Biogenic and green approach for ZnO nanoparticle synthesis via Bacillus licheniformis and their antimicrobial and anticancer potential

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This study reports the biosynthesis of Zinc Oxide nanoparticles (ZnO NPs) through the use of the cell-free extract of Bacillus licheniformis strain FC14167, isolated from soil. The ZnO nanoparticles obtained in this study were characterized using a combination of UV–visible spectroscopy, FTIR, XRD, SEM–EDX, and TEM to assess their optical, structural, and morphological properties. ZnO nanoparticles showed a distinct UV-Vis apeak at 331 nm, FTIR analysis revealed functional groups contributing to nanoparticle synthesis, while XRD patterns verified the crystalline structure and purity. The ZnO NPs exhibited a well-defined hexagonal shape, averaging 36 nm in diameter, evident from SEM and TEM micrographs, and the presence of Zn was confirmed through the EDX spectral analysis. Biosynthesized ZnO NPs were found to possess effective antimicrobial activity and zones of inhibition against certain bacterial and fungal pathogens. Furthermore, the nanoparticles demonstrated cytotoxicity against HT-29 cancer cell lines, resulting to considerable necrotic and abnormal shapes in cancer cells with an IC 50 value determined to be 56.55 µg/mL. The current investigation indicates, biosynthesized ZnO NPs possess substantial anticancer activity and inhibitory effects against HT-29 cancer cell lines. Accordingly, this study conjectures the potential of bacteria-mediated ZnO NPs as effective anticancer agents. Biosynthesis ZnO NPs Bacillus licheniformis HT-29 cell lines Antimicrobial activity Antioxidant activity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Zinc Oxide (ZnO) nanoparticles (NPs) are widely recognized for their unique optical, electrical, and mechanical characteristics [1]. Applications of ZnO nanoparticles span diverse sectors such as UV filtering agents and sunscreens [2], solar cells and photovoltaic devices [3], photocatalysis, wastewater treatment [4, 5], light-emitting diodes (LEDs), optoelectronic devices [6], memory devices, data storage systems [7], food applications, packaging [8] biosensors, biomedical devices, and cancer therapeutics [9–11]. Moreover, ZnO NPs is mainly used as an antimicrobial agent to combat several pathogenic infections [12], and in cosmetics and skincare products [13, 14]. The effectiveness of the NPs highly is shaped by their particle size and morphology. Further, the enhanced surface area to volume ratio enhances their reactivity and antimicrobial activity [15]. Multidrug resistance is a significant global concern, and the potential use of NPs as alternatives to conventional antibiotics is being actively investigated owing to their broad spectrum of activity and limited potential for resistance development [16–18]. Traditionally ZnO NPs are manufactured using chemical and physical processes that rely on hazardous chemicals, posing a major concern for the environment, various life forms, and the ecosystem [19, 20]. These chemicals bind with ZnO NPs, causing toxicity and affecting biocompatibility, limiting their biological applications [21]. This has led researchers to explore alternative, eco-friendly methods for synthesizing ZnO NPs, such as biosynthesis and green chemistry approaches to produce NPs with desirable properties, leveraging chemical synthesis routes as reducing and capping agents [16, 21]. Biogenic synthesis of NPs has proven to be effective, eco-friendly viable replacement for traditional chemical and physical approaches that encompass various methods, including the utilization of plant extracts [22], fruit extracts [23], algae [24], cyanobacteria [25], fungi [26], and bacteria [27]. Notably, bacteria-mediated synthesis offers distinct advantages, such as low cost, high production rate, biocompatibility, genetically tractable, and ease of manipulation of cell growth, rendering it a favourable route for the long-term production of NPs. Further, microorganisms produce functional biomolecule complexes in the supernatant and inside the cell, which convert metal ions into metal NPs [28]. Microbes are ubiquitous and can thrive in a variety of environments, making them ideal candidates for the metal NPs synthesis. Various microbial species includes bacteria, fungi, yeasts, and algae, have been used as natural reducers in producing nanoparticles of metals including gold (Au), silver (Ag), copper (Cu), platinum (Pt), titanium (Ti), and zinc (Zn) [28]. The key advantages of using microbes are simple, large-scale cultivation and downstream processing. Additionally, the cell-free supernatant is rich in metabolites, proteins, and enzymes capable of converting metal ions to NPs and maintain their stability [29]. Microorganisms can produce ZnO NPs via intracellular or extracellular, for instance, Lactobacillus produce intracellularly [30], while fungi ( Aspergillus aeneus ) [31] and yeast ( Pichia fermentans ) produce extracellularly [32]. Generally, not all microbes are capable of synthesizing metallic NPs, since each microbial population has a unique metabolic system suitable for its niche. Therefore, selecting the appropriate microbial strain is crucial for effective NPs synthesis. Among bacteria, lactic acid bacteria have been studied widely because of their safe non-pathogenic, beneficial properties, and food-grade status. Further, Lactobacillus species are among the most studied bacteria for the fabrication of diverse NPs, including Ag, Se, Zn, and Au, with diverse biomedical applications [27]. Also, Lactobacillus can be used to promote the health of humans and animals, which may be an added advantage of its usage in NPs synthesis for biomedical applications. The genus Lactobacillus is a Gram-positive bacterium surrounded by a dense cell wall composed of peptidoglycan, lipoteichoic acid, collagen, and polysaccharides [33]. Functional moieties in Te layers have negative electrokinetic charge that attract metal cations, act as ligands, allowing them to function as active sites for metal ion biosorption and bio-reduction, and help NPs formation [27, 34]. However, the exact mechanism behind ZnO NPs formation remains unclear and requires further investigation. In this study, the cell-free extract of Bacillus licheniformis strain FC14167 isolated from soil served as biological medium to produce ZnO NPs. Their successful formation were validated via several physicochemical analyses. Additionally, antimicrobial and antioxidant evaluations were also performed to assess examine their suitability for biomedical uses. 2. Materials and Methods 2.1 ZnO-NPs Synthesis by Bacillus licheniformis The present work employed the Bacillus licheniformis strain with the GenBank accession number PQ591714. Details on the isolation and identification of the strain will be published separately since they are out of the scope of the present study. After 24h of growth of B. licheniformis in nutrient broth (NB) at 37°C, the culture was centrifuged at 10,000 rpm for 5 min to separate cell to obtain cell-free supernatant. The supernatant then supplemented with 0.1M zinc sulphate heptahydrate and incubated overnight in a shaking incubator at room temperature. The nanoparticle synthesis was evidenced by the development of a white precipitate accumulating at the flask’s bottom. After synthesis, ZnO NPs were separated by centrifugation at 14,000 rpm, duration of 10 min and repeated twice, subsequently calcified at 120°C [35]. 2.2 Analysis of structural and chemical characteristics of ZnO NPs The successful formation of ZnO NPs was verified through UV–Vis diffuse reflectance spectroscopic (DRS) analysis (UV-3092, Lab India) covering a wavelength range from 200 to 700 nm with measurements taken every 1 nm. The diffraction pattern of the powder sample was recorded collecting data across a 2θ range of 10° to 80° under 35 kV and 30 mA current (X'Pert PRO-PANalytical XRD). Based on Scherrer’s equation, mean crystallite size was calculated using the XRD data. Fourier transform infrared spectroscopic spectrum was recorded between 400 and 4000 cm − 1 with 4 cm − 1 intervals to identify the functional organic molecules existing in culture supernatant accountable for the reduction or stabilization of ZnO nanoparticles (Jasco FT/IR-4700 Spectrometer Type-A, Japan). Field Emission Scanning Electron Microscopy (FESEM) (Zeiss Sigma 300, Carl Zeiss AG, Germany), operated at 5 kV, was used to examine the morphology, structural characteristics, and particle size of the synthesized nanoparticles. Energy-dispersive X-ray spectroscopy (EDX), coupled with FESEM, was employed for elemental analysis. For detailed morphological and structural examination, Transmission Electron Microscopy (TEM) was performed (FEI Tecnai G2 20 S-TWIN) with a 200 kV electron source, either LaB₆ or tungsten emitter, and a point resolution of 0.24 nm. 2.3 Assessment of the anti-microbial effect of ZnO NPs The antibacterial potential of ZnO NPs prepared from bacterial cell-free supernatant was investigated against four pathogenic bacterial species, such as Klebsiella oxytoca, Staphylococcus epidermidis, Escherichia coli, and Pseudomonas aeruginosa through well diffusion method. Similarly, ZnO NPs was also tested for their antifungal efficacy against pathogenic fungal strains of Candida albicans, C. kuresi, Aspergillus niger with concentrations ranging from 100 to 400 µl. Muller-Hinton agar (MHA) plates were inoculated with overnight-grown pathogenic bacterial cultures using sterile swabs to ensure even distribution. Using a sterile gel puncher, wells measuring 5 mm in diameter were created in MHA plates. ZnO NPs suspensions at concentrations of 100, 200, 300, and 400 µl were carefully added to each well. Following 24 hours of incubation at 37°C, diameters of the clear zones around each well were measured in millimeters, and the antimicrobial activity was assessed by referencing a standard interpretation chart [36]. The experiments were carried out in three independent replicates, and data are presented as the mean values with corresponding standard deviations. 2.4 Assessment of the antioxidant effect of ZnO NPs To assess antioxidant potential, ZnO NPs were subjected to a 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay following the protocol established by Shimada et al.'s method [37]. Approximately 3 µL of DPPH 1.0 mM dissolved in methanol was added to 1 ml of ZnO nanoparticle suspensions across a concentration range of 5 to 25 µg/mL. The reaction mixtures were incubated in the absence of light at room temperature for 30 min. After incubation, absorbance was recorded at 517 nm employing UV-Visible microplate reader, with methanol as control. The scavenging ability was calculated as per the equation given below: 2.5 Cytotoxicity of ZnO NPs To assess cytotoxicity, the MTT assay was carried out on the HT-29 human colon cancer cell line treated with synthesized ZnO nanoparticles, with cells sourced from NCCS, Pune. [38]. Briefly, 10,000 cells per well were cultured into a 96-well microplate and incubated for 24 hours in culture medium consisted of DMEM, fortified with 10% FBS and 1% antibiotic solution, maintained at 37°C in a humidified 5% CO₂ atmosphere. After treatment with different concentrations of ZnO NPs for 24 hours, MTT solution was added, followed by a 2-hour incubation. The medium was carefully discarded, and 100 µL of DMSO was added to solubilize the formazan crystals, and absorbance readings were taken at 540 nm and 660 nm (iMark, Biorad, USA). 2.6 Statistical evaluation Results are expressed as mean ± SD from three independent experiments. Statistical significance was evaluated using one-way ANOVA with Tukey’s multiple comparison test (p < 0.05). IC₅₀ values were obtained via non-linear regression using GraphPad Prism (evaluation version). 3. Results and Discussion 3.1. Spectroscopic study of ZnO nanoparticles 3.1.1 UV-Visible Spectral Analysis ZnO nanoparticles were efficiently produced in this study through a biosynthetic approach employing the cell free culture supernatant of Bacillus licheniformis , which simultaneously contributed to the reduction and surface functionalization. Occurrence of the ZnO NPs synthesis was visually indicated by a noticeable color transition upon adding zinc ions to the cell-free supernatant. In contrast, no change was observed in the control samples without zinc ions, which confirmed the contribution played by the bacterial extract on facilitating the biosynthesis of ZnO NPs. As illustrated in Fig. 1 , the synthesized ZnO nanoparticles exhibited a sharp UV-Vis absorption peak at 331 nm, indicative of effective NPs synthesis. An observable blue shift in the absorption spectrum suggests that particles are not significantly larger than the exciting Bohr radius. It has been established that the characteristic absorption peaks of green-synthesized ZnO nanoparticles occur within the 330–390 nm range [15, 39, 40]. 3.1.2 FT-IR study FTIR spectroscopy was used to explore the molecular interactions at the interface of zinc oxide and the bioactive organic compounds in the bacterial cell-free extract. It also helped identify the organic functional moieties and biomolecules responsible for the biological synthesis of ZnO NPs and the FTIR spectrum is shown in Fig. 2 . A distinct spectral band appeared with a value of 799 cm⁻¹ indicated Zn–O–Zn stretching vibration, confirming the ZnO fabrication. (Fig. 2 ). A peak observed with a value of 1020 cm⁻¹ was attributed to C–O stretch vibrations. Meanwhile, the band appearing at 1535 cm⁻¹ corresponded to C = C stretch, C–N–H bend, and amide group functionalities. Additionally, the absorption bands at 2856 and 2918 cm⁻¹ were associated with C–H bond stretch [39–41]. The observation of these peaks suggested the presence of surface-bound molecules in the bacterial cell-free extract. An absorption peak appearing at 2532 cm⁻¹ was assigned to the stretching vibrations of hydroxyl (–OH) groups. The FTIR spectrum obtained for ZnO NPs is characterized by the existence of a prominent aromatic ring and carboxylic acid, confirming the role of bioactive molecules in involved in biosynthesis of ZnO NPs [40]. 3.2 Structural elements analysis The XRD patterns of the synthesized ZnO NPs are presented in Fig. 3 . Based on XRD results, ZnO NPs were found to be pure and crystalline nature. The XRD pattern (Fig. 3 ) revealed conspicuous peaks corresponding to lattice planes of (100), (-110), (101), (002), (102), (110), and (103) at Bragg's angles of 31.7°, 56.6°, 36.2°, 34.4°, 62.8°, and 47.5°, respectively. As indicated by the data the biofabricated NPs have a face-centred hexagonal crystal structure was confirmed. The obtained peaks were matched with corresponding lattice planes according to the JCPDS reference pattern (Card No: 96-230-117). Additionally, estimated mean diameter of the nanoparticles was around 36 nm based on the Debye–Scherrer equation. 3.3 Morphological studies ZnO NP’s morphological features were examined by employing a FE-SEM (Fig. 4 a and b). The SEM micrographic images captured at different magnifications revealed well-defined hexagonal NPs averaging 36 nm in diameter, confirming their morphology. In addition, TEM micrographic images revealed that the hexagonal nanoparticles form a cluster-like configuration having an average size of about ~ 36 nm (Fig. 4 c). ZnO NP’s SAED pattern showed clear diffraction rings, confirming their crystalline phase (Fig. 4 d). The EDX spectrum showed two distinct and significant zinc peaks at 1.2 keV, Indicating that zinc was present in the fabricated nanoparticles. Figure 5 illustrates the EDX spectra of the ZnO NPs synthesized in this study exhibited distinct peaks for zinc (Zn) and oxygen (O). These results further validated the elemental composition and purity of the biosynthesized ZnO nanoparticle (Fig. 4 e). 3.4. Antimicrobial efficiencies of biosynthesized ZnO NPs The well diffusion method was employed to assess the in vitro antibacterial efficacy of the synthesized ZnO NPs. The results demonstrated that biosynthesized ZnO NPs showed superior anti-bacterial efficacy (Fig. 5 a). At the highest dose of 400 µL, the ZnO nanoparticles produced the largest clearance zone i.e., 13 ± 1 mm against K. oxytoca , followed by noticeable inhibitory effects against S. epidermidis and P. aeruginosa . The findings clearly demonstrate that the elevating dose of ZnO NPs led to a stronger zone of inhibition. The synthesized ZnO NPs showed significant active against a diverse group of bacteria, such as representatives from both Gram-positive and Gram-negative classes. According to previous findings, ZnO nanoparticles produced through green techniques display stronger antibacterial effect against Gram-negative bacteria in comparison to Gram-positive strains. The discrepancy in susceptibility is often ascribed with the structural composition of the bacterial cell walls. The thick, peptidoglycan-rich layer found in Gram-positive bacteria can act as a barrier, reducing the penetration and overall effectiveness of ZnO nanoparticles [42–44]. The biosynthesized ZnO nanoparticles further examined for their antifungal effect against several fungal strains, including Aspergillus niger , Candida krusei , and Candida albicans . The antifungal activity is dose-dependent. At the highest ZnO nanoparticle concentration tested, inhibition zones reached 12.68 ± 0.58 mm for C. albicans and 11.68 ± 0.58 mm for A. niger , indicating significant antifungal activity. (Fig. 5 b). Our results are consistent with previous reports of [45, 46]. Their distinct electronic properties allow ZnO nanoparticles to interrupt electron transport pathways in biological environments, thereby generating reactive oxygen species (ROS) [47]. ZnO nanoparticles exert their antibacterial effects is thought to occur by means of several mechanisms and the exact mechanism remains elusive. Firstly, nanoparticles can affect microbial cells by interacting with their membranes through electrostatic forces, allowing them to penetrate it. Once inside, ZnO nanoparticles may induce oxidative stress by generating ROS which results in cellular destruction [48]. Furthermore, ZnO NPs possess surface properties that can catalyze the generation of hydrogen peroxide (H 2 O 2 ), which can diffuse into the bacterial membrane, leading to cell damage and death [49, 50]. 3.10. Antioxidant activity of ZnO NPs This study showed that biosynthesized ZnO NPs displayed notable antioxidant potential, particularly showing strong hydrogen peroxide scavenging capacity. In relation to ascorbic acid, scavenging property of ZnO NPs is found to be less; however, no significant changes were observed. The antioxidant effect showed a concentration-dependent pattern with higher concentrations yielding stronger activity with ZnO nanoparticles (Fig. 6). The observed antioxidant potential of the biosynthesized ZnO nanoparticles may result from the functional groups and bioactive constituents present in the bacterial cell-free supernatant. The bioactive components in the supernatant provide hydrogen atoms, which prevent free radical reactions [40]. This study reveals that the bacterial cell-free supernatant contains bio-components that enhance the antioxidant activity. Based on these results, biosynthesized ZnO NPs may serve as promising candidates for antioxidant therapeutic applications. 3.11. Cytotoxicity of ZnO NPs against HT-29 cells To determine the cytotoxic efficacy of ZnO nanoparticles, an MTT test was conducted against human colon cancer cell lines. The finding indicated that the anticancer activity of ZnO NPs on HT-29 cells was increased with concentration, exhibiting comparable cytotoxic effects at low doses and following a dose response (Fig. 7 ). In this investigation, the dose of ZnO NPs required to reduce cell viability by 50% (IC₅₀) was calculated as 56.55 µg/m against HT-29 cells. Previously, the reported IC 50 values for ZnO NPs against HT-29 cells are 54.16 g/mL [40], 45.82 g/mL [51], and > 500 µg/mL [52]. The IC₅₀ values of ZnO NPs synthesized biologically and chemically were 135.9 µg/mL [53] and 131 µg/mL [54], respectively, against HT-29 cells. Nanoparticle concentration was a key factor influencing their activity against cancer cells, potentially passing through ion channels in the cell membrane and interacting with nitrogenous bases in DNA as well as various intracellular proteins. ZnO NPs are internalized through endocytic pathways, with uptake and the levels of accumulation depending on size, shape, and cell type [55]. Upon entry, ZnO NPs dissolve and release Zn²⁺ ions that disrupt mitochondrial function by disturbing the flow of electrons through the electron transport chain. This induces levated ROS levels, which induce oxidative stress and lead to the deterioration of mitochondrial structures and DNA integrity, and ultimately triggering apoptosis-mediated cytotoxicity [56, 57]. 4. Conclusion This present study reports the effective fabrication of ZnO nanoparticles using bacterial cell-free supernatant offering a novel and sustainable route for nanoparticle production. Detailed analysis of the ZnO NPs revealed a sharp UV-visible distinct spectral peak occurring at a defined wavelength, validating their purity, whereas FTIR spectra showed characteristic bioactive organic moieties involved in nanoparticle formation. ZnO nanoparticles are well-defined hexagonal NPs averaging 36 nm in diameter with pure crystalline form confirmed through XRD, SEM, and TEM analyses. Furthermore, the anticancer investigation suggested that ZnO NPs induced dose-dependent cytotoxicity. Collectively, these findings suggest that ZnO NPs derived from bacterial cell-free supernatant may serve as an effective cancer therapy agent. This study presents valuable insights into the biological uses and potential of ZnO NPs, encompassing production methods, antibacterial activity, antioxidant activity, and anticancer activity. As a low-cost, low-toxic, and multifunctional material, ZnO NPs hold great promise for future biological applications. Despite these promising findings, ongoing research efforts are crucial to fully address the remaining gaps and limitations, including the targeted delivery of ZnO NPs to multiple cells and live animals. While ZnO NPs have demonstrated strong antimicrobial and anticancer efficacy, additional studies are required for translating these findings into practical applications. Future prospective : Despite the advantages of ZnO NPs, several challenges remain unaddressed to unlock the full potential of ZnO NPs. Scalability is a significant concern, as producing high-quality ZnO NPs on a large scale with consistent properties remains a hurdle. Given, the potential toxicity of ZnO NPs is a concern, further research is essential to evaluate their possible health and ecological risks. The functionalization of ZnO NPs with other materials and compounds is a viable technique for improving their properties and uses. Overall, ZnO NPs hold vast potential in various fields, and with ongoing scientific progress, current obstacles may be overcome, unlocking new uses and paving the path for their increased adoption in cosmetics and personal care products. Declarations Declaration of competing interest The author declares that no competing financial interests or personal ties could have influenced the work presented in this paper. Ethical approval Not applicable. Conflicts of interest The authors declare no conflict of interest. Funding This study received no external support. Author Contribution Author ContributionSP-Priyadharsini Shanumuganandam, TS-Sathiamoorthi Thangavelu. SP, TS -All authors contributed to the study conception and design. SP- performed the literature review, data collection, and analysis. contributed to the methodology and validation of results. TS-supervised the project and provided critical feedback. SP, TS All authors discussed the results, reviewed, and approved the final manuscript."Consent for publication:All authors agree to published the paper in your esteemed journal. Acknowledgements All the authors sincerely acknowledge the support provided by RUSA 2.0 and the Research Instrumentation Facilities of the Department of Microbiology, Alagappa University, Karaikudi, Tamil Nadu. The authors also wish to express their appreciation to the Central Instrumentation Centre (CIC) at Madurai Kamaraj University, Madurai, Tamil Nadu for assistance with TEM analysis, Bannari Amman Institute of Technology, Sathyamangalam, Tamil Nadu for conducting EDX and SEM analyses, and Vellore Institute of Technology, Vellore, Tamil Nadu for performing the XRD analysis. Data availability statement No supplementary data for this work can be provided. References Sharma DK, Shukla S, Sharma KK, Kumar V: A review on ZnO: Fundamental properties and applications . Materials Today: Proceedings 2022, 49 :3028–3035. Schneider SL, Lim HW: A review of inorganic UV filters zinc oxide and titanium dioxide . 2019, 35 (6):442–446. Otalora C, Botero MA, Ordoñez G: ZnO compact layers used in third-generation photovoltaic devices: a review . Journal of Materials Science 2021, 56 (28):15538–15571. 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Ezhilarasi AA, Vijaya JJ, Kaviyarasu K, Maaza M, Ayeshamariam A, Kennedy LJ: Green synthesis of NiO nanoparticles using Moringa oleifera extract and their biomedical applications: Cytotoxicity effect of nanoparticles against HT-29 cancer cells . Journal of Photochemistry and Photobiology B: Biology 2016, 164 :352–360. Boskabadi SH, Zafar BS, Ali N, and Tabrizi MH: The green-synthesized zinc oxide nanoparticle as a novel natural apoptosis inducer in human breast (MCF7 and MDA-MB231) and colon (HT-29) cancer cells . Inorganic and Nano-Metal Chemistry 2020, 51 (5):733–743. Paranthaman S, Shivakumar CS, Kalaipriya S, Venkatesh HN, Gireesha J, Pasha S, Shazly GA, Anandan S, Shivamallu C, Kollur SP: One-pot green synthesis of zinc oxide nanoparticles using Morus laevigata aqueous extract and evaluation of its anticancer potential against HT-29 cell line . 2024, 47 (1). 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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-7440837","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":511617879,"identity":"67fba2a7-f034-4366-a6d1-5bbdb29eeb9a","order_by":0,"name":"Priyadharsini Shanumuganandam","email":"","orcid":"","institution":"Alagappa University","correspondingAuthor":false,"prefix":"","firstName":"Priyadharsini","middleName":"","lastName":"Shanumuganandam","suffix":""},{"id":511617880,"identity":"7d7d72fe-bdf6-41a9-bfcd-ce1ce916d24e","order_by":1,"name":"Sathiamoorthi Thangavelu","email":"data:image/png;base64,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","orcid":"","institution":"Alagappa University","correspondingAuthor":true,"prefix":"","firstName":"Sathiamoorthi","middleName":"","lastName":"Thangavelu","suffix":""}],"badges":[],"createdAt":"2025-08-23 11:08:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7440837/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7440837/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":90878391,"identity":"ef69955e-998d-4c9b-9590-cd2fdf4695e4","added_by":"auto","created_at":"2025-09-09 09:22:19","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":19619,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUV–Vis spectrum of ZnO NPs synthesized using cell-free supernatant of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eB. licheniformis\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-7440837/v1/eeac92cb9b4dbeda3d8a3f15.png"},{"id":90879292,"identity":"ee03af23-d354-4952-b4b3-97389676d050","added_by":"auto","created_at":"2025-09-09 09:30:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":44201,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFTIR spectrum of ZnO NPs synthesized using cell-free extract of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eB. licheniformis\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-7440837/v1/cdd09300a6f1ca2938fedb38.png"},{"id":90878393,"identity":"d83d0e34-fcb9-4d55-aa57-f9a5d83f5937","added_by":"auto","created_at":"2025-09-09 09:22:19","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":36074,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eX-ray diffraction pattern of biosynthesized ZnO NPs\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-7440837/v1/aba1b9b502d0880beb5de1f9.png"},{"id":90879293,"identity":"0e26da81-21bd-4415-b35c-93e182fd86de","added_by":"auto","created_at":"2025-09-09 09:30:19","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":819347,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMorphological analysis (FE-SEM and TEM) of ZnO nanoparticles. \u003c/strong\u003e(a) FE-SEM image at 40× Magnification (b) FE-SEM image at 20× Magnification (c) SAED Pattern (d) D-space value of ZnO NPs (e) Elemental analysis of biosynthesized ZnO NPs.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-7440837/v1/ea8d1d18ac0f5664dd0c9c52.png"},{"id":90878395,"identity":"33ef0095-534e-48ac-9c82-116ba523a6ed","added_by":"auto","created_at":"2025-09-09 09:22:19","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":60159,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluation of the antimicrobial effect of biosynthesized ZnO nanoparticles. against pathogenic (a) bacterial and (b) fungal strains at varying concentrations. Data are expressed as mean ± SD from three independent experiments.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-7440837/v1/16c76355e74928e9812ee226.png"},{"id":90878394,"identity":"78ad8cad-85fd-4fc3-b999-81882b6220af","added_by":"auto","created_at":"2025-09-09 09:22:19","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":41629,"visible":true,"origin":"","legend":"\u003cp\u003eAntioxidant activity of biosynthesized ZnO nanoparticles determined by the DPPH assay. Data are expressed as mean ± standard deviation from three independent replicates.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-7440837/v1/c0236ce491f7b279d87b4d66.png"},{"id":90878397,"identity":"db72a5d8-12e3-4fad-a91b-be7f2952d88e","added_by":"auto","created_at":"2025-09-09 09:22:19","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":593360,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCytotoxic activity of ZnO NPs against HT-29 human colon cancer cells evaluated through MTT assay.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-7440837/v1/05862dc33ebe45f98e4c37ea.png"},{"id":90880106,"identity":"2a89be29-ff18-40c9-9b5f-94fe08cedc4c","added_by":"auto","created_at":"2025-09-09 09:38:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5081010,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7440837/v1/d9763a46-3a98-419f-b896-db4abf766f49.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Biogenic and green approach for ZnO nanoparticle synthesis via Bacillus licheniformis and their antimicrobial and anticancer potential","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eZinc Oxide (ZnO) nanoparticles (NPs) are widely recognized for their unique optical, electrical, and mechanical characteristics [1]. Applications of ZnO nanoparticles span diverse sectors such as UV filtering agents and sunscreens [2], solar cells and photovoltaic devices [3], photocatalysis, wastewater treatment [4, 5], light-emitting diodes (LEDs), optoelectronic devices [6], memory devices, data storage systems [7], food applications, packaging [8] biosensors, biomedical devices, and cancer therapeutics [9\u0026ndash;11]. Moreover, ZnO NPs is mainly used as an antimicrobial agent to combat several pathogenic infections [12], and in cosmetics and skincare products [13, 14]. The effectiveness of the NPs highly is shaped by their particle size and morphology. Further, the enhanced surface area to volume ratio enhances their reactivity and antimicrobial activity [15]. Multidrug resistance is a significant global concern, and the potential use of NPs as alternatives to conventional antibiotics is being actively investigated owing to their broad spectrum of activity and limited potential for resistance development [16\u0026ndash;18].\u003c/p\u003e\u003cp\u003eTraditionally ZnO NPs are manufactured using chemical and physical processes that rely on hazardous chemicals, posing a major concern for the environment, various life forms, and the ecosystem [19, 20]. These chemicals bind with ZnO NPs, causing toxicity and affecting biocompatibility, limiting their biological applications [21]. This has led researchers to explore alternative, eco-friendly methods for synthesizing ZnO NPs, such as biosynthesis and green chemistry approaches to produce NPs with desirable properties, leveraging chemical synthesis routes as reducing and capping agents [16, 21]. Biogenic synthesis of NPs has proven to be effective, eco-friendly viable replacement for traditional chemical and physical approaches that encompass various methods, including the utilization of plant extracts [22], fruit extracts [23], algae [24], cyanobacteria [25], fungi [26], and bacteria [27]. Notably, bacteria-mediated synthesis offers distinct advantages, such as low cost, high production rate, biocompatibility, genetically tractable, and ease of manipulation of cell growth, rendering it a favourable route for the long-term production of NPs. Further, microorganisms produce functional biomolecule complexes in the supernatant and inside the cell, which convert metal ions into metal NPs [28].\u003c/p\u003e\u003cp\u003eMicrobes are ubiquitous and can thrive in a variety of environments, making them ideal candidates for the metal NPs synthesis. Various microbial species includes bacteria, fungi, yeasts, and algae, have been used as natural reducers in producing nanoparticles of metals including gold (Au), silver (Ag), copper (Cu), platinum (Pt), titanium (Ti), and zinc (Zn) [28]. The key advantages of using microbes are simple, large-scale cultivation and downstream processing. Additionally, the cell-free supernatant is rich in metabolites, proteins, and enzymes capable of converting metal ions to NPs and maintain their stability [29]. Microorganisms can produce ZnO NPs via intracellular or extracellular, for instance, \u003cem\u003eLactobacillus\u003c/em\u003e produce intracellularly [30], while fungi (\u003cem\u003eAspergillus aeneus\u003c/em\u003e) [31] and yeast (\u003cem\u003ePichia fermentans\u003c/em\u003e) produce extracellularly [32]. Generally, not all microbes are capable of synthesizing metallic NPs, since each microbial population has a unique metabolic system suitable for its niche. Therefore, selecting the appropriate microbial strain is crucial for effective NPs synthesis.\u003c/p\u003e\u003cp\u003eAmong bacteria, lactic acid bacteria have been studied widely because of their safe non-pathogenic, beneficial properties, and food-grade status. Further, \u003cem\u003eLactobacillus\u003c/em\u003e species are among the most studied bacteria for the fabrication of diverse NPs, including Ag, Se, Zn, and Au, with diverse biomedical applications [27]. Also, \u003cem\u003eLactobacillus\u003c/em\u003e can be used to promote the health of humans and animals, which may be an added advantage of its usage in NPs synthesis for biomedical applications. The genus \u003cem\u003eLactobacillus\u003c/em\u003e is a Gram-positive bacterium surrounded by a dense cell wall composed of peptidoglycan, lipoteichoic acid, collagen, and polysaccharides [33]. Functional moieties in Te layers have negative electrokinetic charge that attract metal cations, act as ligands, allowing them to function as active sites for metal ion biosorption and bio-reduction, and help NPs formation [27, 34]. However, the exact mechanism behind ZnO NPs formation remains unclear and requires further investigation. In this study, the cell-free extract of \u003cem\u003eBacillus licheniformis\u003c/em\u003e strain FC14167 isolated from soil served as biological medium to produce ZnO NPs. Their successful formation were validated via several physicochemical analyses. Additionally, antimicrobial and antioxidant evaluations were also performed to assess examine their suitability for biomedical uses.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 ZnO-NPs Synthesis by \u003cem\u003eBacillus licheniformis\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eThe present work employed the \u003cem\u003eBacillus licheniformis\u003c/em\u003e strain with the GenBank accession number PQ591714. Details on the isolation and identification of the strain will be published separately since they are out of the scope of the present study. After 24h of growth of \u003cem\u003eB. licheniformis\u003c/em\u003e in nutrient broth (NB) at 37\u0026deg;C, the culture was centrifuged at 10,000 rpm for 5 min to separate cell to obtain cell-free supernatant. The supernatant then supplemented with 0.1M zinc sulphate heptahydrate and incubated overnight in a shaking incubator at room temperature. The nanoparticle synthesis was evidenced by the development of a white precipitate accumulating at the flask\u0026rsquo;s bottom. After synthesis, ZnO NPs were separated by centrifugation at 14,000 rpm, duration of 10 min and repeated twice, subsequently calcified at 120\u0026deg;C [35].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Analysis of structural and chemical characteristics of ZnO NPs\u003c/h2\u003e\u003cp\u003eThe successful formation of ZnO NPs was verified through UV\u0026ndash;Vis diffuse reflectance spectroscopic (DRS) analysis (UV-3092, Lab India) covering a wavelength range from 200 to 700 nm with measurements taken every 1 nm. The diffraction pattern of the powder sample was recorded collecting data across a 2θ range of 10\u0026deg; to 80\u0026deg; under 35 kV and 30 mA current (X'Pert PRO-PANalytical XRD). Based on Scherrer\u0026rsquo;s equation, mean crystallite size was calculated using the XRD data. Fourier transform infrared spectroscopic spectrum was recorded between 400 and 4000 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e with 4 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e intervals to identify the functional organic molecules existing in culture supernatant accountable for the reduction or stabilization of ZnO nanoparticles (Jasco FT/IR-4700 Spectrometer Type-A, Japan). Field Emission Scanning Electron Microscopy (FESEM) (Zeiss Sigma 300, Carl Zeiss AG, Germany), operated at 5 kV, was used to examine the morphology, structural characteristics, and particle size of the synthesized nanoparticles. Energy-dispersive X-ray spectroscopy (EDX), coupled with FESEM, was employed for elemental analysis. For detailed morphological and structural examination, Transmission Electron Microscopy (TEM) was performed (FEI Tecnai G2 20 S-TWIN) with a 200 kV electron source, either LaB₆ or tungsten emitter, and a point resolution of 0.24 nm.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Assessment of the anti-microbial effect of ZnO NPs\u003c/h2\u003e\u003cp\u003eThe antibacterial potential of ZnO NPs prepared from bacterial cell-free supernatant was investigated against four pathogenic bacterial species, such as \u003cem\u003eKlebsiella oxytoca, Staphylococcus epidermidis, Escherichia coli, and Pseudomonas aeruginosa\u003c/em\u003e through well diffusion method. Similarly, ZnO NPs was also tested for their antifungal efficacy against pathogenic fungal strains of \u003cem\u003eCandida albicans, C. kuresi, Aspergillus niger\u003c/em\u003e with concentrations ranging from 100 to 400 \u0026micro;l. Muller-Hinton agar (MHA) plates were inoculated with overnight-grown pathogenic bacterial cultures using sterile swabs to ensure even distribution. Using a sterile gel puncher, wells measuring 5 mm in diameter were created in MHA plates. ZnO NPs suspensions at concentrations of 100, 200, 300, and 400 \u0026micro;l were carefully added to each well. Following 24 hours of incubation at 37\u0026deg;C, diameters of the clear zones around each well were measured in millimeters, and the antimicrobial activity was assessed by referencing a standard interpretation chart [36]. The experiments were carried out in three independent replicates, and data are presented as the mean values with corresponding standard deviations.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Assessment of the antioxidant effect of ZnO NPs\u003c/h2\u003e\u003cp\u003eTo assess antioxidant potential, ZnO NPs were subjected to a 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay following the protocol established by Shimada et al.'s method [37]. Approximately 3 \u0026micro;L of DPPH 1.0 mM dissolved in methanol was added to 1 ml of ZnO nanoparticle suspensions across a concentration range of 5 to 25 \u0026micro;g/mL. The reaction mixtures were incubated in the absence of light at room temperature for 30 min. After incubation, absorbance was recorded at 517 nm employing UV-Visible microplate reader, with methanol as control.\u003c/p\u003e\u003cp\u003eThe scavenging ability was calculated as per the equation given below:\u003c/p\u003e\u003cp\u003e\u003cimg src=\"https://myfiles.space/user_files/127393_c7e80a1c9bb65875/127393_custom_files/img1757408879.png\" style=\"width: 609px;\"\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Cytotoxicity of ZnO NPs\u003c/h2\u003e\u003cp\u003eTo assess cytotoxicity, the MTT assay was carried out on the HT-29 human colon cancer cell line treated with synthesized ZnO nanoparticles, with cells sourced from NCCS, Pune. [38]. Briefly, 10,000 cells per well were cultured into a 96-well microplate and incubated for 24 hours in culture medium consisted of DMEM, fortified with 10% FBS and 1% antibiotic solution, maintained at 37\u0026deg;C in a humidified 5% CO₂ atmosphere. After treatment with different concentrations of ZnO NPs for 24 hours, MTT solution was added, followed by a 2-hour incubation. The medium was carefully discarded, and 100 \u0026micro;L of DMSO was added to solubilize the formazan crystals, and absorbance readings were taken at 540 nm and 660 nm (iMark, Biorad, USA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Statistical evaluation\u003c/h2\u003e\u003cp\u003eResults are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD from three independent experiments. Statistical significance was evaluated using one-way ANOVA with Tukey\u0026rsquo;s multiple comparison test (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). IC₅₀ values were obtained via non-linear regression using GraphPad Prism (evaluation version).\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Spectroscopic study of ZnO nanoparticles\u003c/h2\u003e\u003cdiv id=\"Sec11\" class=\"Section3\"\u003e\u003ch2\u003e3.1.1 UV-Visible Spectral Analysis\u003c/h2\u003e\u003cp\u003eZnO nanoparticles were efficiently produced in this study through a biosynthetic approach employing the cell free culture supernatant of \u003cem\u003eBacillus licheniformis\u003c/em\u003e, which simultaneously contributed to the reduction and surface functionalization. Occurrence of the ZnO NPs synthesis was visually indicated by a noticeable color transition upon adding zinc ions to the cell-free supernatant. In contrast, no change was observed in the control samples without zinc ions, which confirmed the contribution played by the bacterial extract on facilitating the biosynthesis of ZnO NPs. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the synthesized ZnO nanoparticles exhibited a sharp UV-Vis absorption peak at 331 nm, indicative of effective NPs synthesis. An observable blue shift in the absorption spectrum suggests that particles are not significantly larger than the exciting Bohr radius. It has been established that the characteristic absorption peaks of green-synthesized ZnO nanoparticles occur within the 330\u0026ndash;390 nm range [15, 39, 40].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section3\"\u003e\u003ch2\u003e3.1.2 FT-IR study\u003c/h2\u003e\u003cp\u003eFTIR spectroscopy was used to explore the molecular interactions at the interface of zinc oxide and the bioactive organic compounds in the bacterial cell-free extract. It also helped identify the organic functional moieties and biomolecules responsible for the biological synthesis of ZnO NPs and the FTIR spectrum is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. A distinct spectral band appeared with a value of 799 cm⁻\u0026sup1; indicated Zn\u0026ndash;O\u0026ndash;Zn stretching vibration, confirming the ZnO fabrication. (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). A peak observed with a value of 1020 cm⁻\u0026sup1; was attributed to C\u0026ndash;O stretch vibrations. Meanwhile, the band appearing at 1535 cm⁻\u0026sup1; corresponded to C\u0026thinsp;=\u0026thinsp;C stretch, C\u0026ndash;N\u0026ndash;H bend, and amide group functionalities. Additionally, the absorption bands at 2856 and 2918 cm⁻\u0026sup1; were associated with C\u0026ndash;H bond stretch [39\u0026ndash;41]. The observation of these peaks suggested the presence of surface-bound molecules in the bacterial cell-free extract. An absorption peak appearing at 2532 cm⁻\u0026sup1; was assigned to the stretching vibrations of hydroxyl (\u0026ndash;OH) groups. The FTIR spectrum obtained for ZnO NPs is characterized by the existence of a prominent aromatic ring and carboxylic acid, confirming the role of bioactive molecules in involved in biosynthesis of ZnO NPs [40].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Structural elements analysis\u003c/h2\u003e\u003cp\u003eThe XRD patterns of the synthesized ZnO NPs are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Based on XRD results, ZnO NPs were found to be pure and crystalline nature. The XRD pattern (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) revealed conspicuous peaks corresponding to lattice planes of (100), (-110), (101), (002), (102), (110), and (103) at Bragg's angles of 31.7\u0026deg;, 56.6\u0026deg;, 36.2\u0026deg;, 34.4\u0026deg;, 62.8\u0026deg;, and 47.5\u0026deg;, respectively. As indicated by the data the biofabricated NPs have a face-centred hexagonal crystal structure was confirmed. The obtained peaks were matched with corresponding lattice planes according to the JCPDS reference pattern (Card No: 96-230-117). Additionally, estimated mean diameter of the nanoparticles was around 36 nm based on the Debye\u0026ndash;Scherrer equation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Morphological studies\u003c/h2\u003e\u003cp\u003eZnO NP\u0026rsquo;s morphological features were examined by employing a FE-SEM (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea and b). The SEM micrographic images captured at different magnifications revealed well-defined hexagonal NPs averaging 36 nm in diameter, confirming their morphology. In addition, TEM micrographic images revealed that the hexagonal nanoparticles form a cluster-like configuration having an average size of about\u0026thinsp;~\u0026thinsp;36 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). ZnO NP\u0026rsquo;s SAED pattern showed clear diffraction rings, confirming their crystalline phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). The EDX spectrum showed two distinct and significant zinc peaks at 1.2 keV, Indicating that zinc was present in the fabricated nanoparticles. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e illustrates the EDX spectra of the ZnO NPs synthesized in this study exhibited distinct peaks for zinc (Zn) and oxygen (O). These results further validated the elemental composition and purity of the biosynthesized ZnO nanoparticle (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.4. Antimicrobial efficiencies of biosynthesized ZnO NPs\u003c/h2\u003e\u003cp\u003eThe well diffusion method was employed to assess the \u003cem\u003ein vitro\u003c/em\u003e antibacterial efficacy of the synthesized ZnO NPs. The results demonstrated that biosynthesized ZnO NPs showed superior anti-bacterial efficacy (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). At the highest dose of 400 \u0026micro;L, the ZnO nanoparticles produced the largest clearance zone i.e., 13\u0026thinsp;\u0026plusmn;\u0026thinsp;1 mm against \u003cem\u003eK. oxytoca\u003c/em\u003e, followed by noticeable inhibitory effects against \u003cem\u003eS. epidermidis\u003c/em\u003e and \u003cem\u003eP. aeruginosa\u003c/em\u003e. The findings clearly demonstrate that the elevating dose of ZnO NPs led to a stronger zone of inhibition. The synthesized ZnO NPs showed significant active against a diverse group of bacteria, such as representatives from both Gram-positive and Gram-negative classes. According to previous findings, ZnO nanoparticles produced through green techniques display stronger antibacterial effect against Gram-negative bacteria in comparison to Gram-positive strains. The discrepancy in susceptibility is often ascribed with the structural composition of the bacterial cell walls. The thick, peptidoglycan-rich layer found in Gram-positive bacteria can act as a barrier, reducing the penetration and overall effectiveness of ZnO nanoparticles [42\u0026ndash;44].\u003c/p\u003e\u003cp\u003eThe biosynthesized ZnO nanoparticles further examined for their antifungal effect against several fungal strains, including \u003cem\u003eAspergillus niger\u003c/em\u003e, \u003cem\u003eCandida krusei\u003c/em\u003e, and \u003cem\u003eCandida albicans\u003c/em\u003e. The antifungal activity is dose-dependent. At the highest ZnO nanoparticle concentration tested, inhibition zones reached 12.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58 mm for \u003cem\u003eC. albicans\u003c/em\u003e and 11.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58 mm for \u003cem\u003eA. niger\u003c/em\u003e, indicating significant antifungal activity. (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Our results are consistent with previous reports of [45, 46]. Their distinct electronic properties allow ZnO nanoparticles to interrupt electron transport pathways in biological environments, thereby generating reactive oxygen species (ROS) [47].\u003c/p\u003e\u003cp\u003eZnO nanoparticles exert their antibacterial effects is thought to occur by means of several mechanisms and the exact mechanism remains elusive. Firstly, nanoparticles can affect microbial cells by interacting with their membranes through electrostatic forces, allowing them to penetrate it. Once inside, ZnO nanoparticles may induce oxidative stress by generating ROS which results in cellular destruction [48]. Furthermore, ZnO NPs possess surface properties that can catalyze the generation of hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), which can diffuse into the bacterial membrane, leading to cell damage and death [49, 50].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.10. Antioxidant activity of ZnO NPs\u003c/h2\u003e\u003cp\u003eThis study showed that biosynthesized ZnO NPs displayed notable antioxidant potential, particularly showing strong hydrogen peroxide scavenging capacity. In relation to ascorbic acid, scavenging property of ZnO NPs is found to be less; however, no significant changes were observed. The antioxidant effect showed a concentration-dependent pattern with higher concentrations yielding stronger activity with ZnO nanoparticles (Fig.\u0026nbsp;6). The observed antioxidant potential of the biosynthesized ZnO nanoparticles may result from the functional groups and bioactive constituents present in the bacterial cell-free supernatant. The bioactive components in the supernatant provide hydrogen atoms, which prevent free radical reactions [40]. This study reveals that the bacterial cell-free supernatant contains bio-components that enhance the antioxidant activity. Based on these results, biosynthesized ZnO NPs may serve as promising candidates for antioxidant therapeutic applications.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.11. Cytotoxicity of ZnO NPs against HT-29 cells\u003c/h2\u003e\u003cp\u003eTo determine the cytotoxic efficacy of ZnO nanoparticles, an MTT test was conducted against human colon cancer cell lines. The finding indicated that the anticancer activity of ZnO NPs on HT-29 cells was increased with concentration, exhibiting comparable cytotoxic effects at low doses and following a dose response (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003e). In this investigation, the dose of ZnO NPs required to reduce cell viability by 50% (IC₅₀) was calculated as 56.55 \u0026micro;g/m against HT-29 cells. Previously, the reported IC\u003csub\u003e50\u003c/sub\u003e values for ZnO NPs against HT-29 cells are 54.16 g/mL [40], 45.82 g/mL [51], and \u0026gt;\u0026thinsp;500 \u0026micro;g/mL [52]. The IC₅₀ values of ZnO NPs synthesized biologically and chemically were 135.9 \u0026micro;g/mL [53] and 131 \u0026micro;g/mL [54], respectively, against HT-29 cells.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eNanoparticle concentration was a key factor influencing their activity against cancer cells, potentially passing through ion channels in the cell membrane and interacting with nitrogenous bases in DNA as well as various intracellular proteins. ZnO NPs are internalized through endocytic pathways, with uptake and the levels of accumulation depending on size, shape, and cell type [55]. Upon entry, ZnO NPs dissolve and release Zn\u0026sup2;⁺ ions that disrupt mitochondrial function by disturbing the flow of electrons through the electron transport chain. This induces levated ROS levels, which induce oxidative stress and lead to the deterioration of mitochondrial structures and DNA integrity, and ultimately triggering apoptosis-mediated cytotoxicity [56, 57].\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eThis present study reports the effective fabrication of ZnO nanoparticles using bacterial cell-free supernatant offering a novel and sustainable route for nanoparticle production. Detailed analysis of the ZnO NPs revealed a sharp UV-visible distinct spectral peak occurring at a defined wavelength, validating their purity, whereas FTIR spectra showed characteristic bioactive organic moieties involved in nanoparticle formation. ZnO nanoparticles are well-defined hexagonal NPs averaging 36 nm in diameter with pure crystalline form confirmed through XRD, SEM, and TEM analyses. Furthermore, the anticancer investigation suggested that ZnO NPs induced dose-dependent cytotoxicity. Collectively, these findings suggest that ZnO NPs derived from bacterial cell-free supernatant may serve as an effective cancer therapy agent. This study presents valuable insights into the biological uses and potential of ZnO NPs, encompassing production methods, antibacterial activity, antioxidant activity, and anticancer activity. As a low-cost, low-toxic, and multifunctional material, ZnO NPs hold great promise for future biological applications. Despite these promising findings, ongoing research efforts are crucial to fully address the remaining gaps and limitations, including the targeted delivery of ZnO NPs to multiple cells and live animals. While ZnO NPs have demonstrated strong antimicrobial and anticancer efficacy, additional studies are required for translating these findings into practical applications.\u003c/p\u003e\u003cp\u003e\u003cb\u003eFuture prospective\u003c/b\u003e:\u003c/p\u003e\u003cp\u003eDespite the advantages of ZnO NPs, several challenges remain unaddressed to unlock the full potential of ZnO NPs. Scalability is a significant concern, as producing high-quality ZnO NPs on a large scale with consistent properties remains a hurdle. Given, the potential toxicity of ZnO NPs is a concern, further research is essential to evaluate their possible health and ecological risks. The functionalization of ZnO NPs with other materials and compounds is a viable technique for improving their properties and uses. Overall, ZnO NPs hold vast potential in various fields, and with ongoing scientific progress, current obstacles may be overcome, unlocking new uses and paving the path for their increased adoption in cosmetics and personal care products.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eDeclaration of competing interest\u003c/h2\u003e\n\u003cp\u003eThe author declares that no competing financial interests or personal ties could have influenced the work presented in this paper.\u003c/p\u003e\n\u003ch2\u003eEthical approval\u003c/h2\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003ch2\u003eConflicts of interest\u003c/h2\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis study received no external support.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eAuthor ContributionSP-Priyadharsini Shanumuganandam, TS-Sathiamoorthi Thangavelu. SP, TS -All authors contributed to the study conception and design. SP- performed the literature review, data collection, and analysis. contributed to the methodology and validation of results. TS-supervised the project and provided critical feedback. SP, TS All authors discussed the results, reviewed, and approved the final manuscript.\u0026quot;Consent for publication:All authors agree to published the paper in your esteemed journal.\u003c/p\u003e\n\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eAll the authors sincerely acknowledge the support provided by RUSA 2.0 and the Research Instrumentation Facilities of the Department of Microbiology, Alagappa University, Karaikudi, Tamil Nadu. The authors also wish to express their appreciation to the Central Instrumentation Centre (CIC) at Madurai Kamaraj University, Madurai, Tamil Nadu for assistance with TEM analysis, Bannari Amman Institute of Technology, Sathyamangalam, Tamil Nadu for conducting EDX and SEM analyses, and Vellore Institute of Technology, Vellore, Tamil Nadu for performing the XRD analysis.\u003c/p\u003e\n\u003ch2\u003eData availability statement\u003c/h2\u003e\n\u003cp\u003eNo supplementary data for this work can be provided.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSharma DK, Shukla S, Sharma KK, Kumar V: \u003cb\u003eA review on ZnO: Fundamental properties and applications\u003c/b\u003e. \u003cem\u003eMaterials Today: Proceedings\u003c/em\u003e 2022, \u003cb\u003e49\u003c/b\u003e:3028\u0026ndash;3035.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSchneider SL, Lim HW: \u003cb\u003eA review of inorganic UV filters zinc oxide and titanium dioxide\u003c/b\u003e. 2019, \u003cb\u003e35\u003c/b\u003e(6):442\u0026ndash;446.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOtalora C, Botero MA, Ordo\u0026ntilde;ez G: \u003cb\u003eZnO compact layers used in third-generation photovoltaic devices: a review\u003c/b\u003e. \u003cem\u003eJournal of Materials Science\u003c/em\u003e 2021, \u003cb\u003e56\u003c/b\u003e(28):15538\u0026ndash;15571.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEl Golli A, Contreras S, Dridi C: \u003cb\u003eBio-synthesized ZnO nanoparticles and sunlight-driven photocatalysis for environmentally-friendly and sustainable route of synthetic petroleum refinery wastewater treatment\u003c/b\u003e. \u003cem\u003eScientific Reports\u003c/em\u003e 2023, \u003cb\u003e13\u003c/b\u003e(1):20809.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHussain RT, Hossain MS, Shariffuddin JH: \u003cb\u003eGreen synthesis and photocatalytic insights: A review of zinc oxide nanoparticles in wastewater treatment\u003c/b\u003e. \u003cem\u003eMaterials Today Sustainability\u003c/em\u003e 2024, \u003cb\u003e26\u003c/b\u003e:100764.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChandran S, Ganesan AP, Asthana N, Pandey SS, Singh KRB, Natarajan A: \u003cb\u003eProliferating optoelectronic properties of doped ZnO nanoparticles\u003c/b\u003e. \u003cem\u003eJournal of Molecular Structure\u003c/em\u003e 2024, \u003cb\u003e1311\u003c/b\u003e:138310.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJaafar AH, Lowe C, Gee A, Kemp NT: \u003cb\u003eOptoelectronic Switching Memory Based on ZnO Nanoparticle/Polymer Nanocomposites\u003c/b\u003e. \u003cem\u003eACS Applied Polymer Materials\u003c/em\u003e 2023, \u003cb\u003e5\u003c/b\u003e(4):2367\u0026ndash;2373.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZare M, Namratha K, Ilyas S, Sultana A, Hezam A, L S, Surmeneva MA, Surmenev RA, Nayan MB, Ramakrishna S \u003cem\u003eet al\u003c/em\u003e: \u003cb\u003eEmerging Trends for ZnO Nanoparticles and Their Applications in Food Packaging\u003c/b\u003e. \u003cem\u003eACS Food Science \u0026amp; 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In: \u003cem\u003eInternational Journal of Molecular Sciences.\u003c/em\u003e vol. 23; 2022.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMoghimipour E, Rezaei M, Ramezani Z, Kouchak M, Amini M, Angali KA, Dorkoosh FA, Handali S: \u003cb\u003eTransferrin targeted liposomal 5-fluorouracil induced apoptosis via mitochondria signaling pathway in cancer cells\u003c/b\u003e. \u003cem\u003eLife Sciences\u003c/em\u003e 2018, \u003cb\u003e194\u003c/b\u003e:104\u0026ndash;110.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"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":"Biosynthesis, ZnO NPs, Bacillus licheniformis, HT-29 cell lines, Antimicrobial activity, Antioxidant activity","lastPublishedDoi":"10.21203/rs.3.rs-7440837/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7440837/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMicrobial synthesis of nanoparticles has gained attention as an eco-friendly, efficient, and cost-effective alternative to conventional chemical and physical approaches. This study reports the biosynthesis of Zinc Oxide nanoparticles (ZnO NPs) through the use of the cell-free extract of \u003cem\u003eBacillus licheniformis\u003c/em\u003e strain FC14167, isolated from soil. The ZnO nanoparticles obtained in this study were characterized using a combination of UV\u0026ndash;visible spectroscopy, FTIR, XRD, SEM\u0026ndash;EDX, and TEM to assess their optical, structural, and morphological properties. ZnO nanoparticles showed a distinct UV-Vis apeak at 331 nm, FTIR analysis revealed functional groups contributing to nanoparticle synthesis, while XRD patterns verified the crystalline structure and purity. The ZnO NPs exhibited a well-defined hexagonal shape, averaging 36 nm in diameter, evident from SEM and TEM micrographs, and the presence of Zn was confirmed through the EDX spectral analysis. Biosynthesized ZnO NPs were found to possess effective antimicrobial activity and zones of inhibition against certain bacterial and fungal pathogens. Furthermore, the nanoparticles demonstrated cytotoxicity against HT-29 cancer cell lines, resulting to considerable necrotic and abnormal shapes in cancer cells with an IC\u003csub\u003e50\u003c/sub\u003e value determined to be 56.55 \u0026micro;g/mL. The current investigation indicates, biosynthesized ZnO NPs possess substantial anticancer activity and inhibitory effects against HT-29 cancer cell lines. Accordingly, this study conjectures the potential of bacteria-mediated ZnO NPs as effective anticancer agents.\u003c/p\u003e","manuscriptTitle":"Biogenic and green approach for ZnO nanoparticle synthesis via Bacillus licheniformis and their antimicrobial and anticancer potential","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-09 09:22:14","doi":"10.21203/rs.3.rs-7440837/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":"f21dda72-fe16-4d37-a6ba-298d7bb73c8e","owner":[],"postedDate":"September 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-09T09:22:14+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-09 09:22:14","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7440837","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7440837","identity":"rs-7440837","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}