustainable production. Sustainability , 14 (3), 1784. Yadav, S. K., Kumar, A., & Mehta, N. (2023). Synthesis and characterization of nanostructured graphene-doped selenium. RSC advances , 13 (20), 13564–13574. Mahanty, S., Bakshi, M., Ghosh, S., Chatterjee, S., Bhattacharyya, S., Das, P., …Chaudhuri, P. (2019). Green synthesis of iron oxide nanoparticles mediated by filamentous fungi isolated from Sundarban mangrove ecosystem, India. BioNanoScience , 9 , 637–651. Matouk, Z., Islam, M., Gutiérrez, M., Pireaux, J. J., & Achour, A. (2022). X-ray photoelectron spectroscopy (XPS) analysis of ultrafine au nanoparticles supported over reactively sputtered TiO2 films. Nanomaterials , 12 (20), 3692. Kozlov, N. K., Natashina, U. A., Tamarov, K. P., Gongalsky, M. B., Solovyev, V. V,Kudryavtsev, A. A., … Osminkina, L. A. (2017). Recycling of silicon: from industrial waste to biocompatible nanoparticles for nanomedicine. Materials Research Express , 4 (9), 095026. Moreno-Martín, G., Espada-Bernabé, E., Gómez-Gómez, B., León-González, M. E., & Madrid, Y. (2023). Evaluation of the transformation of selenite and selenium nanoparticles to seleno-amino acids produced by Escherichia coli and Staphylococcus aureus by using liquid chromatography-inductively coupled plasma mass spectrometry and single-particle-inductively coupled plasma mass spectrometry and different sample treatments. Spectrochimica Acta Part B: Atomic Spectroscopy , 200 , 106611. Dhanjal, S., & Cameotra, S. S. (2010). Aerobic biogenesis of selenium nanospheres by Bacillus cereus isolated from coalmine soil. Microbial cell factories , 9 (1), 52. Wadhwani, S. A., Shedbalkar, U. U., Singh, R., & Chopade, B. A. (2016). Biogenic selenium nanoparticles: current status and future prospects. Applied microbiology and biotechnology , 100 (6), 2555–2566. Vasanthakumar, S., Manikandan, M., & Arumugam, M. (2024). Green synthesis, characterization and functional validation of bio-transformed selenium nanoparticles. Biochemistry and Biophysics Reports , 39 , 101760. Saravanan, K., Madhaiyan, M., Periyasamy, P., Manivannan, P., Bayrakdar, A., & Balakrishnan, V. (2025). Green synthesis and detailed characterization of selenium nanoparticles derived from Alangium salviifolium (Lf) Wangerin. Chemical Physics Impact , 10 , 100876. Chang, J. L., Liao, C. W., Arthisree, D., Senthil Kumar, A., & Zen, J. M. (2022). A size-controlled graphene oxide materials obtained by one-step electrochemical exfoliation of carbon fiber cloth for applications to in situ gold nanoparticle formation and electrochemical sensors—a preliminary study. Biosensors , 12 (6), 360. Song, C., Li, X., Wang, S., & Meng, Q. (2016). Enhanced conversion and stability of biosynthetic selenium nanoparticles using fetal bovine serum. RSC advances , 6 (106), 103948–103954. Sun, L., Zhang, R., Wang, Y., & Chen, W. (2014). Plasmonic Ag@ AgCl nanotubes fabricated from copper nanowires as high-performance visible light photocatalyst. ACS Applied Materials & Interfaces , 6 (17), 14819–14826. Han, D. S., Batchelor, B., & Abdel-Wahab, A. (2013). XPS analysis of sorption of selenium (IV) and selenium (VI) to mackinawite (FeS). Environmental Progress & Sustainable Energy , 32 (1), 84–93. Ruiz-Fresneda, M. A., Eswayah, A. S., Romero-González, M., Gardiner, P. H. E., Solari, P. L., & Merroun, M. L. (2020). Chemical and structural characterization of Se IV biotransformations by Stenotrophomonas bentonitica into Se 0 nanostructures and volatiles Se species. Environmental Science: Nano , 7 (7), 2140–2155. Singh, A., Dey, P., Mihara, H., Prakash, R., & Prakash, N. T. (2025). Enhanced antimicrobial properties of cotton gauze functionalized with biosynthesized selenium nanoparticles. Polymer Bulletin , 82 (9), 3885–3908. Roy, N., Nivedya, T., Paira, P., & Chakrabarty, R. (2025). Selenium-based nanomaterials: green and conventional synthesis methods, applications, and advances in dye degradation. RSC advances , 15 (4), 3008–3025. Alsehli, B. R., Al-Hakkani, M. F., Alluhayb, A. H., Saleh, S. M., Mohamed, H., Hassane, A. M. A., & Hassan, M. H. A. (2025). Facile Mycosynthesis of Selenium Nanoparticles by Aspergillus candidus Extract: Unveiling Characterization, Potent Antimicrobial Properties, and Cytotoxic Activities. BioNanoScience , 15 (2), 234. Likidtaveesin, S., Changtong, N., Jarussophon, N., & Chunhachart, O. (2025). Biosynthesis of Selenium Nanoparticles and Selenium-Enriched g-Polyglutamic Acid (Se-PGA) by Bacillus subtilis. Trends in Sciences , 22 (4), 9391. Srivastava, N., & Mukhopadhyay, M. (2015). Biosynthesis and structural characterization of selenium nanoparticles using Gliocladium roseum. Journal of Cluster Science , 26 (5), 1473–1482. Hashem, A. H., & Salem, S. S. (2022). Green and ecofriendly biosynthesis of selenium nanoparticles using Urtica dioica (stinging nettle) leaf extract: Antimicrobial and anticancer activity. Biotechnology journal , 17 (2), 2100432. Alagesan, V., & Venugopal, S. (2019). Green synthesis of selenium nanoparticle using leaves extract of Withania somnifera and its biological applications and photocatalytic activities. Bionanoscience , 9 (1), 105–116. Gharieb, M. M., Soliman, A. M., & Omara, M. S. (2025). Biosynthesis of selenium nanoparticles by potential endophytic fungi Penicillium citrinum and Rhizopus arrhizus: characterization and maximization. Biomass Conversion and Biorefinery , 15 (2), 2319–2328. Elshikiby, L. A., Baka, Z. A. M., & El-Zahed, M. M. (2025). Biological activities of optimized biosynthesized selenium nanoparticles using Proteus mirabilis PQ350419 alone or combined with chitosan and ampicillin against common multidrug-resistant bacteria. Microbial Cell Factories , 24 (1), 159. Shawky, M., Kalaba, M. H., & El-Sherbiny, G. M. (2025). Combined impact of biosynthesized selenium nanoparticles and imipenem against carbapenem-resistant Pseudomonas aeruginosa and their associated virulence factors. BMC microbiology , 25 (1), 235. Babajide, A. M., Adebami, G. E., & Adebayo-Tayo, B. C. (2025). Screening of rhizobacteria from monkey pod trees for plant growth promoters and evaluating the antifungal potential of the biosynthesized selenium nanoparticles. Scientific Reports , 15 (1), 16797. Dwivedi, S., AlKhedhairy, A. A., Ahamed, M., & Musarrat, J. (2013). Biomimetic synthesis of selenium nanospheres by bacterial strain JS-11 and its role as a biosensor for nanotoxicity assessment: a novel Se-bioassay. PloS one , 8(3), e57404. Sharma, G., Sharma, A. R., Bhavesh, R., Park, J., Ganbold, B., Nam, J. S., & Lee, S. S. (2014). Biomolecule-mediated synthesis of selenium nanoparticles using dried Vitis vinifera (raisin) extract. Molecules , 19 (3), 2761–2770. Shar, A. H., Lakhan, M. N., Wang, J., Ahmed, M., Alali, K. T., Ahmed, R., … Dayo,A. Q. (2019). Facile synthesis and characterization of selenium nanoparticles by the hydrothermal approach. Dig J Nanomater Biostruct , 14 , 867–872. Zhang, S. Y., Zhang, J., Liu, Y., Ma, X., & Chen, H. Y. (2005). Electrochemical synthesis of selenium nanotubes by using CTAB soft-template. Electrochimica acta , 50 (22), 4365–4370. Triantis, T., Troupis, A., Gkika, E., Alexakos, G., Boukos, N., Papaconstantinou, E., & Hiskia, A. (2009). Photocatalytic synthesis of Se nanoparticles using polyoxometalates. Catalysis Today , 144 (1–2), 2–6. Forootanfar, H., Adeli-Sardou, M., Nikkhoo, M., Mehrabani, M., Amir-Heidari, B., Shahverdi, A. R., & Shakibaie, M. (2014). Antioxidant and cytotoxic effect of biologically synthesized selenium nanoparticles in comparison to selenium dioxide. Journal of Trace Elements in Medicine and Biology , 28 (1), 75–79. Tugarova, A. V., Mamchenkova, P. V., Khanadeev, V. A., & Kamnev, A. A. (2020). Selenite reduction by the rhizobacterium Azospirillum brasilense, synthesis of extracellular selenium nanoparticles and their characterisation. New biotechnology , 58 , 17–24. Anu, K., Singaravelu, G., Murugan, K., & Benelli, G. (2017). Green-synthesis of selenium nanoparticles using garlic cloves (Allium sativum): biophysical characterization and cytotoxicity on vero cells. Journal of Cluster Science , 28 (1), 551–563. Arunthirumeni, M., Veerammal, V., & Shivakumar, M. S. (2022). Biocontrol efficacy of mycosynthesized selenium nanoparticle using Trichoderma sp. on insect pest Spodoptera litura. Journal of Cluster Science , 33 (4), 1645–1653. Abdel-Moneim, A. M. E., El-Saadony, M. T., Shehata, A. M., Saad, A. M., Aldhumri, S. A., Ouda, S. M., & Mesalam, N. M. (2022). Antioxidant and antimicrobial activities of Spirulina platensis extracts and biogenic selenium nanoparticles against selected pathogenic bacteria and fungi. Saudi Journal of Biological Sciences , 29 (2), 1197–1209. Additional Declarations No competing interests reported. Supplementary Files SupplemantaryInformation.docx Cite Share Download PDF Status: Posted Version 1 posted 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. 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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-7492605","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":508287058,"identity":"d1b3126a-7836-4b93-9b0c-48d59975742b","order_by":0,"name":"Saibal Ghosh","email":"","orcid":"","institution":"Tocklai Tea Research Institute, Tea Research Association","correspondingAuthor":false,"prefix":"","firstName":"Saibal","middleName":"","lastName":"Ghosh","suffix":""},{"id":508287059,"identity":"8abfaaab-9b1b-467f-ba96-c80e1683b4d3","order_by":1,"name":"Shouvik Mahanty","email":"","orcid":"","institution":"Indian Institute of Science 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5","display":"","copyAsset":false,"role":"figure","size":142528,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig 4\u003c/strong\u003e. XPS high-resolution spectra of Selenium nanoparticle (Se 3d) from the purified SeNPs.\u003c/p\u003e","description":"","filename":"41.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7492605/v1/35804a8a2a2d0c36eff69420.jpg"},{"id":90485916,"identity":"023d0890-be17-4a3a-8325-412664511800","added_by":"auto","created_at":"2025-09-03 08:51:31","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":981684,"visible":true,"origin":"","legend":"\u003cp\u003eFig. 5. Biosynthesized a) FESEM images b) EDX spectrum c i to iii) TEM images.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7492605/v1/d31eddfb5f3cf7fdf7d193bf.png"},{"id":91179789,"identity":"5a96c6d7-a1ae-4eb9-838c-40b59f43c6a3","added_by":"auto","created_at":"2025-09-12 12:38:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2530579,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7492605/v1/6750be1d-df42-462e-9538-61fd158fe392.pdf"},{"id":90485922,"identity":"cfc4ec90-4d01-4568-94b6-8388a6aa4f49","added_by":"auto","created_at":"2025-09-03 08:51:31","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1099544,"visible":true,"origin":"","legend":"","description":"","filename":"SupplemantaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-7492605/v1/1bdc2068b3ced15404a60545.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Eco-Friendly Synthesis and Characterization of Crystalline Selenium Nanoparticles via Bacillus cereus","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eIn the recent era, agricultural production increasingly necessitates additional inputs aimed at enhancing crop yield, sustaining plant quality and growth, prolonging the post-harvest longevity of products, and augmenting the market value of plant foods by improving their nutritional attributes and sensory qualities [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. To meet agricultural demands, synthetic fertilizers and toxic pesticides have been widely used, but their overapplication has led to health risks and environmental pollution. As a sustainable alternative, nanotechnology-based solutions, such as nanobiostimulants, nano-fertilizers, and nano-pesticides, are emerging. These innovations offer reduced toxicity compared to conventional products while enhancing crop productivity, disease management, and post-harvest preservation [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In this scenario, gold (Au) and silver (Ag) are often used in nanoparticle (NP) production; however, their usage may be limited by toxicity at certain quantities. Hence, biologically generated Au and AgNPs have enhanced stability and less phytotoxicity, making them advantageous for agricultural applications [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Aside from these few metalloid and metal oxide NPs, such as SiO\u003csub\u003e2\u003c/sub\u003e, ZnO, and CuO, which have been shown to enhance plant nutrition and bolster disease resistance [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eSelenium (Se) is a crucial trace element present as over 25 types of selenoenzymes and selenoproteins in the human body, playing a vital role in sustaining normal physiological activities. Excessive intake or deficiency of Se, whether in organic or inorganic forms, can result in significant harm due to its inherent toxicity. Selenium nanoparticles (SeNPs), the nano-form of Se, exhibit reduced toxicity and enhanced biocompatibility relative to their counterparts [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Selenium is a constituent of several essential enzymes, including thioredoxin reductase and glutathione reductase, and is crucial for sustaining normal human metabolism. Although not essential for plants, selenium is advantageous for crops. At appropriate concentrations, selenium may promote plant development, augment antioxidant capacity, and mitigate damage inflicted by environmental stressors in plants [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. According to Garcia Marquez et al.[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] plant foods are the principal source of Se for humans, with a recommended daily intake of 60\u0026ndash;70 \u0026micro;g/day for adults; nevertheless, excessive consumption of Se (\u0026gt;\u0026thinsp;400 \u0026micro;g/day) may result in detrimental health consequences. Consequently, Se is used as a fertilizer to modulate crop development and yield Se-enriched food. The SeNPs have enhanced bioavailability and reduced toxicity compared to selenite and selenate, suggesting they are viable alternatives to conventional inorganic Se species. Numerous studies indicate that SeNPs function as bio-stimulators and antioxidants, enhancing food quality and providing resistance against phytopathogen assaults [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe synthesis of SeNPs is primarily accomplished through chemical and physical methods. Nonetheless, these methods are expensive, complex, and consistently produce toxic by-products, significantly limiting the broader application of SeNPs. Consequently, it is imperative to investigate environmentally friendly methods for the synthesis of SeNPs. Recently, the biosynthesis methods for the preparation of SeNPs have garnered significant attention [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eUnder the action of selenite reductase and selenate reductase, Selenate (SeO\u003csub\u003e4\u003c/sub\u003e\u0026sup2;\u003csup\u003e\u0026minus;\u003c/sup\u003e) is converted to its red allotrope, selenium (Se\u003csup\u003e0\u003c/sup\u003e). This typically results in the accumulation of SeNPs on the cell surface or in the culture medium. The biogenic synthesis of SeNPs may be facilitated by microorganisms, including bacteria, fungi, and plant parts (root, shoot, and leaf), offering a straightforward, safe, and eco-friendly method for their preparation [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The size and shape of SeNPs can be effectively regulated by adjusting the pH, incubation temperature, metal ion concentration, reaction time, and the quantity of organic matter in the culture medium [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Presently, many bacterial isolates have been recognized for their ability to synthesize SeNPs, owing to their ease of handling, rapid growth rate, and low-cost maintenance, making them ideal organisms for the green synthesis of nanoparticles [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Several studies have indicated that certain bacterial isolates, including \u003cem\u003eLactobacillus acidophilus\u003c/em\u003e [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], \u003cem\u003eBacillus cereus\u003c/em\u003e [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], \u003cem\u003eBacillus licheniformis\u003c/em\u003e [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], and \u003cem\u003ePseudomonas alcaliphila\u003c/em\u003e [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], are capable of producing Bio-SeNPs, with applications in antimicrobial, anticancer, bioremediation, and enhancement of plant quality. Agricultural soil associated with mica mines creates a forest that serves as a detritus-based niche, functioning as a hub for bacterial diversity. The genera \u003cem\u003eCandidatus solibacter\u003c/em\u003e, \u003cem\u003eCandidatus koribacter\u003c/em\u003e, \u003cem\u003eSphingomonas\u003c/em\u003e, \u003cem\u003eGemmatimonas\u003c/em\u003e, \u003cem\u003eBacillus\u003c/em\u003e, \u003cem\u003eSorangium, Serratia\u003c/em\u003e, \u003cem\u003ePseudomonas\u003c/em\u003e, and \u003cem\u003eAzoarcus\u003c/em\u003e constituted a significantly diverse bacterial community observed [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Numerous mining locations are recognized for containing selenium, a trace metal inherently found in the Earth's crust [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The content fluctuates according to rock type, averaging around 0.09 mg/kg in the top continental crust. Shales generally possess high concentrations (about 0.3 mg/kg), whereas granites and limestones have around 0.025 mg/kg, and sandstones have even lower amounts, around 0.01 mg/kg. Coal often exhibits elevated selenium concentrations, sometimes reaching several mg/kg [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe current work successfully synthesized Bio-SeNPs using rhizospheric bacteria isolated from mica-rich agricultural soil in Giridih, Jharkhand, India. The study presents a comprehensive analysis of the characterization of Bio-SeNPs using diverse tools.\u003c/p\u003e"},{"header":"2 Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Moist soil sample collection\u003c/h2\u003e\u003cp\u003eSoil samples from the rhizosphere of rice were obtained from three blocks (Deori, Gawan, and Tisri) in the Giridih district of Jharkhand, India Fig.\u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. The predominant operating mica mines in Giridih are located within these three blocks of the district. Samples were obtained from rice fields next to mica mines, where leachate and surface runoff from the mines accumulate naturally owing to the lower elevation. Three soil samples were randomly taken from each mine, and all samples were homogenized to form a single soil sample for isolation. Samples were maintained in sterile plastic bags, appropriately labeled, transported in an ice box, and promptly transferred to the laboratory for further processing.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Isolation of Bacterial Strain Transforming Selenium (Se\u003csup\u003e0\u003c/sup\u003e)\u003c/h2\u003e\u003cp\u003eSerial dilution and nutrient agar plating were used for the isolation of bacterial strains that convert selenium. Commercial Aleksandrow broth medium (CAB- HiMedia Laboratories, M-1997) was used for screening of bacterial isolation, which is mica-rich selective medium. The enrichment technique, followed by a serial dilution technique (in 0.87% normal sterile saline solution) was used for the isolation. In the enrichment process, 5 g of soil was inoculated into sterile CAB broth media and incubated at 28\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C under shaking conditions for seven days. After incubation, serially diluted (10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e) enriched bacterial isolates were plated on CAB media with agar-agar (3%) followed by incubation at 28\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C for seven days [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The pure culture of the colonies forming a clear halo zone was further established in Aleksandrow broth media. 21 Screened bacterial isolates were stained (gram-stain) for approximate detection of purity of each of the isolates, and pure colonies (n\u0026thinsp;=\u0026thinsp;30) were transferred to sterile slants on nutrient agar medium (HiMedia) and sterile glycerol stock medium for long-term preservation at -20 \u0026deg; C. The morphological parameters of chosen isolates, including pigmentation, Gram reaction, margin, colony height, optical density, and slime generation, were previously examined by our study [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Molecular Characterization of Selected Isolate\u003c/h2\u003e\u003cp\u003eThe molecular identification of chosen bacterial isolates was conducted as previously described by Ghosh et al. [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Genomic DNA was extracted from a single colony of bacterial isolates for confirmation of molecular identification using a DNA isolation kit (NucleoSpin Microbial DNA, MACHEREY-NAGEL). Genomic DNA was preserved at -20\u0026deg;C for further use. The amplification of 16S ribosomal DNA was performed with universal bacterial primer sets, namely forward primer 27f (5\u0026acute;-AGAGTTTGATCCTGGCTCAG-3\u0026acute;) and reverse primer 1492r (5\u0026acute;-TACGGTTACCTTGTTACGACTT-3\u0026acute;). The PCR amplification procedure consisted of a 25 \u0026micro;l mixture containing 45 ng of genomic DNA, 5 U/\u0026micro;l of Taq polymerase (Takara Bio Inc.), 2.5 \u0026micro;l of 10 \u0026times; buffer, 2.5 mM dNTP mixture, 1.5 mM MgCl\u003csub\u003e2\u003c/sub\u003e, and 10 pmol/\u0026micro;l of each primer. The PCR reaction was conducted under the following parameters: initial denaturation at 94\u0026deg;C for 4 minutes, followed by 30 cycles comprising denaturation at 94\u0026deg;C for 30 seconds, annealing at 54\u0026deg;C for 1 minute, extension at 72\u0026deg;C for 1 minute, and a final extension at 72\u0026deg;C for 7 minutes. To verify amplification, 5 \u0026micro;l of the PCR result was subjected to 1% agarose gel electrophoresis and then analyzed using a gel documentation system (Bio-Rad). A 100 bp plus ladder (GeNetBio Corp, Korea) was used to ascertain the product size. Upon confirmation, the amplified PCR products were purified using gel electrophoresis with a PCR purification kit, following the manufacturer's instructions (Macherey-Nagel), and stored at -20\u0026deg;C for further analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Synthesis of Bio-SeNPs\u003c/h2\u003e\u003cp\u003eNutrient broth medium was made and autoclaved for 20 minutes at 121\u0026deg;C. Subsequently, a sterilized sodium selenate (Na\u003csub\u003e2\u003c/sub\u003eSeO\u003csub\u003e4\u003c/sub\u003e, HIMEDIA-GRM7516-100G) solution was added from a 10,000 mg/L stock solution to achieve a concentration of 300 mg/L. The flasks were inoculated with a 10% inoculum of a 24-hour-old culture of a previously identified strain (\u003cem\u003eBacillus cereus\u003c/em\u003e), followed by incubation for 48 hours in an orbital shaker at 200 rpm and 30\u0026deg;C. Upon completion of the incubation period, the culture medium's hue changed to red, signifying the production of nano-selenium. The fermentation broth underwent centrifugation for 20 minutes at 5000 rpm, after which the supernatant was discarded. The residual pellets were subjected to three consecutive washes with 0.9% NaCl solution, thereafter treated with 37% HCl, maintained at room temperature for two days, and then centrifuged and washed several times with autoclaved distilled water to eliminate HCl. The acquired red precipitate was desiccated at 70\u0026deg;C and used for further analysis [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e illustrates the flow chart for the production of Bio-SeNPs.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Characterization of the Bio-Synthesis SeNPs\u003c/h2\u003e\u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\u003ch2\u003e2.5.1. UV-Visible Absorption Spectroscopic Analysis\u003c/h2\u003e\u003cp\u003eThe nanoparticle was characterized using a UV-Vis spectrophotometer (Thermo Scientific, GENESYS 180 UV-VIS spectrophotometer) throughout a wavelength range of 200 to 800 nm at 1-nm intervals, employing a 1cm thick quartz cuvette. The reaction's progress was observed by the visible alteration in the color of the mixture.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\u003ch2\u003e2.5.2. X-Ray Diffraction (XRD) Analysis\u003c/h2\u003e\u003cp\u003eThe crystalline phase of the Bio-SeNPs was determined using XRD measurements conducted using a Philips X-ray diffractometer (SmartLab, RIGAKU). The diffraction pattern of a thin layer of synthesized NPs on a glass substrate was recorded from 10\u0026deg; to 80\u0026deg; (2θ) with a step size of 0.02\u0026deg;, using Cu Kα radiation (k\u0026thinsp;=\u0026thinsp;1.542 \u0026Aring;) at 40 kV and 30 mA. The crystal phase was ascertained by juxtaposing the computed interplanar spacing values and the associated diffraction peak intensities with the theoretical values from the Joint Committee on Powder Diffraction Standards-International Centre for Diffraction Data (JCPDS-ICCD) database [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section3\"\u003e\u003ch2\u003e2.5.3. Fourier Transform Infrared (FTIR) Spectroscopic Analysis\u003c/h2\u003e\u003cp\u003eFTIR spectroscopy to used identify and characterize biologically synthesized Se nanoparticles measuring a sample absorbed infrared (IR) radiation. Using the attenuated total reflection (ATR) technique, spectra were obtained with a Thermo Scientific Nicolet iS5, scanning the range of 3500 to 400 cm⁻\u0026sup1; at a resolution of 4 cm⁻\u0026sup1; [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section3\"\u003e\u003ch2\u003e2.5.4. X-ray photoelectron spectroscopy (XPS) analysis\u003c/h2\u003e\u003cp\u003eX-ray photoelectron spectroscopy (XPS) measurements for surface chemistry investigations were conducted using a K-Alpha instrument (PHI 5000 VersaProbe III) with Al Kα monochromatic X-ray (1486.6 eV) radiation and a spot size of 300 \u0026times; 300 \u0026micro;m\u0026sup2;. The spectrometer has a flood cannon for charge compensation, with any charge-induced energy shift rectified by anchoring the C 1s line at 284.4 eV. The peak-fitting approach included subtracting a Shirley-type background from the spectra and fitting the peaks using symmetric Gaussian functions [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section3\"\u003e\u003ch2\u003e2.5.5. Scanning Electron Microscope (SEM) Analysis\u003c/h2\u003e\u003cp\u003eA high-resolution scanning electron microscope SUPRA 55 VP- 4132 CARL ZEISS With attachment \u0026ndash; ED was used to ascertain the dimensions and morphology of the produced nano-selenium particles. Two distinct imaging modalities were employed\u0026mdash;bright field (with an electron accelerating voltage of 200 kV, using LaB6 as the electron source) and diffraction pattern imaging (DPI). A 4 k x 4 k resolution eagle CCD camera was used to capture transmitted electron pictures. The TEM Imaging \u0026amp; Analysis program was used to evaluate the EDX peak spectrum [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section3\"\u003e\u003ch2\u003e2.5.6. Transmission Electron Microscopy (TEM) Analysis\u003c/h2\u003e\u003cp\u003eFor TEM, 5\u0026micro;l of the sample solution was deposited on carbon-coated copper grids and stored in desiccators. Upon drying, the grids were examined using a JEOL JEM 2100 HR. The size distribution for the polydispersity index (PDI) of SeNPs was assessed using the following Eq.\u0026nbsp;(\u003cspan refid=\"Equ1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$\\:PDI=\\:{\\left(\\sigma\\:/\\text{D}\\right)}^{2}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003ewhere D represents the mean diameter of the Bio-SeNPs and σ denotes the standard deviation (SD).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"3 Results and Discussion","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.1. General features and molecular identification of the Potential Bacterial Isolates\u003c/h2\u003e\u003cp\u003eThe bacterial strain was isolated from mica-contaminated rice rhizospheric agricultural soil. A previous study by Ghosh et al.[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] reported the morphological characterization of the isolate as follows: pigmentation \u0026ndash; white, Gram reaction \u0026ndash; positive, margin \u0026ndash; smooth, opacity \u0026ndash; translucent to opaque, and slime production \u0026ndash; high. Molecular identification of the bacterial isolate was carried out using the 16S ribosomal DNA region. The isolate was identified as \u003cem\u003eBacillus\u003c/em\u003e (strain: \u003cem\u003eBacillus cereus strain\u003c/em\u003e YB1806). It was previously reported that this bacterial isolate can solubilize potassium and phosphorus from non-exchangeable forms of these elements and also possesses nitrogen-fixing ability. The selected strain can also promote plant growth by producing indole-3-acetic acid (IAA), gibberellic acid (GA₃), hydrogen cyanide (HCN), ammonia (NH₃), and siderophores. Based on all the previous characteristics, the \u003cem\u003eBacillus cereus\u003c/em\u003e isolate showed the potential to reduce Se⁴⁺ to Se⁰ through bacterial enzyme- and metabolite-mediated mechanisms, and the results were positive. The visual identification of changes in colour of the broth Fig. S (a \u0026amp; b) after 48 hr of incubation at room temperature (30\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C) was considered as a positive result for the formation of Bio-SeNPs. The process of biosynthesizing selenium nanoparticles (SeNPs) through bacterial isolates entails a bioreduction mechanism, wherein bacteria convert soluble selenium salts, such as sodium selenite (SeO₃\u0026sup2;⁻) or sodium selenate (SeO₄\u0026sup2;⁻), into elemental selenium (Se⁰). This method is environmentally sustainable, economically viable, and safe, in contrast to traditional chemical and physical approaches[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. A UV-VIS spectrophotometer was used to further monitor the Bio-SeNPs that were produced. Both the negative control (Na₂SeO₄ solution) and the positive control (just nutrient broth) showed no discernible color change throughout the process.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Characterization of Bio-SeNPs\u003c/h2\u003e\u003cdiv id=\"Sec17\" class=\"Section3\"\u003e\u003ch2\u003e3.2.1. UV-Visible Spectra Analysis of the SeNPs Suspension\u003c/h2\u003e\u003cp\u003eUV-VIS spectroscopy was used to assess the optical characteristics of the bio-synthesized SeNP suspensions Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e (a \u0026amp; c). The UV\u0026ndash;VIS spectrum of the SeNP suspensions showed an increase in absorbance intensity in the shorter UV range. The bio-synthesized SeNPs produced by \u003cem\u003eBacillus cereus\u003c/em\u003e exhibited a maximum absorbance at 282 nm, which might be due to a plasmon resonance spike at approximately 2 nm, a characteristic feature of selenium nanoparticles Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e (c). Additionally, no prominent peaks were observed for the bacterial broth Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e (a) and selenium salt within the studied wavelength range Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e (b). The findings align with earlier studies, including the research by Dhanjal and Cameotra [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], which illustrated the aerobic biogenesis of selenium nanospheres by \u003cem\u003eBacillus cereus\u003c/em\u003e and analyzed their optical properties through UV\u0026ndash;VIS spectroscopy. Their research established that the appearance of red coloration and a broad UV\u0026ndash;VIS peak serve as a reliable indicator of SeNP synthesis.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section3\"\u003e\u003ch2\u003e3.2.4. XRD Analysis\u003c/h2\u003e\u003cp\u003eXRD analysis was conducted to elucidate the crystalline characteristics of SeNPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.). The XRD patterns confirmed the crystalline structure of synthesized SeNPs exhibit a prominent diffraction peak at 2θ\u0026thinsp;=\u0026thinsp;23.51\u0026deg;, 29.70\u0026deg;, 41.30\u0026deg;, 43.64\u0026deg;, 45.3\u0026deg;, 48.10\u0026deg;, 51.72\u0026deg; and 56.14\u0026deg; corresponding to the (100), (101), (110), (102), (111), (200), (201) and (112) planes respectively. The result further confirmed the hexagonal structure of SeNP according to the JCPDS reference code 06\u0026ndash;0362. This diffraction pattern is characteristic of hexagonal SeNPs, recognized for their thermodynamic stability and distinctive physicochemical features [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The results align with prior research indicating that SeNPs synthesized by several green and chemical methods exhibited comparable peak positions and phase identities [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section3\"\u003e\u003ch2\u003e3.2.3. FTIR Characterisation of Bio-SeNPs\u003c/h2\u003e\u003cp\u003eFTIR spectroscopy analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003e) was conducted to examine the presence of biomolecules in synthesized SeNPs. The FTIR spectra of SeNP span from 4000 to 400 cm\u003csup\u003e-1\u003c/sup\u003e. The spectra reveal absorption peaks at 3400 cm\u003csup\u003e-1\u003c/sup\u003e and 2920 cm\u003csup\u003e-1\u003c/sup\u003e, corresponding to the stretching vibrations of O-H and aliphatic C-H, respectively. The prominent peak at 1642 cm\u003csup\u003e-1\u003c/sup\u003e is attributed to the CO\u0026ndash;NH stretching vibration associated with amide-I, a defining feature of proteins, while the peak at 1546 cm\u003csup\u003e-1\u003c/sup\u003e relates to the N\u0026ndash;H bending vibration linked to amide-II, another characteristic section of proteins. The absorption peaks at 1380 cm\u003csup\u003e-1\u003c/sup\u003e, 1224 cm\u003csup\u003e-1\u003c/sup\u003e, and 1064 cm\u003csup\u003e-1\u003c/sup\u003e are indicative of symmetrical C\u0026ndash;H stretching, C\u0026ndash;O stretching vibrations of the carboxyl group, and C\u0026ndash;O\u0026ndash;C bending vibrations, respectively, within the typical area of polysaccharides. The absorption spectra of SeNPs exhibit significant peaks at 689 cm⁻\u0026sup1; and 480 cm⁻\u0026sup1;, indicating Se\u0026ndash;Se/Se\u0026ndash;O bond vibrations that affirm the biogenesis of SeNPs [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section3\"\u003e\u003ch2\u003e3.2.5. XPS analysis\u003c/h2\u003e\u003cp\u003eXPS technique has been used to analyse the surface functional group of SeNP. The XPS survey spectrum, recorded between 0 and 1100 eV (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e), represents the core binding energies of C, N, O, Cl and Se in SeNP (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The high-resolution spectra of C, N, O, Cl and Se have also been recorded to analyse the surface electronic property. The high resolution of the C 1s spectrum has been carefully deconvoluted, which consists of three peaks at 284.8, 285.7 and 288.6 eV corresponding to C\u0026ndash;C or C\u0026ndash;H in amino acid chains, C\u0026ndash;O or C\u0026ndash;H in alcohol, amine, ether or amide and C\u0026thinsp;=\u0026thinsp;O or O\u0026ndash;C\u0026ndash;C in carbonyl, amide or hemiacetal bindings, respectively Fig. S3(b) [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The Gaussian fitted O 1s peak is deconvolved into two peaks at 532.2 and 533.3 eV. These two peaks are assigned as C\u0026thinsp;=\u0026thinsp;O and C\u0026ndash;O binding, respectively Fig. S3 (c) [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The presence of C\u0026thinsp;=\u0026thinsp;O and C\u0026ndash;O bonds in O 1s and C 1s confirms the C and O bonding, which plays a pivotal role in the formation of SeNP. The deconvoluted N 1s and Cl 2p scans are fitted with a Gaussian profile, amine group detected at 399.3 eV, protonated amine group detected at 400.5 eV and Cl 2p\u003csub\u003e1/2\u003c/sub\u003e detected at 199.9 eV, respectively Fig. S3(d) [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In case of Se 3d XPS spectrum has been deconvoluted with care and shows two deconvoluted peaks at 55.3 and 56.5 eV and assigned as Se 3d\u003csub\u003e3/2\u003c/sub\u003e and Se 3d\u003csub\u003e5/2\u003c/sub\u003e spin-orbit coupling, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e) [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. From the literature survey, it is noted that 3d\u003csub\u003e5/2\u003c/sub\u003e of metallic selenium Se\u003csup\u003e0\u003c/sup\u003e is expected at 55.1 eV, whereas Se\u003csup\u003e\u0026minus;\u0026thinsp;II\u003c/sup\u003e, Se\u003csup\u003eII\u003c/sup\u003e, Se\u003csup\u003eIV\u003c/sup\u003e, Se\u003csup\u003eIV\u003c/sup\u003e are expected approximately at binding energies\u0026thinsp;\u0026lt;\u0026thinsp;55, 57.7, 59.4, and 61 eV, respectively [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The binding energy at 55.3 eV in the NP confirmed the presence of Se\u003csup\u003e0\u003c/sup\u003e. Also, it has been noted that a slightly higher binding energy is formed due to polarised leading of Se\u003csup\u003e0\u003c/sup\u003e. It has been observed selenium signal's initial intensity is substantially lower as a result of the data being noisy. From here, it has been concluded that in our SeNP, selenium is a combination of polarized Se\u003csup\u003e0\u003c/sup\u003e and Se\u003csup\u003eII\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section3\"\u003e\u003ch2\u003e3.2.4 SEM and TEM analysis\u003c/h2\u003e\u003cp\u003eThe biosynthesized selenium nanoparticles (SeNPs) were thoroughly characterized to assess their shape, structure, and composition using Field Emission Scanning Electron Microscopy (FESEM), High-Resolution Transmission Electron Microscopy (HRTEM), and Energy Dispersive X-ray Spectroscopy (EDX) (Fig.\u0026nbsp;6). FESEM images showed that the SeNPs primarily had a spherical shape, with some appearing quasi-spherical and elliptical (Fig.\u0026nbsp;6a). This diversity is common in biologically synthesized nanoparticles, where factors like biomolecular capping agents influence particle growth [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. These agents, which include fungal polysaccharides, proteins, or enzymatic cofactors, play an important role in controlling nucleation and growth during synthesis. Overall, the nanoparticles were well-dispersed, though some areas showed moderate aggregation, likely due to biomolecular bridging or incomplete electrostatic stabilization. The particle sizes, measured from the FESEM images using ImageJ software (n\u0026thinsp;=\u0026thinsp;50), ranged from 48 to 115 nm, with an average size of 82.4\u0026thinsp;\u0026plusmn;\u0026thinsp;15.6 nm. This size range is ideal for medical and environmental uses because it balances the surface area-to-volume ratio with colloidal stability [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The particles appeared smooth and uniformly electron-dense, suggesting a consistent selenium core without significant pores or surface defects. The brightness seen in the FESEM images is consistent with selenium\u0026rsquo;s high atomic number (Z\u0026thinsp;=\u0026thinsp;34), which improves backscattered electron yield and provides excellent imaging contrast, reinforcing the elemental identity of the particles [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eEnergy Dispersive X-ray Spectroscopy (EDX) analysis was performed to examine the elemental makeup of the SeNPs and confirm the purity and uniformity of the selenium incorporation. The EDX spectrum (Fig.\u0026nbsp;6b) had a dominant sharp peak at about 11.22 keV, matching the Kα emission line of selenium and confirming it as the main element in the nanoparticle core. The peak had a narrow full-width at half maximum (FWHM), indicating high purity and no significant overlap or interference from secondary phases. Quantitative analysis through peak integration showed that selenium content was around 91.6 wt%, confirming the strong presence of elemental Se in the material. The high signal-to-noise ratio and the sharp Gaussian profile of the peak reflect efficient selenium incorporation and a strong detector response, typical of well-prepared crystalline selenium.\u003c/p\u003e\u003cp\u003eIn addition to selenium, the spectrum showed minor peaks related to oxygen (~\u0026thinsp;0.52 keV) and carbon (~\u0026thinsp;0.27 keV), likely from organic matter in the synthesis medium or the carbon support grid used during sample preparation [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Oxygen might be part of a thin layer on the nanoparticle surface or linked to capping molecules that contain hydroxyl or carboxyl groups. These results align with earlier studies on biosynthesized selenium, where organic functional groups from fungal extracts slightly oxidize the nanoparticle surface but do not alter the core elemental structure. Trace amounts of sulfur (~\u0026thinsp;2.31 keV) and phosphorus (~\u0026thinsp;2.01 keV) were also found, likely from leftover biomolecules that contain thiol or phosphate groups (Hashem et al. 2022)[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Importantly, no signals were detected for heavy metals or transition elements like Cu, Zn, or Fe, confirming the chemical purity of the synthesis process and ruling out co-precipitation or contamination [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eTo confirm and improve the structural insights from FESEM, HRTEM analysis was conducted. The high-resolution TEM images revealed individual SeNPs with sharp contrast and well-defined edges, confirming the particulate nature of the synthesized material. At low magnification, the particles maintained a nearly spherical shape with minimal aggregation, which may relate to grid preparation and drop-casting of well-dispersed colloidal samples. The average diameter from the TEM images was about 76.1\u0026thinsp;\u0026plusmn;\u0026thinsp;10.3 nm (Fig.\u0026nbsp;6ci-ciii), which aligns with the FESEM measurements, reinforcing the accuracy and consistency of the particle sizing. At higher magnification, the SeNPs showed a strong contrast between the selenium-rich core and the surrounding organic material, indicating a surface-bound organic layer. This organic coating, likely made of fungal proteins, amino acids, and other metabolites, acts as a stabilizing shell, preventing particles from merging and improving colloidal stability in water [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Although the shell is thin (~\u0026thinsp;1.5\u0026ndash;2.0 nm), it is vital for modulating surface interactions, especially in medical applications where surface properties influence biocompatibility and targeting.\u003c/p\u003e\u003cp\u003eThe nanoparticles appeared solid and crystalline with no signs of hollowness or core-shell separation. No voids, cracks, or amorphous boundaries were seen in the SeNP cores (Fig.\u0026nbsp;6ci-ciii), supporting the idea that nucleation was driven by a uniform and quick reduction of selenium oxyanions (e.g., SeO₃\u0026sup2;⁻ or SeO₄\u0026sup2;⁻) to elemental selenium (Se⁰) with the help of fungal reductants [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. The particle size distribution, while not perfectly uniform, showed a relatively narrow range, suggesting a diffusion-limited growth process instead of burst nucleation or Ostwald ripening, which are more common in chemically synthesized systems [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. This level of control, achieved without toxic reducing agents or surfactants, highlights the effectiveness of the biological synthesis process in creating nanomaterials ready for use [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe compositional purity is particularly critical in assessing the potential uses of the SeNPs in sensitive biological fields, such as drug delivery or antimicrobial coatings, where impurities can seriously affect toxicity or biological function [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Additionally, the consistent elemental makeup across various sampling points suggests a high level of uniformity in the synthesized batch [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Although complete elemental mapping wasn\u0026rsquo;t conducted, the strong selenium peak across multiple EDX spots supports the idea of even Se distribution.\u003c/p\u003e\u003cp\u003eThe combined structural and compositional data obtained through FESEM, TEM, and EDX strongly confirms the successful synthesis of selenium nanoparticles through a green, biologically mediated approach. Their structural integrity, near-spherical shape, nanoscale size, and elemental purity indicate that the fungal synthesis process not only facilitated the reduction of selenium oxyanions but also directed the growth and stabilization of selenium nuclei into uniform nanostructures. The measured particle size range (45\u0026ndash;115 nm) is beneficial, providing a high surface area while avoiding excessive aggregation or rapid settling. The organic layer seen in the TEM plays a dual role: it stabilizes the nanoparticles colloidally and offers a functional surface that can be used for further attachment to targeting ligands or biomolecules in therapeutic uses.\u003c/p\u003e\u003cp\u003eFurthermore, the stability and composition of the SeNPs suggest excellent suitability for redox-sensitive applications, such as oxidative stress management, reactive oxygen species (ROS) removal, or targeted cancer treatment, where selenium\u0026rsquo;s redox-active nature offers therapeutic benefits. The thin organic shell also hints at possible bio-interaction behaviors, such as improved cellular uptake or enzyme-like functions. In environmental cleanup, these particles can act as effective electron donors or surface agents for immobilizing heavy metals or breaking down stubborn pollutants. Overall, the characterization confirms the success of the biological synthesis method in producing high-quality, compositionally pure, and structurally stable selenium nanoparticles suitable for various biomedical and environmental applications.\u003c/p\u003e\u003cp\u003eThe studies analyzed for the comparison of various synthesis processes are enumerated in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. This comparative study examined the physical, chemical, and green synthesis of SeNPs, focusing on the size and shape of the resulting nanoparticles. Various approaches, including microbial, fungal, green, and algal biosynthesis, have been examined within biosynthetic processes. Previous investigations, as illustrated in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, indicate that hydrothermal methods Shar et al. [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e] produced hexagonal nanoparticles measuring 169 nm, whereas electrochemical methods Zhang et al. [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e] yielded tubular nanoparticles of 200 nm. Photocatalysis Triantis et al. [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e] exhibited no discernible shape, with nanoparticle sizes ranging from 4 to 110 nm. Among different biosynthesis processes, microbial [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e], green synthesis [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e], fungal [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e], and algal [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e] methods have demonstrated the capability to synthesize selenium nanoparticles with sizes ranging from 40 to 400 nm. Considering the above-mentioned points, the present study highlights the biosynthesis of SeNPs by bacterial isolates (\u003cem\u003eBacillus cereus\u003c/em\u003e) from mica-contaminated rhizospheric agricultural soil of Giridih district, India. In this study, Bacillus cereus produced nanoparticles measuring 282 nm, which falls within the reported range of 40\u0026ndash;400 nm, confirming that the synthesized nanoparticles are SeNPs.\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 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eComparative comparison of the synthesis of Bio-SeNPs by various methodologies.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\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\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSynthesis method\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eProcess\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eShape\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSize (nm)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003ereference\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePhysical/Chemical\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHydrothermal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ehexagonal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e169.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eElectrochemical\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003etube\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e200\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePhotocatalysis\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e40\u0026ndash;110\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBiosynthesis\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMicrobial\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003espherical\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e80\u0026ndash;220\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFungal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003evarious\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e~\u0026thinsp;137nm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGreen synthesis\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003espherical\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e40\u0026ndash;100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAlgal synthesis\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003espherical\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e45\u0026ndash;80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMicrobial\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003espherical\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e400\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\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\u003c/div\u003e\u003c/div\u003e"},{"header":"4 Conclusion","content":"\u003cp\u003eThis study demonstrates a green, scalable, and sustainable method for making selenium nanoparticles (SeNPs) using a strain of Bacillus cereus, which we isolated from selenium-contaminated rice field soil. The biosynthetic method in this work relies on the natural ability of the bacterial strain to reduce selenite (Se⁴⁺) ions to elemental selenium (Se⁰) nanoparticles at room temperature. This process does not require harmful chemicals or extreme conditions. Microbial synthesis reduces environmental impact and improves the biocompatibility and functionality of the resulting nanostructures. The reddish color of the reaction mixture indicated the formation of elemental selenium, which was confirmed by a distinct UV-Visible absorption peak at 282 nm, a common marker for selenium nanostructures, showing successful synthesis.\u003c/p\u003e\u003cp\u003eDetailed analyses using FESEM and TEM showed that the biosynthesized SeNPs were mostly spherical and evenly sized, with smooth surfaces. This indicates effective nucleation and controlled growth in biological conditions. FESEM images displayed uniform particle formation with little agglomeration, while TEM provided exact size measurements, estimating the average particle size at about 76.1\u0026thinsp;\u0026plusmn;\u0026thinsp;10.3 nm. These nanoscale dimensions are ideal for many biomedical applications since they balance cellular uptake efficiency and lower toxicity. Additionally, a faint organic layer around the SeNPs, seen in high-resolution TEM images, suggests the presence of biomolecular capping agents, likely made up of extracellular proteins, enzymes, or metabolites from the bacteria.\u003c/p\u003e\u003cp\u003eWe evaluated elemental composition and purity using energy-dispersive X-ray spectroscopy (EDX), which confirmed selenium as the principal component with a strong peak at approximately 11.22 keV. Quantitative analysis indicated that selenium content reached up to 91.6 wt%, with small amounts of carbon and oxygen, coming from organic capping agents or the carbon-coated sample grid. No signs of toxic heavy metals or synthetic impurities were found, confirming the high purity and environmentally friendly nature of the biosynthesized nanomaterial. The EDX results support the successful reduction of selenite to Se⁰ and exclude the possibility of external elemental contamination during synthesis.\u003c/p\u003e\u003cp\u003eFourier-transform infrared spectroscopy (FTIR) further examined the surface chemistry, revealing various functional groups linked to protein and carbohydrate biomolecules. We observed key absorption bands at 3400 cm⁻\u0026sup1; (O\u0026ndash;H and N\u0026ndash;H stretching), 1642 cm⁻\u0026sup1; (amide I), and 1546 cm⁻\u0026sup1; (amide II), suggesting that bacterial proteins acted as both reducing and stabilizing agents. Additional peaks corresponding to C\u0026ndash;H, C\u0026thinsp;=\u0026thinsp;O, and C\u0026ndash;N vibrations imply the presence of polysaccharides and amino acids on the nanoparticle surface. This organic capping is vital for preventing aggregation and improving the biocompatibility and functionality of SeNPs in biological contexts.\u003c/p\u003e\u003cp\u003eX-ray diffraction (XRD) analysis provided insights into crystallinity and phase composition. The diffractogram showed characteristic peaks for the (100), (101), (110), and (201) planes of crystalline selenium, matching the standard JCPDS card No. 06-0362. The sharpness and intensity of these peaks confirm the high crystallinity of the nanoparticles, which is important for their physical properties, redox activity, and electronic structure. The XRD pattern did not show amorphous selenium or any secondary phases, reinforcing the effectiveness of the biosynthetic method in producing phase-pure SeNPs.\u003c/p\u003e\u003cp\u003eTo gain more information on elemental oxidation states and surface bonding, we performed X-ray photoelectron spectroscopy (XPS). The high-resolution Se 3d spectrum displayed a doublet at 55.3 eV and 56.5 eV, indicating the Se⁰ oxidation state. The lack of peaks at higher binding energies suggests there were no residual selenite or selenate species, confirming complete reduction. Additional spectra for carbon (C 1s) and oxygen (O 1s) showed peaks consistent with C\u0026ndash;C, C\u0026thinsp;=\u0026thinsp;O, and O\u0026ndash;H groups, reflecting the complex organic layer around the particles. The organic functionality on the surface offers opportunities for further biofunctionalization, such as ligand conjugation or drug loading.\u003c/p\u003e\u003cp\u003eBy integrating these analytical techniques, we gain a comprehensive understanding of the biosynthesized selenium nanoparticles: they are uniform, crystalline, highly pure, and surface-functionalized nanostructures produced through a low-cost and eco-friendly microbial process. This synthesis method marks a significant shift from traditional physical and chemical approaches, which are often energy-intensive, toxic, and hard to scale. In contrast, the microbial method does not require external reducing agents or capping chemicals, operates under mild conditions, and yields nanoparticles with excellent biocompatibility and a minimal environmental impact.\u003c/p\u003e\u003cp\u003eBeyond synthesis and characterization, the properties of these SeNPs make them promising for various applications. Their nanoscale size and organic coating boost their potential for targeted drug delivery, antioxidant therapy, antimicrobial coatings, and cancer treatment. Furthermore, their redox activity and stability make them suitable for catalytic and sensing applications, especially in biomedical and environmental fields. From an ecological standpoint, the green synthesis method aligns with sustainable nanotechnology principles and offers a practical strategy for utilizing microbial diversity in bioremediation and producing valuable nanomaterials.\u003c/p\u003e\u003cp\u003eIn summary, this study confirms the feasibility of microbial biosynthesis for high-quality selenium nanoparticles and highlights the importance of detailed physicochemical characterization in assessing their application potential. The \u003cem\u003eBacillus cereus\u003c/em\u003e-mediated approach described here presents a reliable, reproducible, and environmentally friendly method for producing selenium nanostructures, linking green chemistry, nanotechnology, and biotechnology. Future research can further explore optimizing these SeNPs for uses in therapeutics, diagnostics, and environmental detoxification.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgment:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors acknowledge the financial support from SERB, Govt. of India (SERB Sanction Order No: PDF/2023/000288) and Tokclai Tea Research Institute, India for providing all the laboratory facilities.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSG:\u003c/strong\u003e Conceptualization, methodology, data analysis, original draft preparation, writing, investigation, reviewing and editing; \u003cstrong\u003eSM:\u003c/strong\u003e Data analysis, original draft preparation, writing, investigation, reviewing and editing; \u003cstrong\u003eSD:\u003c/strong\u003e Laboratory analysis, methodology, reviewing and editing; \u003cstrong\u003eSP:\u003c/strong\u003e Data analysis, writing and reviewing; \u003cstrong\u003eGKD:\u003c/strong\u003e Resources and reviewing; \u003cstrong\u003eRP:\u003c/strong\u003e Supervision, resources, reviewing and editing; \u003cstrong\u003eSM (Majumder):\u003c/strong\u003e Supervision, conceptualization, reviewing and editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank the Anusandhan National Research Foundation (SERB), Govt. of India, under the project (SERB Sanction Order No: PDF/2023/000288) for the necessary funding.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo datasets were generated or analysed during the current study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclarations\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEthics Approval: Not applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eResearch Involving Humans and Animals Statement: None.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eInformed Consent: None\u003c/p\u003e\n\u003cp\u003eConflict of interest: The authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGarza-Garc\u0026iacute;a, J. J. O., Hern\u0026aacute;ndez-D\u0026iacute;az, J. A., Le\u0026oacute;n-Morales, J. M., Vel\u0026aacute;zquez-Ju\u0026aacute;rez, G., Zamudio-Ojeda, A., Arratia-Quijada, J., \u0026amp; Garc\u0026iacute;a-Morales, S. (2023). Selenium nanoparticles based on Amphipterygium glaucum extract with antibacterial, antioxidant, and plant biostimulant properties. \u003cem\u003eJournal of nanobiotechnology\u003c/em\u003e, \u003cem\u003e21\u003c/em\u003e(1), 252.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHofmann, T., Lowry, G. V., Ghoshal, S., Tufenkji, N., Brambilla, D., Dutcher, J. R., \u0026amp; Landry, M. P. (2020). Technology readiness and overcoming barriers to sustainably implement nanotechnology-enabled plant agriculture. \u003cem\u003eNature Food\u003c/em\u003e, \u003cem\u003e1\u003c/em\u003e(7), 416\u0026ndash;425.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSong, J., Yu, S., Yang, R., Xiao, J., \u0026amp; Liu, J. (2023). Opportunities for the use of selenium nanoparticles in agriculture. \u003cem\u003eNanoImpact\u003c/em\u003e, \u003cem\u003e31\u003c/em\u003e, 100478.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSun, Y., Shi, Y., Jia, H., Ding, H., Yue, T., \u0026amp; Yuan, Y. (2021). Biosynthesis of selenium nanoparticles of Monascus purpureus and their inhibition to Alicyclobacillus acidoterrestris. \u003cem\u003eFood Control\u003c/em\u003e, \u003cem\u003e130\u003c/em\u003e, 108366.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRamakrishnan, M., Arivalagan, J., Satish, L., Mohan, M., Christyraj, J. R. S. S., Chandran, S. A., \u0026amp; Kalishwaralal, K. (2022). Selenium: a potent regulator of ferroptosis and biomass production. \u003cem\u003eChemosphere\u003c/em\u003e, \u003cem\u003e306\u003c/em\u003e, 135531.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGarc\u0026iacute;a M\u0026aacute;rquez, V., Moreno, M., Mendoza, \u0026Aacute;. B., A., \u0026amp; Mac\u0026iacute;as, M., J (2020). Ionic selenium and nanoselenium as biofortifiers and stimulators of plant metabolism. \u003cem\u003eAgronomy\u003c/em\u003e, \u003cem\u003e10\u003c/em\u003e(9), 1399.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKumar, A., \u0026amp; Prasad, K. S. (2021). Role of nano-selenium in health and environment. \u003cem\u003eJournal of Biotechnology\u003c/em\u003e, \u003cem\u003e325\u003c/em\u003e, 152\u0026ndash;163.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYang, R., Li, Q., Zhou, W., Yu, S., \u0026amp; Liu, J. (2022). Speciation analysis of selenium nanoparticles and inorganic selenium species by dual-cloud point extraction and ICP-MS determination. \u003cem\u003eAnalytical Chemistry\u003c/em\u003e, \u003cem\u003e94\u003c/em\u003e(47), 16328\u0026ndash;16336.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHussain, A., Lakhan, M. N., Hanan, A., Soomro, I. A., Ahmed, M., Bibi, F., \u0026amp; Zehra, I. (2023). Recent progress on green synthesis of selenium nanoparticles\u0026ndash;a review. \u003cem\u003eMaterials Today Sustainability\u003c/em\u003e, \u003cem\u003e23\u003c/em\u003e, 100420.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSarkar, J., Mridha, D., Davoodbasha, M. A., Banerjee, J., Chanda, S., Ray, K., \u0026amp; Sarkar, J. (2023). A state-of-the-art systemic review on selenium nanoparticles: Mechanisms and factors influencing biogenesis and its potential applications. \u003cem\u003eBiological Trace Element Research\u003c/em\u003e, \u003cem\u003e201\u003c/em\u003e(10), 5000\u0026ndash;5036.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMedina-Cruz, D., Truong, L. B., Sotelo, E., Mart\u0026iacute;nez, L., Gonz\u0026aacute;lez, M. U., Huttel, Y., \u0026amp; Cholula-D\u0026iacute;az, J. L. (2023). Bacterial-mediated selenium nanoparticles as highly selective antimicrobial agents with anticancer properties. \u003cem\u003eRSC Sustainability\u003c/em\u003e, \u003cem\u003e1\u003c/em\u003e(6), 1436\u0026ndash;1448.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAlam, H., Khatoon, N., Khan, M. A., Husain, S. A., Saravanan, M., \u0026amp; Sardar, M. (2020). Synthesis of selenium nanoparticles using probiotic bacteria Lactobacillus acidophilus and their enhanced antimicrobial activity against resistant bacteria. \u003cem\u003eJournal of Cluster Science\u003c/em\u003e, \u003cem\u003e31\u003c/em\u003e, 1003\u0026ndash;1011.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKora, A. J. (2018). Bacillus cereus, selenite-reducing bacterium from contaminated lake of an industrial area: A renewable nanofactory for the synthesis of selenium nanoparticles. \u003cem\u003eBioresources and Bioprocessing\u003c/em\u003e, \u003cem\u003e5\u003c/em\u003e(1), 1\u0026ndash;12.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAli, E. N., El-Sonbaty, S. M., \u0026amp; Salem, F. M. (2013). Evaluation of selenium nanoparticles as a potential chemopreventive agent against lung carcinoma. \u003cem\u003eInt J Pharm Biol Sci\u003c/em\u003e, \u003cem\u003e2\u003c/em\u003e(4), 38\u0026ndash;46.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang, W., Chen, Z., Liu, H., Zhang, L., Gao, P., \u0026amp; Li, D. (2011). Biosynthesis and structural characteristics of selenium nanoparticles by Pseudomonas alcaliphila. \u003cem\u003eColloids and Surfaces B: Biointerfaces\u003c/em\u003e, \u003cem\u003e88\u003c/em\u003e(1), 196\u0026ndash;201.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChakraborty, S., Ghosh, S., Banerjee, S., Kumar, S., \u0026amp; Bhattacharyya, P. (2024). Elucidating the synergistic effect of acidity and metalloid poisoning on the microbiome through metagenomics and machine learning approaches. \u003cem\u003eEnvironmental Research\u003c/em\u003e, \u003cem\u003e243\u003c/em\u003e, 117885.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEtteieb, S., Magdouli, S., Zolfaghari, M., \u0026amp; Brar, S. (2020). Monitoring and analysis of selenium as an emerging contaminant in mining industry: A critical review. \u003cem\u003eScience of the Total Environment\u003c/em\u003e, \u003cem\u003e698\u003c/em\u003e, 134339.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLopes, G., \u0026Aacute;vila, F. W., \u0026amp; Guilherme, L. R. G. (2017). Selenium behavior in the soil environment and its implication for human health. \u003cem\u003eCi\u0026ecirc;ncia e Agrotecnologia\u003c/em\u003e, \u003cem\u003e41\u003c/em\u003e, 605\u0026ndash;615.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGhosh, S., Mondal, S., Banerjee, S., Mukherjee, A., \u0026amp; Bhattacharyya, P. (2023). Temporal Dynamics of Potassium Release from Waste Mica as Influenced by Potassium Mobilizing Bacteria. \u003cem\u003eJournal of Pure and Applied Microbiology\u003c/em\u003e. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.22207/JPAM.17.1.17\u003c/span\u003e\u003cspan address=\"10.22207/JPAM.17.1.17\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGhazi, A. A., El-Nahrawy, S., El-Ramady, H., \u0026amp; Ling, W. (2022). Biosynthesis of nano-selenium and its impact on germination of wheat under salt stress for sustainable production. \u003cem\u003eSustainability\u003c/em\u003e, \u003cem\u003e14\u003c/em\u003e(3), 1784.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYadav, S. K., Kumar, A., \u0026amp; Mehta, N. (2023). Synthesis and characterization of nanostructured graphene-doped selenium. \u003cem\u003eRSC advances\u003c/em\u003e, \u003cem\u003e13\u003c/em\u003e(20), 13564\u0026ndash;13574.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMahanty, S., Bakshi, M., Ghosh, S., Chatterjee, S., Bhattacharyya, S., Das, P., \u0026hellip;Chaudhuri, P. (2019). Green synthesis of iron oxide nanoparticles mediated by filamentous fungi isolated from Sundarban mangrove ecosystem, India. \u003cem\u003eBioNanoScience\u003c/em\u003e, \u003cem\u003e9\u003c/em\u003e, 637\u0026ndash;651.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMatouk, Z., Islam, M., Guti\u0026eacute;rrez, M., Pireaux, J. J., \u0026amp; Achour, A. (2022). X-ray photoelectron spectroscopy (XPS) analysis of ultrafine au nanoparticles supported over reactively sputtered TiO2 films. \u003cem\u003eNanomaterials\u003c/em\u003e, \u003cem\u003e12\u003c/em\u003e(20), 3692.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKozlov, N. K., Natashina, U. A., Tamarov, K. P., Gongalsky, M. B., Solovyev, V. V,Kudryavtsev, A. A., \u0026hellip; Osminkina, L. A. (2017). Recycling of silicon: from industrial waste to biocompatible nanoparticles for nanomedicine. \u003cem\u003eMaterials Research Express\u003c/em\u003e, \u003cem\u003e4\u003c/em\u003e(9), 095026.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMoreno-Mart\u0026iacute;n, G., Espada-Bernab\u0026eacute;, E., G\u0026oacute;mez-G\u0026oacute;mez, B., Le\u0026oacute;n-Gonz\u0026aacute;lez, M. E., \u0026amp; Madrid, Y. (2023). Evaluation of the transformation of selenite and selenium nanoparticles to seleno-amino acids produced by Escherichia coli and Staphylococcus aureus by using liquid chromatography-inductively coupled plasma mass spectrometry and single-particle-inductively coupled plasma mass spectrometry and different sample treatments. \u003cem\u003eSpectrochimica Acta Part B: Atomic Spectroscopy\u003c/em\u003e, \u003cem\u003e200\u003c/em\u003e, 106611.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDhanjal, S., \u0026amp; Cameotra, S. S. (2010). Aerobic biogenesis of selenium nanospheres by Bacillus cereus isolated from coalmine soil. \u003cem\u003eMicrobial cell factories\u003c/em\u003e, \u003cem\u003e9\u003c/em\u003e(1), 52.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWadhwani, S. A., Shedbalkar, U. U., Singh, R., \u0026amp; Chopade, B. A. (2016). Biogenic selenium nanoparticles: current status and future prospects. \u003cem\u003eApplied microbiology and biotechnology\u003c/em\u003e, \u003cem\u003e100\u003c/em\u003e(6), 2555\u0026ndash;2566.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVasanthakumar, S., Manikandan, M., \u0026amp; Arumugam, M. (2024). Green synthesis, characterization and functional validation of bio-transformed selenium nanoparticles. \u003cem\u003eBiochemistry and Biophysics Reports\u003c/em\u003e, \u003cem\u003e39\u003c/em\u003e, 101760.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSaravanan, K., Madhaiyan, M., Periyasamy, P., Manivannan, P., Bayrakdar, A., \u0026amp; Balakrishnan, V. (2025). Green synthesis and detailed characterization of selenium nanoparticles derived from Alangium salviifolium (Lf) Wangerin. \u003cem\u003eChemical Physics Impact\u003c/em\u003e, \u003cem\u003e10\u003c/em\u003e, 100876.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChang, J. L., Liao, C. W., Arthisree, D., Senthil Kumar, A., \u0026amp; Zen, J. M. (2022). A size-controlled graphene oxide materials obtained by one-step electrochemical exfoliation of carbon fiber cloth for applications to in situ gold nanoparticle formation and electrochemical sensors\u0026mdash;a preliminary study. \u003cem\u003eBiosensors\u003c/em\u003e, \u003cem\u003e12\u003c/em\u003e(6), 360.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSong, C., Li, X., Wang, S., \u0026amp; Meng, Q. (2016). Enhanced conversion and stability of biosynthetic selenium nanoparticles using fetal bovine serum. \u003cem\u003eRSC advances\u003c/em\u003e, \u003cem\u003e6\u003c/em\u003e(106), 103948\u0026ndash;103954.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSun, L., Zhang, R., Wang, Y., \u0026amp; Chen, W. (2014). Plasmonic Ag@ AgCl nanotubes fabricated from copper nanowires as high-performance visible light photocatalyst. \u003cem\u003eACS Applied Materials \u0026amp; Interfaces\u003c/em\u003e, \u003cem\u003e6\u003c/em\u003e(17), 14819\u0026ndash;14826.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHan, D. S., Batchelor, B., \u0026amp; Abdel-Wahab, A. (2013). XPS analysis of sorption of selenium (IV) and selenium (VI) to mackinawite (FeS). \u003cem\u003eEnvironmental Progress \u0026amp; Sustainable Energy\u003c/em\u003e, \u003cem\u003e32\u003c/em\u003e(1), 84\u0026ndash;93.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRuiz-Fresneda, M. A., Eswayah, A. S., Romero-Gonz\u0026aacute;lez, M., Gardiner, P. H. E., Solari, P. L., \u0026amp; Merroun, M. L. (2020). Chemical and structural characterization of Se IV biotransformations by Stenotrophomonas bentonitica into Se 0 nanostructures and volatiles Se species. \u003cem\u003eEnvironmental Science: Nano\u003c/em\u003e, \u003cem\u003e7\u003c/em\u003e(7), 2140\u0026ndash;2155.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSingh, A., Dey, P., Mihara, H., Prakash, R., \u0026amp; Prakash, N. T. (2025). Enhanced antimicrobial properties of cotton gauze functionalized with biosynthesized selenium nanoparticles. \u003cem\u003ePolymer Bulletin\u003c/em\u003e, \u003cem\u003e82\u003c/em\u003e(9), 3885\u0026ndash;3908.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRoy, N., Nivedya, T., Paira, P., \u0026amp; Chakrabarty, R. (2025). Selenium-based nanomaterials: green and conventional synthesis methods, applications, and advances in dye degradation. \u003cem\u003eRSC advances\u003c/em\u003e, \u003cem\u003e15\u003c/em\u003e(4), 3008\u0026ndash;3025.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAlsehli, B. R., Al-Hakkani, M. F., Alluhayb, A. H., Saleh, S. M., Mohamed, H., Hassane, A. M. A., \u0026amp; Hassan, M. H. A. (2025). Facile Mycosynthesis of Selenium Nanoparticles by Aspergillus candidus Extract: Unveiling Characterization, Potent Antimicrobial Properties, and Cytotoxic Activities. \u003cem\u003eBioNanoScience\u003c/em\u003e, \u003cem\u003e15\u003c/em\u003e(2), 234.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLikidtaveesin, S., Changtong, N., Jarussophon, N., \u0026amp; Chunhachart, O. (2025). Biosynthesis of Selenium Nanoparticles and Selenium-Enriched g-Polyglutamic Acid (Se-PGA) by Bacillus subtilis. \u003cem\u003eTrends in Sciences\u003c/em\u003e, \u003cem\u003e22\u003c/em\u003e(4), 9391.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSrivastava, N., \u0026amp; Mukhopadhyay, M. (2015). Biosynthesis and structural characterization of selenium nanoparticles using Gliocladium roseum. \u003cem\u003eJournal of Cluster Science\u003c/em\u003e, \u003cem\u003e26\u003c/em\u003e(5), 1473\u0026ndash;1482.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHashem, A. H., \u0026amp; Salem, S. S. (2022). Green and ecofriendly biosynthesis of selenium nanoparticles using Urtica dioica (stinging nettle) leaf extract: Antimicrobial and anticancer activity. \u003cem\u003eBiotechnology journal\u003c/em\u003e, \u003cem\u003e17\u003c/em\u003e(2), 2100432.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAlagesan, V., \u0026amp; Venugopal, S. (2019). Green synthesis of selenium nanoparticle using leaves extract of Withania somnifera and its biological applications and photocatalytic activities. \u003cem\u003eBionanoscience\u003c/em\u003e, \u003cem\u003e9\u003c/em\u003e(1), 105\u0026ndash;116.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGharieb, M. M., Soliman, A. M., \u0026amp; Omara, M. S. (2025). Biosynthesis of selenium nanoparticles by potential endophytic fungi Penicillium citrinum and Rhizopus arrhizus: characterization and maximization. \u003cem\u003eBiomass Conversion and Biorefinery\u003c/em\u003e, \u003cem\u003e15\u003c/em\u003e(2), 2319\u0026ndash;2328.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eElshikiby, L. A., Baka, Z. A. M., \u0026amp; El-Zahed, M. M. (2025). Biological activities of optimized biosynthesized selenium nanoparticles using Proteus mirabilis PQ350419 alone or combined with chitosan and ampicillin against common multidrug-resistant bacteria. \u003cem\u003eMicrobial Cell Factories\u003c/em\u003e, \u003cem\u003e24\u003c/em\u003e(1), 159.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShawky, M., Kalaba, M. H., \u0026amp; El-Sherbiny, G. M. (2025). Combined impact of biosynthesized selenium nanoparticles and imipenem against carbapenem-resistant Pseudomonas aeruginosa and their associated virulence factors. \u003cem\u003eBMC microbiology\u003c/em\u003e, \u003cem\u003e25\u003c/em\u003e(1), 235.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBabajide, A. M., Adebami, G. E., \u0026amp; Adebayo-Tayo, B. C. (2025). Screening of rhizobacteria from monkey pod trees for plant growth promoters and evaluating the antifungal potential of the biosynthesized selenium nanoparticles. \u003cem\u003eScientific Reports\u003c/em\u003e, \u003cem\u003e15\u003c/em\u003e(1), 16797.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDwivedi, S., AlKhedhairy, A. A., Ahamed, M., \u0026amp; Musarrat, J. (2013). Biomimetic synthesis of selenium nanospheres by bacterial strain JS-11 and its role as a biosensor for nanotoxicity assessment: a novel Se-bioassay. \u003cem\u003ePloS one\u003c/em\u003e, 8(3), e57404.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSharma, G., Sharma, A. R., Bhavesh, R., Park, J., Ganbold, B., Nam, J. S., \u0026amp; Lee, S. S. (2014). Biomolecule-mediated synthesis of selenium nanoparticles using dried Vitis vinifera (raisin) extract. \u003cem\u003eMolecules\u003c/em\u003e, \u003cem\u003e19\u003c/em\u003e(3), 2761\u0026ndash;2770.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShar, A. H., Lakhan, M. N., Wang, J., Ahmed, M., Alali, K. T., Ahmed, R., \u0026hellip; Dayo,A. Q. (2019). Facile synthesis and characterization of selenium nanoparticles by the hydrothermal approach. \u003cem\u003eDig J Nanomater Biostruct\u003c/em\u003e, \u003cem\u003e14\u003c/em\u003e, 867\u0026ndash;872.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang, S. Y., Zhang, J., Liu, Y., Ma, X., \u0026amp; Chen, H. Y. (2005). Electrochemical synthesis of selenium nanotubes by using CTAB soft-template. \u003cem\u003eElectrochimica acta\u003c/em\u003e, \u003cem\u003e50\u003c/em\u003e(22), 4365\u0026ndash;4370.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTriantis, T., Troupis, A., Gkika, E., Alexakos, G., Boukos, N., Papaconstantinou, E., \u0026amp; Hiskia, A. (2009). Photocatalytic synthesis of Se nanoparticles using polyoxometalates. \u003cem\u003eCatalysis Today\u003c/em\u003e, \u003cem\u003e144\u003c/em\u003e(1\u0026ndash;2), 2\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eForootanfar, H., Adeli-Sardou, M., Nikkhoo, M., Mehrabani, M., Amir-Heidari, B., Shahverdi, A. R., \u0026amp; Shakibaie, M. (2014). Antioxidant and cytotoxic effect of biologically synthesized selenium nanoparticles in comparison to selenium dioxide. \u003cem\u003eJournal of Trace Elements in Medicine and Biology\u003c/em\u003e, \u003cem\u003e28\u003c/em\u003e(1), 75\u0026ndash;79.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTugarova, A. V., Mamchenkova, P. V., Khanadeev, V. A., \u0026amp; Kamnev, A. A. (2020). Selenite reduction by the rhizobacterium Azospirillum brasilense, synthesis of extracellular selenium nanoparticles and their characterisation. \u003cem\u003eNew biotechnology\u003c/em\u003e, \u003cem\u003e58\u003c/em\u003e, 17\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAnu, K., Singaravelu, G., Murugan, K., \u0026amp; Benelli, G. (2017). Green-synthesis of selenium nanoparticles using garlic cloves (Allium sativum): biophysical characterization and cytotoxicity on vero cells. \u003cem\u003eJournal of Cluster Science\u003c/em\u003e, \u003cem\u003e28\u003c/em\u003e(1), 551\u0026ndash;563.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eArunthirumeni, M., Veerammal, V., \u0026amp; Shivakumar, M. S. (2022). Biocontrol efficacy of mycosynthesized selenium nanoparticle using Trichoderma sp. on insect pest Spodoptera litura. \u003cem\u003eJournal of Cluster Science\u003c/em\u003e, \u003cem\u003e33\u003c/em\u003e(4), 1645\u0026ndash;1653.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAbdel-Moneim, A. M. E., El-Saadony, M. T., Shehata, A. M., Saad, A. M., Aldhumri, S. A., Ouda, S. M., \u0026amp; Mesalam, N. M. (2022). Antioxidant and antimicrobial activities of Spirulina platensis extracts and biogenic selenium nanoparticles against selected pathogenic bacteria and fungi. \u003cem\u003eSaudi Journal of Biological Sciences\u003c/em\u003e, \u003cem\u003e29\u003c/em\u003e(2), 1197\u0026ndash;1209.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Selenium nanoparticles, Soil-borne bacteria, Bio-synthesis, Mica mine","lastPublishedDoi":"10.21203/rs.3.rs-7492605/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7492605/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAmong all the micronutrients, selenium (Se) is highly desirable as a dietary supplement, with the potential to improve germination and seedling development in crops, as well as exhibiting anticancer and antimicrobial properties. Organic and elemental nanoforms of Se demonstrate greater reactivity, higher bioavailability, and lower toxicity compared to inorganic forms. This study proposes the biosynthesis of selenium nanoparticles (SeNPs) using a soil-borne bacterium (\u003cem\u003eBacillus cereus\u003c/em\u003e). The synthesis of SeNPs through rhizospheric bacteria isolated from mica-rich agricultural soil is more environmentally friendly and cost-effective than conventional chemical synthesis methods. The synthesized nanoparticles were purified, dried, and initially characterized by UV-VIS spectroscopy, which showed a prominent peak at 282 nm, a characteristic feature of SeNPs. The crystalline phases were further confirmed by matching the XRD results with the JCPDS reference code 06\u0026ndash;0362. Surface characterization was carried out using FTIR and XPS analyses, and the size and morphology of the particles were finally confirmed by FE-SEM and TEM imaging. The environmentally sustainable biosynthesis of SeNPs by \u003cem\u003eBacillus cereus\u003c/em\u003e from mica-rich rhizospheric soil produces extremely stable, bioavailable, and low-toxicity nanoparticles with potential applications in agriculture.\u003c/p\u003e","manuscriptTitle":"Eco-Friendly Synthesis and Characterization of Crystalline Selenium Nanoparticles via Bacillus cereus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-03 08:51:26","doi":"10.21203/rs.3.rs-7492605/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"49f651be-0987-4117-8c7d-e791b34d2fcc","owner":[],"postedDate":"September 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-11-09T13:53:27+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-03 08:51:26","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7492605","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7492605","identity":"rs-7492605","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}