Biosynthesis and Characterization of Silver Nanoparticles Using Microbes and Remediation of Dyes

Biosynthesis and Characterization of Silver Nanoparticles Using Microbes and Remediation of Dyes | 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 Biosynthesis and Characterization of Silver Nanoparticles Using Microbes and Remediation of Dyes Aditya S, A Vimal, A Kavipraba This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7728875/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 This study explores the eco-friendly biosynthesis of silver nanoparticles (AgNPs) using Bacillus subtilis AVK-21 isolated from water samples, focusing on their characterization and application in photocatalytic degradation of Congo Red dye. Using biomolecules as stabilising and reducing agents, silver nitrate was reduced by microbes to create the AgNPs. The crystalline face-centered cubic structure, spherical shape, and nanoscale size (average of 271.3 nm) of the nanoparticles were validated by thorough characterisation utilising UV-Vis spectroscopy, XRD, FTIR, DLS, zeta potential, SEM, TEM, and EDS. Due to surface plasmon resonance and the reactive oxygen species production, the AgNPs demonstrated strong photocatalytic activity, breaking down 67.01% of Congo Red in the presence of sunlight and 37.80% in the presence of ultraviolet light. The work emphasises the need for more research on scalability and toxicological effects while highlighting the promise of microbially synthesised AgNPs for sustainable environmental remediation, namely in treating dye-polluted effluents. Silver nanoparticles Biosynthesis Photocatalytic Degradation Congo Red Surface Plasmon Resonance Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 1. INTRODUCTION Water pollution from dyes and oils is a major environmental concern, especially in industrial areas where untreated waste is often discharged into water bodies. The textile industry alone accounts for 20% of global wastewater pollution, releasing large quantities of toxic, non-biodegradable dyes into the environment. Azo dyes, in particular, are widely used for their vibrant colors and stability, but they pose serious ecological and health risks. Some common examples include Tartrazine (E102), a yellow dye used in beverages and confectionery, and Sunset Yellow (E110), an orange dye used in snacks and baked goods [ 1 ]. It is estimated that 2,00,000 tons of dyestuff are discharged into the environment annually, with concentrations in textile effluent reaching up to 500 parts per million (ppm) These dyes are resistant to biodegradation and can break down under anaerobic conditions into aromatic amines, many of which are carcinogenic and toxic [ 2 ]. Their presence in water bodies reduces light penetration, impairs photosynthesis, and disrupts aquatic ecosystems. For humans, exposure to azo dye byproducts has been linked to cancer, genetic mutations, and endocrine disruption, while their accumulation in aquatic organisms further magnifies risks through the food chain [ 3 – 8 ]. Conventional methods for dye removal, such as adsorption and photocatalysis, can be expensive and energy-intensive [ 9 ]. Consequently, researchers are exploring alternative solutions, and silver nanoparticles have emerged as a promising candidate for dye degradation due to their unique properties. The production of silver nanoparticles has drawn a lot of interest because of their distinct physicochemical characteristics and wide range of uses in industries like environmental remediation, catalysis, and antibacterial agents [ 10 ]. While traditional physical and chemical methods for nanoparticle synthesis can be effective, they often involve the use of toxic reagents and harsh conditions, which can be environmentally harmful [ 11 ]. The development of sustainable and environmentally acceptable methods for the production of silver nanoparticles has gained attention in recent years, with an emphasis on using biological resources like microorganisms [ 12 – 17 ]. The biosynthesis of silver nanoparticles using microorganisms has emerged as a promising alternative to conventional methods, as it offers several advantages, including the use of renewable and non-toxic precursors, the ability to control the size and shape of the nanoparticles, and the potential for scaling up the production process. Microorganisms, such as bacteria, fungi, and algae, have the ability to reduce silver ions (Ag+) to metallic silver, leading to the formation of silver nanoparticles with diverse morphologies [ 18 – 25 ]. The green synthesis of AgNPs involves three main stages: activation, growth, and termination [ 26 ]. During activation, the reducing agents present in the biological entity initiate the reduction of silver ions. This is followed by the growth phase, where the reduced silver atoms cluster together to form nanoparticles. Finally, the termination phase involves the stabilization of the nanoparticles to prevent further growth and aggregation. This flexibility in preparation and the ability to avoid the utilization of toxic chemicals make green synthesis a preferred method for nanoparticle production [ 27 ]. Green synthesized AgNPs degrade dyes primarily through photocatalytic degradation. This process involves the absorption of light energy by the AgNPs, which generates electron-hole pairs. These excited electrons and holes then react with the dye molecules, leading to their breakdown into less harmful substances [ 28 ]. The efficiency of this process depends on factors such as the size and shape of the AgNPs, the type of dye, and the intensity of light irradiation. Research has indicated that green synthesized AgNPs can efficiently degrade a variety of dyes. The effectiveness of AgNPs in dye degradation can be size-dependent. For example, a study comparing AgNPs synthesized using different concentrations of silver nitrate found that smaller nanoparticles exhibited higher catalytic activity for the degradation of methyl orange [ 29 ]. This is because smaller nanoparticles have a larger surface area to volume ratio, which provides more active sites for the catalytic reaction. In addition to the size of the nanoparticles, the type of dye and the green synthesis method employed also influence the degradation efficiency. For instance, AgNPs synthesized using Citrus reticulata Blanco peel extract completely degraded malachite green dye after 120 hours of incubation in sunlight [ 30 ]. Another study found that AgNPs synthesized using leaf extract of Solena amplexicaulis effectively degraded methylene blue dye under solar irradiation [ 31 ]. This study also showed that the green synthesized NPs reduced methylene blue and 4-nitrophenol significantly [ 32 ]. Furthermore, AgNPs have shown promising results in degrading a mixture of dyes, which is a more realistic scenario mimicking the composition of industrial wastewater. A study by Deepak Gola concluded that chemically synthesized silver nanoparticles (CH-AgNPs) combined with NaBH4 could potentially enhance the dye degradation of orange and blue dyes individually and as a mixture [ 33 ]. Another recent study where researchers explored a novel approach to creating silver nanoparticles by utilizing the natural capabilities of bacteria. They employed three distinct bacterial species – Micrococcus luteus (MBC23), Klebsiella pneumoniae (MBC34) and Enterobacter aerogenes (MBX6) – to synthesize three different types of silver nanoparticles, designated as AgNPs-K, AgNPs-M, and AgNPs-E, respectively. Of the three types, AgNPs-M were the most effective at removing the dye methyl orange from wastewater, eliminating nearly 20% of it within two hours [ 34 ]. A study in Iran investigates a specific type of microalgae used to create silver nanoparticles (AgNPs) and their ability to break down a harmful dye called Methyl Red found in wastewater [ 35 ]. Scientists used the byproducts of a fungus called Fusarium oxysporum to create small silver nanoparticles (SNPs). These nanoparticles were very effective at breaking down a dye called crystal violet when exposed to light. In fact, they could remove almost 99% of the dye from a solution within 4 hours when used at a specific concentration [ 36 ]. 2. MATERIALS AND METHODOLOGY 2.1 MATERIALS Silver Nitrate (AgNO3) powder was purchased from. distilled water was used throughout for solution preparation and washing steps. Sodium hydroxide (NaOH) and hydrochloric acid (HCl) were used for pH adjustments. Nutrient agar and Nutrient broth were used for the cultivation of bacterial isolates. Centrifuges, an incubator shaker, a magnetic stirrer, a UV lamp chamber, and standard sterile glassware such as conical flasks, beakers, and micropipettes were used as needed. 2.2 WATER SAMPLE COLLECTION Water samples were aseptically collected from four isolated, low-human-activity locations in Madurai—Mudalaikulam 1, Mudalaikulam 2, Sholavandhan, and the Vaigai River using 200 mL sterilized plastic containers at a depth of 20–30 cm with proper labelling and stored at 4°C to preserve microbial viability. 2.3 ISOLATION OF MICROBES USING SPREAD PLATE METHOD The water sample bottle was vigorously shaken to homogenize the microbial population. Serial dilutions were performed by transferring 1 mL of the sample into 9 mL of sterile distilled water. Successive dilutions (10 − 1 to 10 − 4) were prepared to achieve a suitable concentration for plating. Using a sterile micropipette, 0.1 mL of the desired dilution was transferred onto the surface of the solidified agar in a petri dish. The sample was spread evenly over the agar surface using a sterile glass spreader or L-shaped rod. After the incubation period of 24–48 hours at 37°C, microbial growth was observed on the nutrient agar plates inoculated with water samples. Distinct microbial colonies were observed on the four sample plates, exhibiting variations in size, shape, color, and texture (Refer Fig. 1 in Online Resource). 2.4 STREAKING FOR PURE CULTURES After incubation, spread plates were examined for isolated colonies based on distinct characteristics such as size, color, shape, texture, and edge definition, and six colonies were selected from four sample plates. The inoculating loop was sterilized by flame, allowed to cool, and then used to gently pick a colony, which was streaked onto a fresh nutrient agar plate in a zigzag pattern across four quadrants to reduce the microbial load and isolate individual colonies, ensuring minimal overlap. The streaked plates were inverted and incubated at 37°C for 24–48 hours, resulting in well-separated colonies with no contamination as shown in Fig. 1 . 2.5 PREPARATION OF BROTH CULTURE Sterile nutrient broth was prepared by dissolving the required amount of powdered medium in distilled water (e.g., 13 g/L), and autoclaved at 121°C for 15 minutes to ensure sterility. A single isolated colony from the streak plate was selected using a sterile loop and inoculated into a sterile 100 mL conical flask containing 100 mL of nutrient broth under aseptic conditions for each of the six samples. The inoculated flasks were incubated at 37°C in a shaker incubator at 120–150 rpm for 18–24 hours to promote uniform growth. After incubation, visible turbidity was observed in all six flasks, indicating microbial growth (Refer Fig. 2 in Online Resource).. No contamination was detected. 2.6 STAINING Two types of staining were performed: Simple Staining and Gram Staining. Simple staining, using Methylene Blue that identifies bacterial cells by enhancing contrast, making cell features like shape, size, and arrangement easier to observe under a microscope. Gram Staining categorizes bacteria into Gram-positive (purple) and Gram-negative (pink/red) based on their cell wall structure. For simple staining, a smear was prepared on a clean slide with a drop of sterile water and a loopful of broth culture, which was air-dried and heat-fixed. Methylene blue was applied for 1–2 minutes, excess stain was washed off, and the slide was air-dried. Under a microscope at 100x magnification, all six samples were examined and found to be bacteria. Streaks 1, 2, and 5 exhibited cocci-shaped cells; Streaks 3 and 4 showed rod-shaped bacteria; and Streak 6 displayed round cells resembling cocci. (Refer Fig. 3 in Online Resource). For Gram staining, a smear was prepared in the same manner, followed by crystal violet for 1 minute, Gram's iodine for 1 minute, ethanol for 15–30 seconds, and Safranin for 1 minute. The slide was examined under a microscope at 100x magnification with immersion oil, and the results were recorded: Gram-positive bacteria appeared purple, and Gram-negative bacteria appeared pink. All six samples were examined, and among the six isolates, three were observed to be Gram-positive (appearing purple) i.e. Streak 3, Streak 5 and Streak 6, while three were Gram-negative (appearing pink) i.e. Streak 1, Streak 2 and Streak 4. 2.7 MICROBIAL IDENTIFICATION BY SEQUENCING Microbial sequencing is a precise method used to identify and characterize microorganisms by analyzing their genetic material. It helps distinguish species by targeting specific genes, such as the 16S ribosomal RNA (16S rRNA) gene for bacteria and archaea or the Internal Transcribed Spacer (ITS) region for fungi (Janda & Abbott, 2007). The sequencing method used for the identification was Sanger sequencing. Out of 6 isolates, a single isolate was considered for the sequencing and further synthesis of Silver nanoparticles. Streak 4 was selected for sequencing as it was having morphologically distinctive characteristics with mucoidal appearance. 16S rRNA was used for the identification of the bacteria. For this purpose, the pure culture of the isolate was submitted to a commercial sequencing service provider, NGS Hub, National Facility for Coastal and Marine Research, Sathyabama Institute of Science and Technology, Chennai, India. The entire sequencing workflow, including genomic DNA extraction, PCR amplification, and sequencing, was carried out by the consultancy. Genomic DNA was extracted from the bacterial sample using a standard phenol-chloroform method or a commercially available DNA extraction kit, as per the consultancy’s protocol. The 16S rRNA gene was then amplified using universal bacterial primers (such as 27F and 1492R), targeting conserved regions of the gene. The amplified products were purified and subjected to Sanger sequencing using cycle sequencing chemistry with fluorescently labeled dideoxynucleotides. The resulting chromatogram files were analyzed for base calling and sequence quality. The final consensus sequence was compared against the NCBI GenBank database using the BLAST (Basic Local Alignment Search Tool) algorithm to identify the isolate based on sequence homology with known bacterial strains. The bacterial species showing the highest similarity percentage and alignment score was considered as the identified strain. The identified strain was subsequently used for the biosynthesis of silver nanoparticles. 2.8 BIOSYNTHESIS AND CHARACTERIZATION OF SILVER NANOPARTICLES Silver nanoparticles were synthesized using microbial processes, wherein biologically active molecules facilitated the reduction of silver ions (Ag⁺) to elemental silver (Ag⁰). Enzymes like nitrate reductase, along with proteins, polysaccharides, and other microbial biomolecules, acted as reducing and stabilizing agents, while functional groups present on these biomolecules provided surface capping to prevent aggregation and maintain colloidal stability. For the synthesis, a selected bacteria was cultured in nutrient broth under optimal growth conditions i.e. 37°C, after which the culture was centrifuged to separate the biomass from the supernatant. The obtained supernatant was then reacted with a 1 mM silver nitrate solution in equal proportion and incubated at room temperature to facilitate nanoparticle formation as shown in Fig. 5 (OnlineResource). The synthesized nanoparticles were purified by multiple centrifugation and washing to eliminate unreacted ions and biomolecular residues. Finally, the purified silver nanoparticles were resuspended in distilled water as shown in Fig. 6 (OnlineResource) and stored at 4°C in dark containers to preserve their stability and prevent light-induced degradation. The synthesized silver nanoparticles were characterized using various analytical techniques to confirm their formation, stability, and physicochemical properties. Surface plasmon resonance (SPR) peak detection was done using UV-visible spectroscopy, a key indicator of nanoparticle synthesis, by measuring absorbance in the 200–800 nm range. The average hydrodynamic diameter, polydispersity index (PDI), and colloidal stability of the nanoparticles were measured using dynamic light scattering (DLS), while Zeta Potential analysis provided insight into surface charge and dispersion stability in suspension. Fourier Transform Infrared (FTIR) spectroscopy identified functional groups responsible for the reduction and capping of nanoparticles, indicating the involvement of microbial biomolecules. Finally, Transmission Electron Microscopy (TEM) provided high-resolution images to assess the shape, size, and dispersion of the nanoparticles, confirming their nanoscale morphology. 2.9 PHOTOCATALYTIC REMEDIATION OF DYE (CONGO RED) USING SILVER NANOPARTICLES The photocatalytic potential of biosynthesized silver nanoparticles was assessed through the degradation of Congo Red dye, a commonly used azo dye. To begin, a stock dye solution was prepared by dissolving 100 mg of Congo Red in 1 liter of deionized water, yielding a concentration of 100 mg/L. From this stock, a working solution of 25 mg/L was obtained by diluting 25 mL of the stock with 75 mL of deionized water. For the degradation experiment, 1 mg of silver nanoparticles (or an optimized concentration) was added to 100 mL of the working dye solution in a clean beaker. The mixture was stirred at 200–300 rpm using a magnetic stirrer to ensure homogeneous dispersion of the nanoparticles. The solution's pH was brought within the range of 3 and 7, depending on the requirement, as pH significantly influences photocatalytic efficiency. The dye-nanoparticle mixture was then exposed to two different light conditions to assess photocatalytic activity: natural sunlight during peak daylight hours and ultraviolet (UV) light at wavelengths such as 254 nm or 365 nm, provided by a UV chamber. These conditions allowed for the evaluation of both solar- and UV-induced degradation pathways. At specific time intervals—0, 10, 20, 30, 60, and 120 minutes—2 mL aliquots of the reaction mixture were withdrawn to monitor the progress of dye degradation. Each sample was centrifuged to remove residual nanoparticles, and the supernatant was analyzed. The degradation of Congo Red was tracked using UV-Visible spectrophotometry by measuring absorbance at 497 nm, which corresponds to the dye’s maximum absorbance wavelength (λmax). The initial absorbance (A₀) and absorbance at each time interval (Aₜ) were recorded and compared to determine the percentage of dye degraded over time. The following formula was used to determine the degradation efficiency: Degradation Efficiency (%) = \(\:\frac{\left(A0-At\right)}{A0}\times\:\:100\) \(\:\) These values were computed for both sunlight and UV exposure conditions, and the results were plotted to provide a comparative understanding of photocatalytic efficiency under different lighting scenarios. This approach highlights the effectiveness of silver nanoparticles in environmental remediation through dye degradation. 3. RESULTS AND DISCUSSION 3.1 MICROBIAL SEQUENCING RESULT Microbial sequencing analysis was conducted to identify the bacterial species present in the sample. 16S rRNA contig sequence of a bacterial isolate has been shown in Fig. 2 . This is a continuous stretch of nucleotide bases (A, T, G, C) obtained from 16S rRNA gene sequencing. The 16S rRNA gene sequencing revealed a high similarity with Bacillus subtilis , confirming its presence. Figure 3 shows that the sequence alignment using BLAST showed a 97.85% identity match with known B. subtilis strain . 3.2 UV-VISIBLE SPECTROMETER ANALYSIS UV-vis spectroscopy is the main and critical instrument for understanding silver nanoparticle formation during the early synthesis stage. It is quite characteristic based on the form, size, and distribution of Ag nanoparticles due to surface plasmon resonance [ 37 ]. The UV-Vis absorption spectra of the synthesized nanoparticles showed a characteristic Surface Plasmon Resonance (SPR) peak at 430 nm, confirming the successful formation of silver nanoparticles as shown in Fig. 4. The observed peak position is consistent with previously reported values for silver nanoparticles, further validating their synthesis. Any slight variations in peak intensity or position could be attributed to factors such as particle size, shape, or stabilizing agents used during synthesis. 3.3 DYNAMIC LIGHT SCATTERING (DLS) ANALYSIS It typically analyzes the hydrodynamic diameter of nanoparticles impacted by capping agents, as well as the presence of an electrical double layer deposited on the surface of nanoparticles, and works best with monodisperse nanoparticles [ 38 ]. The DLS analysis revealed an average hydrodynamic diameter of 269.45 nm with a PDI value of nearly 0, indicating monodisperse nature. The synthesized microbial silver nanoparticles (AgNPs) exhibit a monodisperse size distribution with an average size of 271.3 nm and minimal variation as shown in Fig. 5 . The absence of a reported PDI suggests extremely low polydispersity, meaning the nanoparticles are highly uniform. Overall, the sample appears to be well- synthesized, with a narrow and uniform size distribution, making it suitable for applications requiring consistent nanoparticle size. 3.4 ZETA POTENTIAL ANALYSIS Zeta potential analysis is a crucial technique for evaluating the surface charge and colloidal stability of nanoparticles in suspension. It provides insight into the repulsive or attractive forces between particles, influencing their dispersion, aggregation, and overall stability in a given medium [ 39 ]. A zeta potential of 0.0 mV suggests that the nanoparticles lack a net surface charge, meaning they do not experience significant electrostatic repulsion as shown in Fig. 6 . The electrophoretic mobility being zero further supports that the nanoparticles are not moving in response to the applied electric field, confirming the absence of a measurable charge. Nanoparticles with a zeta potential between − 10 and + 10 mV are considered approximately neutral (Clogston and Patri, 2011). 3.5 TEM ANALYSIS TEM is one of the most sophisticated analytical measurement instruments for visualizing and differentiating nanoparticle size and form. As shown in Fig. 7 , the nanoparticles predominantly exhibit a spherical shape, with some degree of aggregation. The Selected Area Electron Diffraction (SAED) pattern of the AgNPs (Fig. 7 (c)) exhibits distinct concentric diffraction rings, corresponding to the face-centered cubic (FCC) structure of silver. This confirms the crystalline nature of the nanoparticles. Energy Dispersive X-ray Spectroscopy (EDS) was used to determine the elemental composition of the synthesized AgNPs. The EDS spectrum displays strong silver (Ag) peaks as shown in Fig. 8, confirming the successful synthesis of AgNPs. In addition to silver, minor peaks corresponding to C, O, Fe, and Cl were observed. The presence of oxygen and carbon can be attributed to biomolecular capping agents from the microbial extract, which enhance nanoparticle stability. Iron and chlorine impurities may arise from the culture medium or synthesis process. The TEM and EDS analyses confirm the successful biosynthesis of AgNPs with well- defined crystallinity and elemental composition. The observed particle aggregation suggests the necessity for further surface functionalization or pH modification to enhance colloidal stability. The SAED results validate the crystalline structure of silver, aligning with previously reported data on biologically synthesized AgNPs. Statistical analysis was performed using the Chi-Square method to assess the goodness of fit between observed and expected energy values. A Chi-Square test was conducted at a 5% significance level to evaluate the fit of the EDAX data. The alternative hypothesis (HA) proposes a significant difference between the observed and expected energy values, whereas the null hypothesis (H0) asserts that there is no significant difference. To facilitate the Chi-Square analysis, an overlay of the EDS peaks was plotted, allowing for a detailed comparison of the peak intensities corresponding to various elements as shown in Fig. 9. This overlay was essential for evaluating the distribution and accuracy of the detected elements. Table 1 shows the observed and expected energies for each element obtained from overlay of EDX spectrum. The Chi-Square analysis was then applied to determine the statistical significance of the elemental composition, enabling an assessment of the consistency and reliability of the results relative to the expected distribution as shown in Fig. 10 . Table 1 Comparison of detected and expected energy values (in eV) for elements identified in the EDS spectrum of silver nanoparticles Element Detected Energy (eV) Expected Energy (eV) C (Kα) 287.5 277 Si (Kα) 1732.5 1740 Cl (Kα) 2627.5 2620.8 Ag (Lα) 2977.5 2984.3 Ag (Lβ) 3142.5 3150.9 Cu (Kα) 8032.5 8047.8 As shown in Fig. 10 , the results indicate that the critical value is significantly greater than the calculated Chi-Square value. Consequently, we fail to reject the null hypothesis (H0) and conclude that there is no significant difference between the observed and expected energy values. 3.6 FOURIER TRANSFORM INFRARED SPECTROSCOPY ANALYSIS The FTIR analysis was performed to identify the functional groups present on the surface of the microbial silver nanoparticles (AgNPs), which play a essential part in nanoparticle stabilization and capping. The recorded absorption peaks indicate the presence of various biomolecules that might be responsible for the reduction and stabilization of AgNPs. Fourier transform infrared (FTIR) spectroscopy is used to identify the functional groups and metabolites responsible for the reduction and stabilization of silver nanoparticles (AgNPs). It also detects residual chemical moieties on their surface by analyzing how infrared radiation interacts with molecular bonds, producing characteristic stretching and bending vibrations within the 4000–400 cm⁻¹ range. [ 40 – 45 ]. The FTIR spectra reveal the presence of various functional groups, indicating the presence of proteins, polysaccharides, and flavonoids in the biogenic synthesis of AgNPs. The broad absorption peak at 3367 cm⁻¹ corresponds to hydroxyl (O–H) and amine (N–H) stretching, the peak at 1605 cm⁻¹ corresponds to amide (C = O) stretching. Additionally, the absorption peak at 1063 cm⁻¹ corresponds to C = O stretching. The C–H bending mode (836 cm⁻¹) further suggests the presence of aromatic compounds as shown in Table 2 . Table 2. Representative FTIR spectral data indicating the wavenumber positions, peak intensities, and associated functional groups contributing to the reduction and stabilization of silver nanoparticles synthesized using Bacillus subtilis 3.7 PHOTOCATALATYIC DEGRADATION ANALYSIS OF CONGO RED The initial absorbance (A₀) of Congo red at 497 nm was measured as 0.373. The photocatalytic degradation of Congo red under UV and sunlight conditions was analyzed over 2 hours. Under UV light alone, the control flask showed a minor decrease in absorbance from 0.373 to 0.361, corresponding to a 3.22% reduction, indicating that UV exposure alone had a limited effect on dye degradation. In contrast, the test flask treated with 1 mg of AgNPs under UV light exhibited a degradation of 37.8% as shown in Fig. 11 , demonstrating the enhanced photocatalytic activity of AgNPs. Under sunlight conditions, the control flask showed a 6.97% reduction, while the test flask with AgNPs achieved 67.02% degradation as shown in Fig. 12 , confirming the superior efficiency of AgNPs in harnessing solar energy for dye breakdown. The efficiency of photocatalytic activity was plotted as percentage degradation versus time, demonstrating the progressive breakdown of Congo red over the experimental period. These results highlight the role of AgNPs in accelerating dye degradation under both UV and sunlight exposure. Upon exposure to UV light, a noticeable difference in color change was observed between the control and test flasks after 2 hours as shown in Fig. 13 (a). In the control flask, which contained the dye solution without silver nanoparticles, the color remained largely unchanged throughout the duration of the experiment, indicating minimal degradation which was indicated by the shift of initial absorbance from 0.373 to 0.361. In contrast, the test flask containing silver nanoparticles exhibited a gradual fading of color over different time interval and it was collected in Eppendorf tubes which is shown in Fig. 13 (b). For sunlight, there is a drastic change in color between the control and test flasks after 2 hours as shown in Fig. 14 (a). In the control flask, the overall degradation % was 6.97% indicating minimal degradation. In contrast, the test flask had overall degradation of 67.02% with gradual degradation over the time interval which is shown in Fig. 14 (b). Figure 15 shows that there is a progressive increase in degradation efficiency under both conditions; however, a significantly higher degradation rate was observed under sunlight exposure compared to UV light. At 15 minutes, the degradation efficiency was approximately 10% under UV light and 20% under sunlight. As the exposure time increased, the degradation efficiency followed an upward trend, with sunlight reaching nearly 70% at 120 minutes, while UV degradation efficiency remained lower at around 30%. The enhanced photocatalytic activity under sunlight can be attributed to the broader spectral range of solar radiation, which provides higher energy input compared to UV light alone. The interaction of AgNPs with natural sunlight likely generates a greater number of Reactive Oxygen Species (ROS), which play a crucial role in dye degradation. Additionally, the efficient electron transfer mechanism and Surface Plasmon Resonance (SPR) effect of AgNPs under visible light further contribute to their superior photocatalytic performance. 4. CONCLUSION This study demonstrated the eco-friendly microbial synthesis of silver nanoparticles (AgNPs) using Bacillus subtilis AVK-21 isolated from water samples, showcasing the dual advantage of utilizing naturally occurring microbes and minimizing chemical inputs. The AgNPs were comprehensively characterized using multiple analytical techniques—UV-Vis spectroscopy, XRD, FTIR, DLS, zeta potential analysis, SEM, TEM, SAED, and EDX—which confirmed their crystalline nature, surface chemistry, nanoscale size, and stability. Photocatalytic degradation of Congo red dye was assessed under both sunlight and UV light, with AgNPs achieving dye removal efficiencies of 67.01% and 37.80%, respectively. In contrast, the control sample showed only 7.10% degradation. This significant difference underscores the catalytic potential of biosynthesized AgNPs, with enhanced performance because of the surface plasmon resonance effect, efficient electron–hole pair separation, and generation of reactive oxygen species (ROS) under light exposure. Importantly, the study highlights not only the synthesis and functionality of AgNPs but also their practical applicability in environmental remediation. These nanoparticles could potentially be integrated into hybrid treatment systems, such as membrane filters or fixed-bed reactors, for continuous dye degradation in industrial effluents. Moreover, the microbial route offers a scalable and cost-effective alternative for nanoparticle production, especially if waste substrates or effluent-rich microbial communities are employed. Nevertheless, challenges remain in terms of scalability, long-term stability, nanoparticle recovery, and comprehensive toxicological profiling. Addressing these aspects through future interdisciplinary research will be key to transitioning biosynthesized AgNPs from laboratory scale to real-world applications. Overall, this study adds to the expanding field of green nanotechnology and provides encouraging new information about long-term approaches to wastewater treatment and industrial dye pollution. Abbreviations Ag Silver AgNO3 Silver nitrate AgNPs Silver Nanoparticles BLAST Basic Local Alignment Search Tool CH-AgNPs Chemically Synthesized Silver Nanoparticles DLS Dynamic Light Scattering EDS Energy Dispersive X-ray Spectroscopy FCC Face-Centered Cubic FTIR Fourier-Transform Infrared Spectroscopy H2O Water HCl Hydrochloric Acid ITS Internal Transcribed Spacer mV Millivolt NaOH Sodium Hydroxide NPs Nanoparticles PDI Polydispersity Index ppm Parts per million ROS Reactive Oxygen Species RPM Revolution per minute rRNA Ribosomal Ribonucleic Acid SAED Selected Area Electron Diffraction SEM Scanning Electron Microscopy SPR Surface Plasmon Resonance TEM-EDX Transmission Electron Microscopy- Energy Dispersive X-Ray UV-Vis Ultraviolet-Visible Spectroscopy XRD X-ray diffraction Statements and Declarations Data Availability: The 16S rRNA sequence of Bacillus subtilis AVK-21 has been deposited in NCBI under the accession number PV663177 . Conflict of Interest The authors declare that they have no conflict of interest. Funding The authors did not receive support from any organization for the submitted work. No funding was received to assist with the preparation of this manuscript. No funding was received for conducting this study. No funds, grants, or other support was received. Competing interests The authors have no relevant financial or non-financial interests to disclose. Ethics Approval Not applicable. Consent to Participate Not applicable. Consent to Publish Not applicable. Authors Contribution Statement Aditya S and Vimal A performed the experimental work, including microbial isolation, nanoparticle synthesis, characterization, data analysis, and manuscript drafting. Kavipraba A provided supervision, conceptual guidance, and critical revisions as the project guide. 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Angewandte Chemie International Edition, 44(15), 2154–2157. https://doi.org/10.1002/anie.200462208 Zhang, X.-F., Liu, Z.-G., Shen, W., & Gurunathan, S. (2016). Silver Nanoparticles: Synthesis, Characterization, Properties, Applications, and Therapeutic Approaches. International Journal of Molecular Sciences, 17(9), 1534. https://doi.org/10.3390/ijms17091534 Additional Declarations No competing interests reported. Supplementary Files JournalSupplementaryInformation.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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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13:18:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":98277,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUV-vis absorption spectra showing SPR peak at 430 nm confirming the synthesis of AgNPs\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7728875/v1/457972dab870899da2978352.png"},{"id":95835606,"identity":"3541ae49-5cc1-4de2-88ac-5010935b5fa7","added_by":"auto","created_at":"2025-11-13 13:18:16","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":230593,"visible":true,"origin":"","legend":"\u003cp\u003eDynamic Light Scattering (DLS) analysis showing the particle size distribution of silver nanoparticles. The graph illustrates diameter (nm), undersize percentage (%), and frequency (%)\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7728875/v1/db01355d62271b88657a0efe.png"},{"id":95835607,"identity":"f9cdf005-84c7-42f6-9c51-911a47fab816","added_by":"auto","created_at":"2025-11-13 13:18:16","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":338838,"visible":true,"origin":"","legend":"\u003cp\u003eZeta potential profile of silver nanoparticles The plot illustrates the distribution of particle intensity (%) across zeta potential values, revealing a peak at 0 mV, which shows the agglomerative nature of the nanoparticles\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7728875/v1/70236aef6def3651f3be2b47.png"},{"id":96240022,"identity":"dd8f91c2-84e6-40a3-a253-bb7278dfe296","added_by":"auto","created_at":"2025-11-19 07:08:12","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":445519,"visible":true,"origin":"","legend":"\u003cp\u003eTransmission Electron Microscopy (TEM) image of Silver Nanoparticles synthesized from \u003cem\u003eBacillus subtilis AVK-21 \u003c/em\u003eat different scale bars: a) 50 nm \u0026nbsp;b) 100 nm \u0026nbsp;c) Selected Area Electron Diffraction (SAED) pattern of silver nanoparticles.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7728875/v1/9bc127dd5068c414501e3cd8.png"},{"id":96239705,"identity":"496c6938-fd4c-4f4a-8815-c1940f718b9f","added_by":"auto","created_at":"2025-11-19 07:07:24","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":40158,"visible":true,"origin":"","legend":"\u003cp\u003eEnergy Dispersive X-ray Spectroscopy (EDS) spectrum showing the strong signal at ~3keV confirming the elemental composition of silver (Ag-Lβ) in nanoparticles along with other peaks corresponding to elements possibly originating from microbial residues or stabilizing agents.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-7728875/v1/4cd8435a0ced07bd1f10f633.png"},{"id":96239866,"identity":"55e2dc59-b1f4-45d2-aafc-173f5b9bc1b0","added_by":"auto","created_at":"2025-11-19 07:07:51","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":89931,"visible":true,"origin":"","legend":"\u003cp\u003eOverlay of EDS peaks for silver nanoparticles, enabling Chi-square analysis to assess the goodness of fit between observed and expected energy values.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-7728875/v1/a8cf845394f36ee90845e066.png"},{"id":95835608,"identity":"58b6d191-2612-40ac-8950-c010f3bae100","added_by":"auto","created_at":"2025-11-13 13:18:16","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":92994,"visible":true,"origin":"","legend":"\u003cp\u003eStatistical evaluation of elemental data using Chi-square analysis to assess the goodness of fit between observed and expected energy values from the EDS spectrum of silver nanoparticles\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-7728875/v1/6d6814373caea4d9f659797c.png"},{"id":95835612,"identity":"705edff3-ab19-477f-838d-a4f235c0d938","added_by":"auto","created_at":"2025-11-13 13:18:16","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":31770,"visible":true,"origin":"","legend":"\u003cp\u003eTime-dependent degradation of dye in UV light in the presence of biosynthesized silver nanoparticles. Absorbance measured at 497 nm at different time intervals and the decline in absorbance values over time reflects progressive dye degradation, with corresponding percentages calculated relative to the initial absorbance (0.373)\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-7728875/v1/c2e6e690bee49d50a5b1016e.png"},{"id":95835611,"identity":"b55c5bb8-7b54-407e-862c-9c18673b8dab","added_by":"auto","created_at":"2025-11-13 13:18:16","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":69337,"visible":true,"origin":"","legend":"\u003cp\u003eTime-dependent degradation of dye in sunlight in the presence of biosynthesized silver nanoparticles. Absorbance measured at 497 nm at different time intervals and the decline in absorbance values over time reflects progressive dye degradation, with corresponding percentages calculated relative to the initial absorbance (0.373)\u003c/p\u003e","description":"","filename":"12.png","url":"https://assets-eu.researchsquare.com/files/rs-7728875/v1/6a844e6f237ee45687e327e0.png"},{"id":95835620,"identity":"5448a1ea-c840-4491-9a33-3615c02f009c","added_by":"auto","created_at":"2025-11-13 13:18:16","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":328637,"visible":true,"origin":"","legend":"\u003cp\u003ePhotocatalytic degradation of dye by silver nanoparticles under UV light \u0026nbsp;\u003cstrong\u003ea)\u003c/strong\u003e Control and Test flask after 2 hours under UV light \u003cstrong\u003eb)\u003c/strong\u003e Eppendorf tubes containing test samples collected at various time intervals (from left to right) starting with the control sample and ending with the 120 min sample\u003c/p\u003e","description":"","filename":"13.png","url":"https://assets-eu.researchsquare.com/files/rs-7728875/v1/87120b81c239e85e23665a4a.png"},{"id":96240140,"identity":"9a3b7454-79be-4f3c-a22c-50fdf7ec1195","added_by":"auto","created_at":"2025-11-19 07:08:29","extension":"png","order_by":14,"title":"Figure 14","display":"","copyAsset":false,"role":"figure","size":384919,"visible":true,"origin":"","legend":"\u003cp\u003ePhotocatalytic degradation of dye by silver nanoparticles under sunlight \u003cstrong\u003ea)\u003c/strong\u003e Control and Test flask after 2 hours under sunlight \u003cstrong\u003eb)\u003c/strong\u003e Eppendorf tubes containing test samples collected at various time intervals (from left to right) starting with the control sample and ending with the 120 min sample\u003c/p\u003e","description":"","filename":"14.png","url":"https://assets-eu.researchsquare.com/files/rs-7728875/v1/9cdfe42fe846d1ccb3ca6412.png"},{"id":96239583,"identity":"3750c379-08a7-4976-ae26-bf5b34d8222e","added_by":"auto","created_at":"2025-11-19 07:07:01","extension":"png","order_by":15,"title":"Figure 15","display":"","copyAsset":false,"role":"figure","size":29859,"visible":true,"origin":"","legend":"\u003cp\u003eGraph depicting the degradation of Congo Red dye under UV and Sunlight\u003c/p\u003e","description":"","filename":"15.png","url":"https://assets-eu.researchsquare.com/files/rs-7728875/v1/5a2472e52184414afdfe22a6.png"},{"id":99797356,"identity":"51a4d412-54cc-4ff0-98b9-cb280c298e77","added_by":"auto","created_at":"2026-01-08 13:45:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4480381,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7728875/v1/450cf1e4-f827-41b9-82d5-e3d407b3c942.pdf"},{"id":95835662,"identity":"8164174d-8a1b-4351-9fba-2ecec72a7313","added_by":"auto","created_at":"2025-11-13 13:18:17","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":9764300,"visible":true,"origin":"","legend":"","description":"","filename":"JournalSupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-7728875/v1/6d1eb467815359b52400bf7d.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eBiosynthesis and Characterization of Silver Nanoparticles Using Microbes and Remediation of Dyes\u003c/p\u003e","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eWater pollution from dyes and oils is a major environmental concern, especially in industrial areas where untreated waste is often discharged into water bodies. The textile industry alone accounts for 20% of global wastewater pollution, releasing large quantities of toxic, non-biodegradable dyes into the environment. Azo dyes, in particular, are widely used for their vibrant colors and stability, but they pose serious ecological and health risks. Some common examples include Tartrazine (E102), a yellow dye used in beverages and confectionery, and Sunset Yellow (E110), an orange dye used in snacks and baked goods [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. It is estimated that 2,00,000 tons of dyestuff are discharged into the environment annually, with concentrations in textile effluent reaching up to 500 parts per million (ppm) These dyes are resistant to biodegradation and can break down under anaerobic conditions into aromatic amines, many of which are carcinogenic and toxic [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Their presence in water bodies reduces light penetration, impairs photosynthesis, and disrupts aquatic ecosystems. For humans, exposure to azo dye byproducts has been linked to cancer, genetic mutations, and endocrine disruption, while their accumulation in aquatic organisms further magnifies risks through the food chain [\u003cspan additionalcitationids=\"CR4 CR5 CR6 CR7\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Conventional methods for dye removal, such as adsorption and photocatalysis, can be expensive and energy-intensive [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Consequently, researchers are exploring alternative solutions, and silver nanoparticles have emerged as a promising candidate for dye degradation due to their unique properties.\u003c/p\u003e\u003cp\u003eThe production of silver nanoparticles has drawn a lot of interest because of their distinct physicochemical characteristics and wide range of uses in industries like environmental remediation, catalysis, and antibacterial agents [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. While traditional physical and chemical methods for nanoparticle synthesis can be effective, they often involve the use of toxic reagents and harsh conditions, which can be environmentally harmful [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The development of sustainable and environmentally acceptable methods for the production of silver nanoparticles has gained attention in recent years, with an emphasis on using biological resources like microorganisms [\u003cspan additionalcitationids=\"CR13 CR14 CR15 CR16\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The biosynthesis of silver nanoparticles using microorganisms has emerged as a promising alternative to conventional methods, as it offers several advantages, including the use of renewable and non-toxic precursors, the ability to control the size and shape of the nanoparticles, and the potential for scaling up the production process. Microorganisms, such as bacteria, fungi, and algae, have the ability to reduce silver ions (Ag+) to metallic silver, leading to the formation of silver nanoparticles with diverse morphologies [\u003cspan additionalcitationids=\"CR19 CR20 CR21 CR22 CR23 CR24\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The green synthesis of AgNPs involves three main stages: activation, growth, and termination [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. During activation, the reducing agents present in the biological entity initiate the reduction of silver ions. This is followed by the growth phase, where the reduced silver atoms cluster together to form nanoparticles. Finally, the termination phase involves the stabilization of the nanoparticles to prevent further growth and aggregation. This flexibility in preparation and the ability to avoid the utilization of toxic chemicals make green synthesis a preferred method for nanoparticle production [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Green synthesized AgNPs degrade dyes primarily through photocatalytic degradation. This process involves the absorption of light energy by the AgNPs, which generates electron-hole pairs. These excited electrons and holes then react with the dye molecules, leading to their breakdown into less harmful substances [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The efficiency of this process depends on factors such as the size and shape of the AgNPs, the type of dye, and the intensity of light irradiation.\u003c/p\u003e\u003cp\u003eResearch has indicated that green synthesized AgNPs can efficiently degrade a variety of dyes. The effectiveness of AgNPs in dye degradation can be size-dependent. For example, a study comparing AgNPs synthesized using different concentrations of silver nitrate found that smaller nanoparticles exhibited higher catalytic activity for the degradation of methyl orange [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. This is because smaller nanoparticles have a larger surface area to volume ratio, which provides more active sites for the catalytic reaction.\u003c/p\u003e\u003cp\u003eIn addition to the size of the nanoparticles, the type of dye and the green synthesis method employed also influence the degradation efficiency. For instance, AgNPs synthesized using Citrus reticulata Blanco peel extract completely degraded malachite green dye after 120 hours of incubation in sunlight [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Another study found that AgNPs synthesized using leaf extract of Solena amplexicaulis effectively degraded methylene blue dye under solar irradiation [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. This study also showed that the green synthesized NPs reduced methylene blue and 4-nitrophenol significantly [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eFurthermore, AgNPs have shown promising results in degrading a mixture of dyes, which is a more realistic scenario mimicking the composition of industrial wastewater. A study by Deepak Gola concluded that chemically synthesized silver nanoparticles (CH-AgNPs) combined with NaBH4 could potentially enhance the dye degradation of orange and blue dyes individually and as a mixture [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Another recent study where researchers explored a novel approach to creating silver nanoparticles by utilizing the natural capabilities of bacteria. They employed three distinct bacterial species \u0026ndash; Micrococcus luteus (MBC23), Klebsiella pneumoniae (MBC34) and Enterobacter aerogenes (MBX6) \u0026ndash; to synthesize three different types of silver nanoparticles, designated as AgNPs-K, AgNPs-M, and AgNPs-E, respectively. Of the three types, AgNPs-M were the most effective at removing the dye methyl orange from wastewater, eliminating nearly 20% of it within two hours [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. A study in Iran investigates a specific type of microalgae used to create silver nanoparticles (AgNPs) and their ability to break down a harmful dye called Methyl Red found in wastewater [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Scientists used the byproducts of a fungus called Fusarium oxysporum to create small silver nanoparticles (SNPs). These nanoparticles were very effective at breaking down a dye called crystal violet when exposed to light. In fact, they could remove almost 99% of the dye from a solution within 4 hours when used at a specific concentration [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e"},{"header":"2. MATERIALS AND METHODOLOGY","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 MATERIALS\u003c/h2\u003e\u003cp\u003eSilver Nitrate (AgNO3) powder was purchased from. distilled water was used throughout for solution preparation and washing steps. Sodium hydroxide (NaOH) and hydrochloric acid (HCl) were used for pH adjustments. Nutrient agar and Nutrient broth were used for the cultivation of bacterial isolates. Centrifuges, an incubator shaker, a magnetic stirrer, a UV lamp chamber, and standard sterile glassware such as conical flasks, beakers, and micropipettes were used as needed.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 WATER SAMPLE COLLECTION\u003c/h2\u003e\u003cp\u003eWater samples were aseptically collected from four isolated, low-human-activity locations in Madurai\u0026mdash;Mudalaikulam 1, Mudalaikulam 2, Sholavandhan, and the Vaigai River using 200 mL sterilized plastic containers at a depth of 20\u0026ndash;30 cm with proper labelling and stored at 4\u0026deg;C to preserve microbial viability.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 ISOLATION OF MICROBES USING SPREAD PLATE METHOD\u003c/h2\u003e\u003cp\u003eThe water sample bottle was vigorously shaken to homogenize the microbial population. Serial dilutions were performed by transferring 1 mL of the sample into 9 mL of sterile distilled water. Successive dilutions (10\u0026thinsp;\u0026minus;\u0026thinsp;1 to 10\u0026thinsp;\u0026minus;\u0026thinsp;4) were prepared to achieve a suitable concentration for plating.\u003c/p\u003e\u003cp\u003eUsing a sterile micropipette, 0.1 mL of the desired dilution was transferred onto the surface of the solidified agar in a petri dish. The sample was spread evenly over the agar surface using a sterile glass spreader or L-shaped rod. After the incubation period of 24\u0026ndash;48 hours at 37\u0026deg;C, microbial growth was observed on the nutrient agar plates inoculated with water samples. Distinct microbial colonies were observed on the four sample plates, exhibiting variations in size, shape, color, and texture (Refer Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e in Online Resource).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 STREAKING FOR PURE CULTURES\u003c/h2\u003e\u003cp\u003eAfter incubation, spread plates were examined for isolated colonies based on distinct characteristics such as size, color, shape, texture, and edge definition, and six colonies were selected from four sample plates. The inoculating loop was sterilized by flame, allowed to cool, and then used to gently pick a colony, which was streaked onto a fresh nutrient agar plate in a zigzag pattern across four quadrants to reduce the microbial load and isolate individual colonies, ensuring minimal overlap. The streaked plates were inverted and incubated at 37\u0026deg;C for 24\u0026ndash;48 hours, resulting in well-separated colonies with no contamination as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 PREPARATION OF BROTH CULTURE\u003c/h2\u003e\u003cp\u003eSterile nutrient broth was prepared by dissolving the required amount of powdered medium in distilled water (e.g., 13 g/L), and autoclaved at 121\u0026deg;C for 15 minutes to ensure sterility. A single isolated colony from the streak plate was selected using a sterile loop and inoculated into a sterile 100 mL conical flask containing 100 mL of nutrient broth under aseptic conditions for each of the six samples. The inoculated flasks were incubated at 37\u0026deg;C in a shaker incubator at 120\u0026ndash;150 rpm for 18\u0026ndash;24 hours to promote uniform growth. After incubation, visible turbidity was observed in all six flasks, indicating microbial growth (Refer Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e in Online Resource).. No contamination was detected.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 STAINING\u003c/h2\u003e\u003cp\u003eTwo types of staining were performed: Simple Staining and Gram Staining. Simple staining, using Methylene Blue that identifies bacterial cells by enhancing contrast, making cell features like shape, size, and arrangement easier to observe under a microscope. Gram Staining categorizes bacteria into Gram-positive (purple) and Gram-negative (pink/red) based on their cell wall structure.\u003c/p\u003e\u003cp\u003eFor simple staining, a smear was prepared on a clean slide with a drop of sterile water and a loopful of broth culture, which was air-dried and heat-fixed. Methylene blue was applied for 1\u0026ndash;2 minutes, excess stain was washed off, and the slide was air-dried. Under a microscope at 100x magnification, all six samples were examined and found to be bacteria. Streaks 1, 2, and 5 exhibited cocci-shaped cells; Streaks 3 and 4 showed rod-shaped bacteria; and Streak 6 displayed round cells resembling cocci. (Refer Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e in Online Resource).\u003c/p\u003e\u003cp\u003eFor Gram staining, a smear was prepared in the same manner, followed by crystal violet for 1 minute, Gram's iodine for 1 minute, ethanol for 15\u0026ndash;30 seconds, and Safranin for 1 minute. The slide was examined under a microscope at 100x magnification with immersion oil, and the results were recorded: Gram-positive bacteria appeared purple, and Gram-negative bacteria appeared pink. All six samples were examined, and among the six isolates, three were observed to be Gram-positive (appearing purple) i.e. Streak 3, Streak 5 and Streak 6, while three were Gram-negative (appearing pink) i.e. Streak 1, Streak 2 and Streak 4.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 MICROBIAL IDENTIFICATION BY SEQUENCING\u003c/h2\u003e\u003cp\u003eMicrobial sequencing is a precise method used to identify and characterize microorganisms by analyzing their genetic material. It helps distinguish species by targeting specific genes, such as the 16S ribosomal RNA (16S rRNA) gene for bacteria and archaea or the Internal Transcribed Spacer (ITS) region for fungi (Janda \u0026amp; Abbott, 2007). The sequencing method used for the identification was Sanger sequencing.\u003c/p\u003e\u003cp\u003eOut of 6 isolates, a single isolate was considered for the sequencing and further synthesis of Silver nanoparticles. Streak 4 was selected for sequencing as it was having morphologically distinctive characteristics with mucoidal appearance. 16S rRNA was used for the identification of the bacteria. For this purpose, the pure culture of the isolate was submitted to a commercial sequencing service provider, NGS Hub, National Facility for Coastal and Marine Research, Sathyabama Institute of Science and Technology, Chennai, India. The entire sequencing workflow, including genomic DNA extraction, PCR amplification, and sequencing, was carried out by the consultancy.\u003c/p\u003e\u003cp\u003eGenomic DNA was extracted from the bacterial sample using a standard phenol-chloroform method or a commercially available DNA extraction kit, as per the consultancy\u0026rsquo;s protocol. The 16S rRNA gene was then amplified using universal bacterial primers (such as 27F and 1492R), targeting conserved regions of the gene. The amplified products were purified and subjected to Sanger sequencing using cycle sequencing chemistry with fluorescently labeled dideoxynucleotides.\u003c/p\u003e\u003cp\u003eThe resulting chromatogram files were analyzed for base calling and sequence quality. The final consensus sequence was compared against the NCBI GenBank database using the BLAST (Basic Local Alignment Search Tool) algorithm to identify the isolate based on sequence homology with known bacterial strains. The bacterial species showing the highest similarity percentage and alignment score was considered as the identified strain. The identified strain was subsequently used for the biosynthesis of silver nanoparticles.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8 BIOSYNTHESIS AND CHARACTERIZATION OF SILVER NANOPARTICLES\u003c/h2\u003e\u003cp\u003eSilver nanoparticles were synthesized using microbial processes, wherein biologically active molecules facilitated the reduction of silver ions (Ag⁺) to elemental silver (Ag⁰). Enzymes like nitrate reductase, along with proteins, polysaccharides, and other microbial biomolecules, acted as reducing and stabilizing agents, while functional groups present on these biomolecules provided surface capping to prevent aggregation and maintain colloidal stability. For the synthesis, a selected bacteria was cultured in nutrient broth under optimal growth conditions i.e. 37\u0026deg;C, after which the culture was centrifuged to separate the biomass from the supernatant. The obtained supernatant was then reacted with a 1 mM silver nitrate solution in equal proportion and incubated at room temperature to facilitate nanoparticle formation as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003e (OnlineResource). The synthesized nanoparticles were purified by multiple centrifugation and washing to eliminate unreacted ions and biomolecular residues. Finally, the purified silver nanoparticles were resuspended in distilled water as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003e (OnlineResource) and stored at 4\u0026deg;C in dark containers to preserve their stability and prevent light-induced degradation.\u003c/p\u003e\u003cp\u003eThe synthesized silver nanoparticles were characterized using various analytical techniques to confirm their formation, stability, and physicochemical properties. Surface plasmon resonance (SPR) peak detection was done using UV-visible spectroscopy, a key indicator of nanoparticle synthesis, by measuring absorbance in the 200\u0026ndash;800 nm range. The average hydrodynamic diameter, polydispersity index (PDI), and colloidal stability of the nanoparticles were measured using dynamic light scattering (DLS), while Zeta Potential analysis provided insight into surface charge and dispersion stability in suspension. Fourier Transform Infrared (FTIR) spectroscopy identified functional groups responsible for the reduction and capping of nanoparticles, indicating the involvement of microbial biomolecules. Finally, Transmission Electron Microscopy (TEM) provided high-resolution images to assess the shape, size, and dispersion of the nanoparticles, confirming their nanoscale morphology.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9 PHOTOCATALYTIC REMEDIATION OF DYE (CONGO RED) USING SILVER NANOPARTICLES\u003c/h2\u003e\u003cp\u003eThe photocatalytic potential of biosynthesized silver nanoparticles was assessed through the degradation of Congo Red dye, a commonly used azo dye. To begin, a stock dye solution was prepared by dissolving 100 mg of Congo Red in 1 liter of deionized water, yielding a concentration of 100 mg/L. From this stock, a working solution of 25 mg/L was obtained by diluting 25 mL of the stock with 75 mL of deionized water. For the degradation experiment, 1 mg of silver nanoparticles (or an optimized concentration) was added to 100 mL of the working dye solution in a clean beaker. The mixture was stirred at 200\u0026ndash;300 rpm using a magnetic stirrer to ensure homogeneous dispersion of the nanoparticles. The solution's pH was brought within the range of 3 and 7, depending on the requirement, as pH significantly influences photocatalytic efficiency.\u003c/p\u003e\u003cp\u003eThe dye-nanoparticle mixture was then exposed to two different light conditions to assess photocatalytic activity: natural sunlight during peak daylight hours and ultraviolet (UV) light at wavelengths such as 254 nm or 365 nm, provided by a UV chamber. These conditions allowed for the evaluation of both solar- and UV-induced degradation pathways. At specific time intervals\u0026mdash;0, 10, 20, 30, 60, and 120 minutes\u0026mdash;2 mL aliquots of the reaction mixture were withdrawn to monitor the progress of dye degradation. Each sample was centrifuged to remove residual nanoparticles, and the supernatant was analyzed.\u003c/p\u003e\u003cp\u003eThe degradation of Congo Red was tracked using UV-Visible spectrophotometry by measuring absorbance at 497 nm, which corresponds to the dye\u0026rsquo;s maximum absorbance wavelength (λmax). The initial absorbance (A₀) and absorbance at each time interval (Aₜ) were recorded and compared to determine the percentage of dye degraded over time. The following formula was used to determine the degradation efficiency:\u003c/p\u003e\u003cp\u003eDegradation Efficiency (%) =\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{\\left(A0-At\\right)}{A0}\\times\\:\\:100\\)\u003c/span\u003e\u003c/span\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\u003cp\u003eThese values were computed for both sunlight and UV exposure conditions, and the results were plotted to provide a comparative understanding of photocatalytic efficiency under different lighting scenarios. This approach highlights the effectiveness of silver nanoparticles in environmental remediation through dye degradation.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. RESULTS AND DISCUSSION","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1 MICROBIAL SEQUENCING RESULT\u003c/h2\u003e\n \u003cp\u003eMicrobial sequencing analysis was conducted to identify the bacterial species present in the sample. 16S rRNA contig sequence of a bacterial isolate has been shown in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e. This is a continuous stretch of nucleotide bases (A, T, G, C) obtained from 16S rRNA gene sequencing. The 16S rRNA gene sequencing revealed a high similarity with \u003cem\u003eBacillus subtilis\u003c/em\u003e, confirming its presence. Figure \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e shows that the sequence alignment using BLAST showed a 97.85% identity match with known \u003cem\u003eB. subtilis strain\u003c/em\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2 UV-VISIBLE SPECTROMETER ANALYSIS\u003c/h2\u003e\n \u003cp\u003eUV-vis spectroscopy is the main and critical instrument for understanding silver nanoparticle formation during the early synthesis stage. It is quite characteristic based on the form, size, and distribution of Ag nanoparticles due to surface plasmon resonance [\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e]. The UV-Vis absorption spectra of the synthesized nanoparticles showed a characteristic Surface Plasmon Resonance (SPR) peak at 430 nm, confirming the successful formation of silver nanoparticles as shown in Fig. 4. The observed peak position is consistent with previously reported values for silver nanoparticles, further validating their synthesis. Any slight variations in peak intensity or position could be attributed to factors such as particle size, shape, or stabilizing agents used during synthesis.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3 DYNAMIC LIGHT SCATTERING (DLS) ANALYSIS\u003c/h2\u003e\n \u003cp\u003eIt typically analyzes the hydrodynamic diameter of nanoparticles impacted by capping agents, as well as the presence of an electrical double layer deposited on the surface of nanoparticles, and works best with monodisperse nanoparticles [\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e\n \u003cp\u003eThe DLS analysis revealed an average hydrodynamic diameter of 269.45 nm with a PDI value of nearly 0, indicating monodisperse nature. The synthesized microbial silver nanoparticles (AgNPs) exhibit a monodisperse size distribution with an average size of 271.3 nm and minimal variation as shown in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e. The absence of a reported PDI suggests extremely low polydispersity, meaning the nanoparticles are highly uniform. Overall, the sample appears to be well- synthesized, with a narrow and uniform size distribution, making it suitable for applications requiring consistent nanoparticle size.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003e3.4 ZETA POTENTIAL ANALYSIS\u003c/h2\u003e\n \u003cp\u003eZeta potential analysis is a crucial technique for evaluating the surface charge and colloidal stability of nanoparticles in suspension. It provides insight into the repulsive or attractive forces between particles, influencing their dispersion, aggregation, and overall stability in a given medium [\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e]. A zeta potential of 0.0 mV suggests that the nanoparticles lack a net surface charge, meaning they do not experience significant electrostatic repulsion as shown in Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e. The electrophoretic mobility being zero further supports that the nanoparticles are not moving in response to the applied electric field, confirming the absence of a measurable charge. Nanoparticles with a zeta potential between \u0026minus;\u0026thinsp;10 and +\u0026thinsp;10 mV are considered approximately neutral (Clogston and Patri, 2011).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003e3.5 TEM ANALYSIS\u003c/h2\u003e\n \u003cp\u003eTEM is one of the most sophisticated analytical measurement instruments for visualizing and differentiating nanoparticle size and form. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e, the nanoparticles predominantly exhibit a spherical shape, with some degree of aggregation. The Selected Area Electron Diffraction (SAED) pattern of the AgNPs (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e (c)) exhibits distinct concentric diffraction rings, corresponding to the face-centered cubic (FCC) structure of silver. This confirms the crystalline nature of the nanoparticles.\u003c/p\u003e\n \u003cp\u003eEnergy Dispersive X-ray Spectroscopy (EDS) was used to determine the elemental composition of the synthesized AgNPs. The EDS spectrum displays strong silver (Ag) peaks as shown in Fig.\u0026nbsp;8, confirming the successful synthesis of AgNPs. In addition to silver, minor peaks corresponding to C, O, Fe, and Cl were observed. The presence of oxygen and carbon can be attributed to biomolecular capping agents from the microbial extract, which enhance nanoparticle stability. Iron and chlorine impurities may arise from the culture medium or synthesis process.\u003c/p\u003e\n \u003cp\u003eThe TEM and EDS analyses confirm the successful biosynthesis of AgNPs with well- defined crystallinity and elemental composition. The observed particle aggregation suggests the necessity for further surface functionalization or pH modification to enhance colloidal stability. The SAED results validate the crystalline structure of silver, aligning with previously reported data on biologically synthesized AgNPs.\u003c/p\u003e\n \u003cp\u003eStatistical analysis was performed using the Chi-Square method to assess the goodness of fit between observed and expected energy values. A Chi-Square test was conducted at a 5% significance level to evaluate the fit of the EDAX data. The alternative hypothesis (HA) proposes a significant difference between the observed and expected energy values, whereas the null hypothesis (H0) asserts that there is no significant difference. To facilitate the Chi-Square analysis, an overlay of the EDS peaks was plotted, allowing for a detailed comparison of the peak intensities corresponding to various elements as shown in Fig. 9. This overlay was essential for evaluating the distribution and accuracy of the detected elements. Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e shows the observed and expected energies for each element obtained from overlay of EDX spectrum. The Chi-Square analysis was then applied to determine the statistical significance of the elemental composition, enabling an assessment of the consistency and reliability of the results relative to the expected distribution as shown in Fig. \u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003e.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eComparison of detected and expected energy values (in eV) for elements identified in the EDS spectrum of silver nanoparticles\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eElement\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDetected Energy (eV)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eExpected Energy (eV)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC (K\u0026alpha;)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e287.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e277\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSi (K\u0026alpha;)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1732.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1740\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCl (K\u0026alpha;)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2627.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2620.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAg (L\u0026alpha;)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2977.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2984.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAg (L\u0026beta;)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3142.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3150.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCu (K\u0026alpha;)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8032.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8047.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eAs shown in Fig. \u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003e, the results indicate that the critical value is significantly greater than the calculated Chi-Square value. Consequently, we fail to reject the null hypothesis (H0) and conclude that there is no significant difference between the observed and expected energy values.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003e3.6 FOURIER TRANSFORM INFRARED SPECTROSCOPY ANALYSIS\u003c/h2\u003e\n \u003cp\u003eThe FTIR analysis was performed to identify the functional groups present on the surface of the microbial silver nanoparticles (AgNPs), which play a essential part in nanoparticle stabilization and capping. The recorded absorption peaks indicate the presence of various biomolecules that might be responsible for the reduction and stabilization of AgNPs. Fourier transform infrared (FTIR) spectroscopy is used to identify the functional groups and metabolites responsible for the reduction and stabilization of silver nanoparticles (AgNPs). It also detects residual chemical moieties on their surface by analyzing how infrared radiation interacts with molecular bonds, producing characteristic stretching and bending vibrations within the 4000\u0026ndash;400 cm⁻\u0026sup1; range. [\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e\n \u003cp\u003eThe FTIR spectra reveal the presence of various functional groups, indicating the presence of proteins, polysaccharides, and flavonoids in the biogenic synthesis of AgNPs. The broad absorption peak at 3367 cm⁻\u0026sup1; corresponds to hydroxyl (O\u0026ndash;H) and amine (N\u0026ndash;H) stretching, the peak at 1605 cm⁻\u0026sup1; corresponds to amide (C\u0026thinsp;=\u0026thinsp;O) stretching. Additionally, the absorption peak at 1063 cm⁻\u0026sup1; corresponds to C\u0026thinsp;=\u0026thinsp;O stretching. The C\u0026ndash;H bending mode (836 cm⁻\u0026sup1;) further suggests the presence of aromatic compounds as shown in Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u003cstrong\u003eTable 2.\u0026nbsp;\u003c/strong\u003eRepresentative FTIR spectral data indicating the wavenumber positions, peak intensities, and associated functional groups contributing to the reduction and stabilization of silver nanoparticles synthesized using \u003cem\u003eBacillus subtilis\u003c/em\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\u003c/table\u003e\n \u003cp\u003e\u003cem\u003e\u003cimg 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\"\u003e\u003c/em\u003e\u003cbr\u003e\u003c/p\u003e\n \u003c/div\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n \u003ch2\u003e3.7 PHOTOCATALATYIC DEGRADATION ANALYSIS OF CONGO RED\u003c/h2\u003e\n \u003cp\u003eThe initial absorbance (A₀) of Congo red at 497 nm was measured as 0.373. The photocatalytic degradation of Congo red under UV and sunlight conditions was analyzed over 2 hours. Under UV light alone, the control flask showed a minor decrease in absorbance from 0.373 to 0.361, corresponding to a 3.22% reduction, indicating that UV exposure alone had a limited effect on dye degradation. In contrast, the test flask treated with 1 mg of AgNPs under UV light exhibited a degradation of 37.8% as shown in Fig. \u003cspan class=\"InternalRef\"\u003e11\u003c/span\u003e, demonstrating the enhanced photocatalytic activity of AgNPs. Under sunlight conditions, the control flask showed a 6.97% reduction, while the test flask with AgNPs achieved 67.02% degradation as shown in Fig. \u003cspan class=\"InternalRef\"\u003e12\u003c/span\u003e, confirming the superior efficiency of AgNPs in harnessing solar energy for dye breakdown. The efficiency of photocatalytic activity was plotted as percentage degradation versus time, demonstrating the progressive breakdown of Congo red over the experimental period. These results highlight the role of AgNPs in accelerating dye degradation under both UV and sunlight exposure.\u003c/p\u003e\n \u003cp\u003eUpon exposure to UV light, a noticeable difference in color change was observed between the control and test flasks after 2 hours as shown in Fig. \u003cspan class=\"InternalRef\"\u003e13\u003c/span\u003e (a). In the control flask, which contained the dye solution without silver nanoparticles, the color remained largely unchanged throughout the duration of the experiment, indicating minimal degradation which was indicated by the shift of initial absorbance from 0.373 to 0.361. In contrast, the test flask containing silver nanoparticles exhibited a gradual fading of color over different time interval and it was collected in Eppendorf tubes which is shown in Fig. \u003cspan class=\"InternalRef\"\u003e13\u003c/span\u003e (b).\u003c/p\u003e\n \u003cp\u003eFor sunlight, there is a drastic change in color between the control and test flasks after 2 hours as shown in Fig. \u003cspan class=\"InternalRef\"\u003e14\u003c/span\u003e (a). In the control flask, the overall degradation % was 6.97% indicating minimal degradation. In contrast, the test flask had overall degradation of 67.02% with gradual degradation over the time interval which is shown in Fig. \u003cspan class=\"InternalRef\"\u003e14\u003c/span\u003e (b).\u003c/p\u003e\n \u003cp\u003eFigure 15 shows that there is a progressive increase in degradation efficiency under both conditions; however, a significantly higher degradation rate was observed under sunlight exposure compared to UV light. At 15 minutes, the degradation efficiency was approximately 10% under UV light and 20% under sunlight. As the exposure time increased, the degradation efficiency followed an upward trend, with sunlight reaching nearly 70% at 120 minutes, while UV degradation efficiency remained lower at around 30%.\u003c/p\u003e\n \u003cp\u003eThe enhanced photocatalytic activity under sunlight can be attributed to the broader spectral range of solar radiation, which provides higher energy input compared to UV light alone. The interaction of AgNPs with natural sunlight likely generates a greater number of Reactive Oxygen Species (ROS), which play a crucial role in dye degradation. Additionally, the efficient electron transfer mechanism and Surface Plasmon Resonance (SPR) effect of AgNPs under visible light further contribute to their superior photocatalytic performance.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. CONCLUSION","content":"\u003cp\u003eThis study demonstrated the eco-friendly microbial synthesis of silver nanoparticles (AgNPs) using Bacillus subtilis AVK-21 isolated from water samples, showcasing the dual advantage of utilizing naturally occurring microbes and minimizing chemical inputs. The AgNPs were comprehensively characterized using multiple analytical techniques\u0026mdash;UV-Vis spectroscopy, XRD, FTIR, DLS, zeta potential analysis, SEM, TEM, SAED, and EDX\u0026mdash;which confirmed their crystalline nature, surface chemistry, nanoscale size, and stability. Photocatalytic degradation of Congo red dye was assessed under both sunlight and UV light, with AgNPs achieving dye removal efficiencies of 67.01% and 37.80%, respectively. In contrast, the control sample showed only 7.10% degradation. This significant difference underscores the catalytic potential of biosynthesized AgNPs, with enhanced performance because of the surface plasmon resonance effect, efficient electron\u0026ndash;hole pair separation, and generation of reactive oxygen species (ROS) under light exposure. Importantly, the study highlights not only the synthesis and functionality of AgNPs but also their practical applicability in environmental remediation. These nanoparticles could potentially be integrated into hybrid treatment systems, such as membrane filters or fixed-bed reactors, for continuous dye degradation in industrial effluents. Moreover, the microbial route offers a scalable and cost-effective alternative for nanoparticle production, especially if waste substrates or effluent-rich microbial communities are employed.\u003c/p\u003e\u003cp\u003eNevertheless, challenges remain in terms of scalability, long-term stability, nanoparticle recovery, and comprehensive toxicological profiling. Addressing these aspects through future interdisciplinary research will be key to transitioning biosynthesized AgNPs from laboratory scale to real-world applications. Overall, this study adds to the expanding field of green nanotechnology and provides encouraging new information about long-term approaches to wastewater treatment and industrial dye pollution.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"633\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eAg\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSilver\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eAgNO3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSilver nitrate\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eAgNPs\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSilver Nanoparticles\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eBLAST\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eBasic Local Alignment Search Tool\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eCH-AgNPs\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eChemically Synthesized Silver Nanoparticles\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eDLS\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eDynamic Light Scattering\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eEDS\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eEnergy Dispersive X-ray Spectroscopy\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eFCC\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eFace-Centered Cubic\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eFTIR\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eFourier-Transform Infrared Spectroscopy\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eH2O\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eWater\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eHCl\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eHydrochloric Acid\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eITS\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eInternal Transcribed Spacer\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003emV\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eMillivolt\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eNaOH\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSodium Hydroxide\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eNPs\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eNanoparticles\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003ePDI\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003ePolydispersity Index\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eppm\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eParts per million\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eROS\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eReactive Oxygen Species\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eRPM\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eRevolution per minute\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003erRNA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eRibosomal Ribonucleic Acid\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eSAED\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSelected Area Electron Diffraction\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eSEM\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eScanning Electron Microscopy\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eSPR\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSurface Plasmon Resonance\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eTEM-EDX\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eTransmission Electron Microscopy- Energy Dispersive X-Ray\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eUV-Vis\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eUltraviolet-Visible Spectroscopy\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eXRD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eX-ray diffraction\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Statements and Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 16S rRNA sequence of Bacillus subtilis AVK-21 has been deposited in NCBI under the accession number \u003cstrong\u003ePV663177\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors did not receive support from any organization for the submitted work.\u003c/p\u003e\n\u003cp\u003eNo funding was received to assist with the preparation of this manuscript.\u003c/p\u003e\n\u003cp\u003eNo funding was received for conducting this study.\u003c/p\u003e\n\u003cp\u003eNo funds, grants, or other support was received.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Publish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors Contribution Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAditya S and Vimal A performed the experimental work, including microbial isolation, nanoparticle synthesis, characterization, data analysis, and manuscript drafting. Kavipraba A provided supervision, conceptual guidance, and critical revisions as the project guide. All authors (Aditya S, Vimal A, Kavipraba A) contributed to the interpretation of results, approved the final version of the manuscript, and agree to be accountable for all aspects of the work in ensuring the accuracy and integrity of the study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eS. Zafar, D. A. Bukhari, and A. Rehman, \u0026ldquo;Azo dyes degradation by microorganisms \u0026ndash; An efficient and sustainable approach,\u0026rdquo; Saudi Journal of Biological Sciences, vol. 29, no. 12, p. 103437, Sep. 2022, doi: 10.1016/j.sjbs.2022.103437.\u003c/li\u003e\n\u003cli\u003eK.-T. Chung, \u0026ldquo;Azo dyes and human health: A review,\u0026rdquo; Journal of Environmental Science and Health Part C, vol. 34, no. 4, pp. 233\u0026ndash;261, Sep. 2016, doi: 10.1080/10590501.2016.1236602.\u003c/li\u003e\n\u003cli\u003eP. Barciela, A. 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Joicy et al., \u0026ldquo;Photocatalytic degradation of textile dye using green synthesized nanoparticles,\u0026rdquo; Letters in Applied NanoBioScience, vol. 12, no. 4, p. 102, Sep. 2022, doi: 10.33263/lianbs124.102.\u003c/li\u003e\n\u003cli\u003eA. A. Lourthuraj, M. M. Selvam, M. S. Hussain, A.-W. A. Abdel-Warith, E. M. I. Younis, and N. A. Al-Asgah, \u0026ldquo;Dye degradation, antimicrobial and larvicidal activity of silver nanoparticles biosynthesized from Cleistanthus collinus,\u0026rdquo; Saudi Journal of Biological Sciences, vol. 27, no. 7, pp. 1753\u0026ndash;1759, May 2020, doi: 10.1016/j.sjbs.2020.05.008.\u003c/li\u003e\n\u003cli\u003eD. Gola et al., \u0026ldquo;Silver nanoparticles for enhanced dye degradation,\u0026rdquo; Current Research in Green and Sustainable Chemistry, vol. 4, p. 100132, Jan. 2021, doi: 10.1016/j.crgsc.2021.100132.\u003c/li\u003e\n\u003cli\u003eB. 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International Journal of Molecular Sciences, 17(9), 1534. https://doi.org/10.3390/ijms17091534\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"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":"Silver nanoparticles, Biosynthesis, Photocatalytic Degradation, Congo Red, Surface Plasmon Resonance","lastPublishedDoi":"10.21203/rs.3.rs-7728875/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7728875/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study explores the eco-friendly biosynthesis of silver nanoparticles (AgNPs) using \u003cem\u003eBacillus subtilis AVK-21\u003c/em\u003e isolated from water samples, focusing on their characterization and application in photocatalytic degradation of Congo Red dye. Using biomolecules as stabilising and reducing agents, silver nitrate was reduced by microbes to create the AgNPs. The crystalline face-centered cubic structure, spherical shape, and nanoscale size (average of 271.3 nm) of the nanoparticles were validated by thorough characterisation utilising UV-Vis spectroscopy, XRD, FTIR, DLS, zeta potential, SEM, TEM, and EDS. Due to surface plasmon resonance and the reactive oxygen species production, the AgNPs demonstrated strong photocatalytic activity, breaking down 67.01% of Congo Red in the presence of sunlight and 37.80% in the presence of ultraviolet light. The work emphasises the need for more research on scalability and toxicological effects while highlighting the promise of microbially synthesised AgNPs for sustainable environmental remediation, namely in treating dye-polluted effluents.\u003c/p\u003e","manuscriptTitle":"Biosynthesis and Characterization of Silver Nanoparticles Using Microbes and Remediation of Dyes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-13 13:18:11","doi":"10.21203/rs.3.rs-7728875/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":"558e56c9-4201-4a4b-ade1-b60baff0e5d7","owner":[],"postedDate":"November 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-07T11:09:21+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-13 13:18:11","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7728875","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7728875","identity":"rs-7728875","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}