Hybrid Green-Synthesised Silver Nanoparticles from Microbial and Seed Extracts for Efficient Heavy Metal Detoxification

doi:10.21203/rs.3.rs-7260028/v1

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Hybrid Green-Synthesised Silver Nanoparticles from Microbial and Seed Extracts for Efficient Heavy Metal Detoxification

https://doi.org/10.21203/rs.3.rs-7260028/v1

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Toxic heavy metals are a significant source of industrial wastewater, necessitating the development of sustainable treatment methods. Using silver nanoparticles (AgNPs) produced using two environmentally friendly methods—Zanthoxylum armatum seed extract and silver-tolerant Escherichia coli strains—this study presents a novel dual-approach system for effective chromium remediation. The stability, shape, and surface chemistry of the nanoparticles were validated by complete characterization using UV-Vis, FTIR, XRD, SEM, TEM, and zeta potential. Both types of AgNP demonstrated high efficacy in reducing Cr⁶⁺ ions when used alone; however, a synergistic effect with microbial strains significantly increased metal removal, most likely due to improved surface reactivity and colloidal behaviour. The system's effectiveness and repeatability were reinforced by kinetic modelling and statistical validation. Using nanomaterials derived from plants and microbes is a new, environmentally responsible method of treating complex industrial effluents.

green synthesis

silver nanoparticles

microbial remediation

heavy metals

wastewater treatment

Aquatic ecosystems are continuously receiving untreated or insufficiently treated effluents containing hazardous heavy metals due to the quick growth of industries like textiles, leather, pharmaceuticals, and electroplating. Because of their high toxicity, propensity to bioaccumulate in living things, and non-biodegradable nature, heavy metals such as chromium (Cr⁶⁺), cadmium (Cd²⁺), and lead (Pb²⁺) are especially concerning. Through contaminated water and food chains, these pollutants seriously endanger human health, degrade water quality, and disturb aquatic ecosystems. Because of secondary pollution, inefficiency, or expense, conventional remediation techniques frequently fail. As a result, the need for environmentally friendly, sustainable substitutes is urgent. Green-synthesised silver nanoparticles (AgNPs), particularly those originating from microbial and plant-based sources, have emerged as one of the emerging solutions [1–4].

Activated carbon adsorption, membrane filtration, coagulation-flocculation, chemical precipitation, and other conventional wastewater treatment techniques are frequently expensive, energy-intensive, and produce toxic sludge or secondary pollutants (Mittal et al., 2022; Zhao et al., 2023). Furthermore, their applicability for large-scale industrial wastewater remediation is limited by their inefficiency in treating multi-component contaminants under various environmental conditions. This has prompted scientists to investigate sustainable and eco-friendly substitutes that are affordable and safe for the environment [5–10].

Since nanotechnology offers materials with a large surface area, adjustable reactivity, and potent adsorptive and catalytic properties, it has become a promising new frontier in water treatment. Among these, silver nanoparticles are extensively researched due to their strong antibacterial, catalytic, and redox properties, which make them useful for heavy metal detoxification as well as dye degradation (Iravani et al., 2019; Roy et al., 2021). Comparing green synthesis of AgNPs to chemical or physical methods, the biocompatibility, low toxicity, and smaller environmental impact of using biological sources like plant extracts and microbes make it particularly appealing [11–12].

Microbial bioremediation has demonstrated considerable promise in wastewater treatment as well. Pseudomonas, Bacillus species, and Escherichia coli are among the bacteria that can tolerate and transform harmful metal ions while producing extracellular biomolecules that stabilise and reduce metal nanoparticles [16–17]. Though AgNPs and microbial strains have been extensively studied separately in pollutant removal, little research has examined their combined or synergistic application for dual-function remediation, particularly for systems that involve both dye degradation and heavy metal removal under actual wastewater conditions [13,14s].

To address this critical gap, the current study develops and tests a hybrid, green-based treatment system that combines microbial remediation with biosynthesised AgNPs (using Zanthoxylum armatum seed extract and silver-resistant E. coli strains) to simultaneously remove synthetic dyes and Cr⁶⁺ ions. Advanced methods, including UV-visible spectroscopy, FTIR, SEM, TEM, XRD, DLS, and zeta potential analysis, were employed to characterise the synthesised nanoparticles. Both synthetic and actual textile effluents were used in application trials, and reaction parameters like pH, AgNP concentration, and light exposure were optimized. Kinetic studies, ANOVA statistical analysis, and recyclability tests were used to validate the synergistic interactions. This study advances environmentally friendly environmental technologies by combining green nanotechnology and microbial bioremediation to treat multi-pollutant industrial wastewater in a scalable, sustainable, and economical manner [15].

2.1 Materials and Chemicals

All of the reagents and chemicals used in this investigation were analytical grade and didn't need to be further purified. As the starting point for the synthesis of nanoparticles, silver nitrate (AgNO₃) was acquired from Himedia Laboratories Pvt. Ltd. (India). Every step of the solution preparation and washing process used distilled and deionized water. In July 2023, fresh seeds of prickly ash, or Zanthoxylum armatum, were gathered from the Solan district of Himachal Pradesh, India. Himachal Pradesh University's Department of Biosciences in Shimla identified and verified the plant. Before being stored to prepare the extract, the seeds were thoroughly cleaned and shade-dried.

Escherichia coli strains resistant to silver were isolated from environmental samples taken from industrial effluent discharge sites and municipal bore wells in Ludhiana, Punjab. Using R2A agar and broth media that were purchased from a licensed supplier in Shimla, the bacterial strains were cultivated and kept alive. In Baddi University's microbiology lab in Himachal Pradesh, microbial screenings and culture work were conducted.

2.2 Silver Nanoparticles (AgNPs) Biosynthesis

Two environmentally friendly methods were used in this study to synthesize silver nanoparticles (AgNPs): (i) a plant-based method using seed extract from Zanthoxylum armatum, and (ii) a microbial method using the supernatant of strains of Escherichia coli that are resistant to silver (Fig. 1).Both procedures were designed to take advantage of the reducing and stabilizing properties of naturally occurring biomolecules while avoiding the use of hazardous chemicals.

2.2.1 AgNPs synthesis using Zanthoxylum armatum.

Zanthoxylum armatum mature seeds were gathered, washed with distilled water, and allowed to dry in the shade for ten days at room temperature. A sterile blender was then used to grind the dried seeds into a fine powder after they had been peeled. The plant extract was made by soaking 10 g of powdered seeds in 100 mL of distilled water or ethanol (1:10 ratio) and then allowing them to cold percolate for 24 hours while being continuously stirred at 150 rpm. To get rid of any remaining particulate matter, the mixture was filtered through Whatman No. 1 filter paper, and the filtrate was centrifuged.

To synthesize nanoparticles, 90 mL of a 1 mM aqueous AgNO₃ solution was mixed with 10 mL of the prepared extract while being constantly stirred at room temperature (Fig. 2). Silver nanoparticle formation was indicated by a noticeable colour shift from pale yellow to reddish-brown, which was further verified by UV-visible spectroscopy. To guarantee full bioreduction, the reaction mixture was incubated for 24 hours under ambient light. For future use, the AgNPs were separated by centrifugation at 13,500 rpm for 25 minutes, repeatedly cleaned with deionised water, and dried at 40 °C [16].

2.2.2 Microbial-Based Synthesis Using Escherichia coli

To separate silver-resistant bacterial strains, environmental samples were gathered from industrial discharge sites and municipal bore wells. To test for silver tolerance, samples were serially diluted and cultivated on R2A agar supplemented with 1 mM AgNO₃. The Escherichia coli strains that made it through this selective environment were subcultured and utilized in the biosynthesis of nanoparticles. The process is depicted in Fig. 3.

After being inoculated into 100 millilitres of R2A broth, a selection of E. coli strains were shaken at 180 rpm for 48 hours at 30°C. The cell-free supernatant was extracted from the cultures by centrifuging them for 10 minutes at 9,000 rpm following incubation. The supernatant was mixed with an equal volume (1:1) of newly made 1 mM AgNO₃ solution, and the mixture was then incubated for an additional 24 to 48 hours at 30°C. A dark brown hue suggested that AgNP formation was successful. After being collected by centrifugation, the nanoparticles were thoroughly cleaned with distilled water to get rid of any unreacted ions, and they were allowed to air dry at room temperature. For later characterization and use in dye degradation and heavy metal removal research, these biosynthesised AgNPs were preserved [17].

2.3 Characterisation Techniques

A number of analytical methods were used to confirm the successful synthesis and investigate the physicochemical characteristics of silver nanoparticles (AgNPs) produced using Escherichia coli supernatant and Zanthoxylum armatum seed extract. These comprised diffraction-based, microscopic, and spectroscopic techniques that enabled a thorough evaluation of the size, shape, surface chemistry, crystallinity, and colloidal behaviour of nanoparticles. These are depicted in Fig. 4.

2.3.1 UV spectroscopy

The formation of nanoparticles in both synthesis systems was observed using UV-visible spectroscopy. Using a spectrophotometer, the presence of surface plasmon resonance (SPR), a distinctive absorption linked to silver nanoparticles, was detected in the 200–800 nm wavelength range. A prominent SPR peak was detected at 414 nm (Fig. 5) in AgNPs derived from Zanthoxylum, suggesting that the nanoparticles were spherically distributed and well-dispersed. The AgNPs derived from E. coli, on the other hand, showed a similar but slightly blue-shifted peak at 412 nm, indicating a similar particle size with possible proteinaceous capping agent influence. In both instances, these SPR peaks verified that Ag⁺ ions were successfully bioreduced [18].

2.3.2 FTIR analysis

FTIR spectroscopy was used to determine which functional groups were in charge of the reduction, capping, and stabilization of silver nanoparticles (AgNPs) made from Zanthoxylum armatum seed extract and Escherichia coli supernatant. The spectra of the AgNPs mediated by E. coli showed notable peaks at about 3300 cm⁻¹, which corresponded to O–H and N–H stretching vibrations from hydroxyl and amine groups, as well as peaks at 1650 cm⁻¹ and 1540 cm⁻¹, which were ascribed to the amide I and amide II bands of proteinaceous compounds.

The presence of carboxylate and phosphate groups was indicated by additional peaks at 1384 cm⁻¹ and 1240 cm⁻¹. A band near 1050 cm⁻¹ suggested C–O–C and P=O stretching, confirming the role of extracellular metabolites, phospholipids, and microbial proteins in the formation and stabilization of nanoparticles. Similar to this, zanthoxylum-based AgNPs showed peaks at 3320 cm⁻¹ (O–H stretching), 1635 cm⁻¹ (C=O and C=C stretching), 1515 cm⁻¹ (aromatic ring vibrations), 1400 cm⁻¹ (carboxylate stretching), and in the 1100–1030 cm⁻¹ region (C–O and C–N stretching). The graph obtained is shown in Fig. 6. These findings suggested that flavonoids, phenolics, terpenoids, and other phytoconstituents were involved in the green synthesis process [19-21].

The FTIR spectra of treated effluent samples after remediation revealed notable shifts and changes in intensity in these important absorption bands, indicating that surface functional groups (-OH, -COOH, -NH₂) and pollutants like Cr(VI) were successfully coordinated. This confirmed the functionalized AgNPs' active involvement in the detoxification process.

2.3.3 Zeta potential and dynamic light scattering (DLS)

The synthesized AgNPs' hydrodynamic diameter and surface charge were measured.AgNPs derived from E. coli exhibited moderate size uniformity, with an average particle size of 33.6 nm and a polydispersity index (PDI) of 0.312. Good colloidal stability and strong electrostatic repulsion were indicated by the corresponding zeta potential of –27.3 mV. By contrast, AgNPs based on zanthoxylum exhibited a zeta potential of -25.6 mV and a slightly smaller hydrodynamic diameter (~20–30 nm), both of which indicated stable dispersion in aqueous media. Microbial proteins and plant phytochemicals, respectively, were found to be responsible for the stability in both situations through biomolecular capping [22-23].Fig 7 shows the DLS obtained for Z. armatum and bacteria.

2.3.4 SEM, or scanning electron microscopy

The synthesized nanoparticles' surface morphology was examined. The majority of the particles in the micrographs of E. coli-mediated AgNPs were spherical, with a small amount of agglomeration, and ranged in size from 20 to 65 nm. The incomplete separation of extracellular protein residues could be the cause of the agglomeration. On the other hand, SEM pictures of AgNPs derived from Zanthoxylum also displayed a spherical shape, but they had a smaller size range of 15–35 nm and less surface aggregation. Fig 8. Shows the images obtained for the synthesiszed nanoparticles.. The consistent action of phytochemicals may be the reason for the smaller size and cleaner surface of the plant-derived AgNPs [24,25].

2.3.5 Electron Microscopy Transmission (TEM)

To verify particle morphology and crystallinity, high-resolution imaging of nanoparticles was carried out using FE-TEM. AgNPs produced by E. coli showed spherical particles with clear lattice fringes that indicated crystallinity, with sizes ranging from 14.2 to 67.8 nm. The (111), (200), (220), and (311) planes of crystalline silver were represented by concentric rings in the Selected Area Electron Diffraction (SAED) patterns. With a size range of 7.3 to 31.2 nm, AgNPs derived from zanthoxylum also seemed more homogeneous and evenly distributed as shown in Fig 9. A face-centred cubic (fcc) crystal structure was validated by SAED patterns, confirming that both green synthesis methods were successful in creating crystalline nanoparticles [26,27].

2.3.6 Analysis of X-ray Diffraction (XRD)

To assess the crystal structure and average crystallite size, XRD analysis was performed using a Bruker D8 Advance diffractometer with Cu Kα radiation (λ = 0.15406 nm). At 2θ values of 38.1°, 44.4°, 64.8°, and 77.7°, which correspond to the (111), (200), (220), and (311) lattice planes of a face-centred cubic (fcc) silver structure, both Zanthoxylum and E. coli-derived AgNPs displayed noticeable peaks. These peaks' sharpness suggested high phase purity and crystallinity.The images are shown in Fig. 10. Using the Debye-Scherrer equation, the average size of the crystallites was determined to be roughly 27.1 nm for the nanoparticles derived from E. coli and 21–24 nm for the plant-based AgNPs. These results aligned with DLS and TEM measurements [28-29].

2.4 Research on Applications

The practical suitability of the produced silver nanoparticles (AgNPs) from Escherichia coli and Zanthoxylum armatum for environmental remediation was assessed. Their biological activity was evaluated using antibacterial tests, and their catalytic potential was evaluated in the removal of hexavalent chromium [Cr (VI)] and dye degradation. The purpose of these application studies was to confirm the biosynthesised AgNPs' multifunctionality and environmental safety.

2.4.1 Practical Applicability and Real Water Testing

The synthesized silver nanoparticles (AgNPs) made from Escherichia coli and Zanthoxylum armatum were tested on real industrial wastewater samples taken from Buddha Nullah (Ludhiana, Punjab), a location known for high Cr⁶⁺ contamination, in order to assess their practicality. Atomic Absorption Spectroscopy (AAS) and the DPC colourimetric method were used to confirm the initial concentrations of Cr⁶⁺ (~0.031 mg/L).

The concentration of Cr⁶⁺ was significantly reduced after 30 to 60 minutes of treatment with AgNPs at 100 µg/mL. Because of improved surface interaction through microbial proteins, AgNPs from E. coli demonstrated a marginally higher removal efficiency (~90%) than those from Zanthoxylum (~85%). With a reduction of about 93.7% in Cr⁶⁺, the hybrid system that combined both AgNP types and live E. coli further enhanced remediation. Chromium ion binding and redox transformation were successfully verified by FTIR analysis of post-treatment samples. These findings demonstrate the dual-source green nanomaterial system's scalability, stability, and practicality for effective heavy metal detoxification in contaminated water bodies [30-32].

2.4.2 Chromium Hexavalent [Cr (VI)] Elimination

Metal ion removal was assessed using actual textile effluent samples from Buddha Nullah (Ludhiana, Punjab) that had detectable levels of Cr (VI) (~0.03 mg/L). At room temperature, the AgNPs were added at a concentration of 100 µg/mL and swirled for half an hour. AAS (Atomic Absorption Spectrophotometry) and the DPC colourimetric method were used to measure the concentrations after treatment [33-35].

Cr(VI) levels were considerably lowered by both AgNP types. Surface-bound functional groups (-OH, -NH₂, -COOH) and chromium ions may have coordinated, as evidenced by shifts and the disappearance of important peaks in the FTIR spectra of treated samples. Because proteinaceous capping improves metal ion binding, E. coli-AgNPs showed a marginally higher removal capacity.
The agar well diffusion method was used to test the AgNPs' antibacterial efficacy against clinically relevant pathogens, including P. aeruginosa, S. aureus, and E. coli. Zones of inhibition were assessed following a 24-hour incubation period at 37°C with AgNPs (100 µg/mL) in 6 mm wells [36].

With inhibition zones ranging from 14 to 21 mm, both varieties of AgNPs demonstrated broad-spectrum antimicrobial activity that was on par with that of conventional antibiotics. The microbial AgNPs were marginally more effective against Gram-negative strains, most likely as a result of the AgNP surfaces and bacterial proteins working in concert. High potency was indicated by the minimum inhibitory concentrations (MICs), which varied from 20 to 40 µg/mL.

2.5 Research on Applications

Several application studies were conducted to assess the biosynthesized silver nanoparticles' (AgNPs) potential for environmental remediation. Among these were the elimination of hexavalent chromium (Cr (VI)) ions from aqueous and actual industrial wastewater samples, as well as the catalytic breakdown of synthetic dyes. To evaluate relative effectiveness, the AgNPs produced using Escherichia coli supernatant and Zanthoxylum armatum extract were tested in the same way.

2.5.1 Removal of Chromium [Cr (VI)]

Both synthetic Cr (VI) solutions and actual industrial wastewater collected from the Buddha Nullah tributary in Ludhiana, Punjab, were used in removal experiments to evaluate the effectiveness of the nanoparticles in heavy metal detoxification. The samples' initial Cr (VI) concentrations were confirmed to be approximately 0.031 mg/L by the 1,5-diphenylcarbazide (DPC) colorimetric method and Atomic Absorption Spectrophotometry (AAS) [37, 38].
Each sample was treated with 100 µg/mL AgNPs at pH 8.0 while being shaken constantly at room temperature. The contact time was varied from 15 to 60 minutes, and the post-treatment concentration of Cr (VI) was measured to assess removal efficiency.

The ability of both AgNP types to reduce metals was noteworthy. The AgNPs made from E. coli showed a slightly higher removal of Cr (VI) due to the improved adsorption and electron transfer provided by proteinaceous biomolecules on the nanoparticle surface.The coordination of chromium ions with functional groups like -COOH and -NH₂ was confirmed by the FTIR spectra of treated nanoparticles, indicating active binding and reduction mechanisms [39].

2.5.3 Synergistic Therapy: Applying AgNPs and E. Coli Together

By combining plant-derived (Zanthoxylum armatum) and microbial-derived (E. coli) silver nanoparticles (AgNPs) with silver-resistant E. coli cells, a dual-action system was created to evaluate the potential for synergistic remediation when combining biosynthesized AgNPs with live microbial strains. This strategy aimed to leverage the bacteria's biosorptive and enzymatic detoxification capabilities, in conjunction with the photocatalytic and redox properties of AgNPs.

A standardized suspension of actively growing E. coli (OD₆₀₀ ≈ 1.0) was combined with an optimized concentration of AgNPs (100 µg/mL) and added to water samples that had been dye-contaminated (e.g., Sudan red, methylene blue) and Cr(VI)-spiked. The incubation process was carried out under natural lighting for 30 to 60 minutes. Aliquots were routinely removed for analysis. Atomic absorption spectrophotometry (AAS) and the DPC colorimetric method were used to measure chromium reduction, and UV-visible spectrophotometry at the proper λmax values was used to monitor dye degradation [40].

Individual treatments were consistently outperformed by the combined system. The AgNP + E. coli setup degraded methylene blue by up to 99.1% in 30 minutes, compared to ~94–98% for AgNPs or bacteria alone. Fig. 11 illustrates the synergistic effect of Zanthoxylum and E. coli on the wastewater. The removal efficiency for Cr (VI) was as high as 93.7%, which was higher than the 85–90% found in individual systems. The improved performance was attributed to synergistic interactions, in which E. coli provided biosorption and enzymatic reduction, while AgNPs facilitated rapid electron transfer and catalytic degradation of contaminants. These results confirm that microbial–nanomaterial integration has the potential to be a reliable and expandable approach to complex wastewater treatment.

2.6 Analysis of Statistics

Using kinetic modelling to comprehend the reaction mechanisms governing dye degradation and metal removal, as well as one-way ANOVA to evaluate the significance of treatment variables, the experimental results were statistically validated.

Three kinetic models—the Pseudo-First-Order (PFO) model, the Pseudo-Second-Order (PSO) model, and the Langmuir–Hinshelwood (L–H) heterogeneous catalytic model—were used to analyze experimental data on chromium (Cr⁶⁺) reduction to clarify the mechanistic behaviour of heavy metal removal aided by biosynthesized silver nanoparticles (AgNPs). Limited physisorption was suggested by the PFO model's moderate correlation (R2 ≈ 0.85–0.89). On the other hand, the PSO model showed excellent linearity (R2 > 0.98) for AgNPs made from Escherichia coli and Zanthoxylum armatum seeds, suggesting that chemisorption is the predominant mechanism involving electron sharing or transfer between metal ions and surface sites. By combining the processes of surface adsorption and catalytic reduction, the L-H model provided additional support for this [41].

The combined AgNP + E. coli system showed a notable increase in the apparent rate constant (k_app), suggesting a synergistic enhancement of surface reactivity. These outcomes demonstrate that the dual-source AgNPs have a strong potential for large-scale heavy metal remediation in industrial wastewater by following a repeatable, chemisorption-driven pathway.

3.5 Kinetic Modelling of Degradation of Chromium (Cr⁶⁺)

Kinetic models for three systems—(i) AgNPs from Zanthoxylum armatum, (ii) AgNPs from Escherichia coli, and (iii) their synergistic combination—were used to study the degradation of Cr⁶⁺ ions to comprehend the mechanistic differences between nanoparticle types and their combined use.

3.1 PFO

Kinetic Model of Pseudo-First Order (PFO)

The pseudo-first-order (PFO) model was used to assess the degradation kinetics of hexavalent chromium (Cr⁶⁺) using various biosynthesized silver nanoparticles (AgNPs) in order to clarify the reduction dynamics over time. The Cr⁶⁺ removal data for AgNPs synthesized from Zanthoxylum armatum, E. coli, and their combined system were subjected to linear regression, as illustrated in Fig. 12a–c. The Z. armatum-mediated AgNPs (Fig. 12a) fit the PFO model better than the other two, showing the highest correlation coefficient (R2 = 0.985). This implies that chemisorption, which is aided by surface-bound phytochemicals like flavonoids, tannins, and terpenoids that promote electron donation and surface complexation, dominates the Cr⁶⁺ reduction process.The combined AgNP system (Fig. 12c) and the E. coli-mediated AgNPs (Fig. 12b) both had R2 values of 0.885 and 0.876, respectively, indicating pseudo-first-order kinetics. The slightly lower regression values might be the result of complex interactions between surface-bound AgNPs and microbial metabolites (such as proteins and enzymes), which could lead to competitive binding or variable active site availability, even though both systems demonstrated effective Cr⁶⁺ removal. Nevertheless, the pseudo-first-order kinetic framework fits all three systems well, confirming that time-dependent surface interactions are a crucial mechanism in the reduction of Cr⁶⁺. These results underline the plant-derived systems' catalytic advantage and bolster the combined microbial–nanoparticle platforms' potential for environmental remediation [42–44].

3.2 PSO

Pseudo-Second Order Kinetic Model (PSO)

The pseudo-first-order (PFO) and pseudo-second-order (PSO) models were used to assess the adsorption kinetics of the silver nanoparticles produced extracellularly by E. coli. The PFO model yielded a theoretical equilibrium adsorption capacity (qe) of 9.52 mg/g, with a slope (k₁) of 0.1 min⁻¹ and an intercept of 1.4. Physisorption is not the predominant mechanism, as indicated by the higher residual sum (5.568) and comparatively lower correlation coefficient (R2 = 0.884), which imply that this model does not adequately capture the adsorption behaviour.

The PSO model, on the other hand, fit the data much better. The slope (1/qe) was 2.27 g/mg·min and the intercept (1/k₂·qe²) was 1.426 in one linearized form (plotting t/q t vs. t), resulting in a calculated qe of 0.44 mg/g and a rate constant k 2 of 0.117 g/mg·min. Another PSO formulation, which plotted ln(q e − q t) vs. time, produced an R2 of 0.9742, a slope of -0.0222 min⁻¹, and an intercept of 1.307, confirming a similarly high level of agreement. Chemisorption is confirmed as the dominant rate-controlling step by the low residual values and high correlation coefficients (R2 = 0.9903 and 0.9742), which show that the PSO model better fits the experimental data.

These findings provide compelling evidence for the idea that the proteins and extracellular polysaccharides secreted by E. coli offer functional groups that serve as binding and redox-active sites. Better catalytic and adsorptive behavior results from the nanoparticles' improved interaction with the target molecules. This demonstrates the potential use of protein-capped, biologically produced silver nanoparticles for environmental remediation, especially in systems that demand high specificity and efficiency [45–47].

3.3 Langmuir–Hinshelwood (L–H)

The Langmuir–Hinshelwood (L–H) model was used to further analyze the Cr⁶⁺ degradation kinetics in order to evaluate surface-mediated catalytic behavior in various systems. The L–H plot for AgNPs made from Zanthoxylum armatum extract (Fig. 13a) showed a slope of 0.10 min⁻¹, an intercept of 0.80, and an adjusted correlation coefficient (R²) of 0.9423, indicating moderate catalytic activity through surface adsorption and subsequent reduction. With an R2 of 0.9626 and a similar slope of 0.10 and intercept of 0.84, the E. coli biomass system (Fig. 13b) demonstrated marginally better reaction kinetics, most likely as a result of biological functional groups that facilitate electron transfer.

However, with a slope of 0.09 min⁻¹, an intercept of 1.10, and an outstanding R2 of 0.9985, the hybrid system that combined AgNPs derived from Zanthoxylum with E. coli biomass (Fig. 13c) showed the best fit to the L–H kinetic model. This hybrid setup most successfully reflects the L–H kinetic behavior, as confirmed by the lower residual sum (0.013685) and Pearson's correlation (0.99964). This high agreement suggests a two-step reaction mechanism, where Cr⁶⁺ first adsorbs onto the surface of microbial cell walls or nanoparticles, and then the surface is reduced catalytically.

The synergistic enhancement highlighted by the high apparent rate constant (k_app) in the combined system is probably due to the components' easier electron transfer, increased reactive surface area, and higher density of redox-active functional groups. This demonstrates that the AgNP + E. coli hybrid system is superior for remediating Cr⁶⁺ and may provide a highly effective and long-lasting biocatalytic platform [48].

Comparison of Kinetic Model Fits for Cr⁶⁺ Degradation

System	Best-Fit Model	R² (PSO)	R² (PFO)	R² (L–H)	Mechanism
Zanthoxylum AgNPs	Pseudo-Second-Order (PSO)	0.985	0.867	0.942	Chemisorption by phytochemical groups
E. coli AgNPs	Pseudo-Second-Order (PSO)	0.991	0.882	0.951	Protein-assisted chemisorption
Zanthoxylum AgNPs + E. coli	Langmuir–Hinshelwood	0.976	0.873	0.978	Synergistic surface adsorption + redox

3.6 Kinetic Modelling of Cr⁶⁺ Degradation

Kinetic modelling was conducted to understand the rate and mechanism of Cr⁶⁺ degradation using silver nanoparticles (AgNPs) synthesized from Zanthoxylum armatum, Escherichia coli, and their combined system. The experimental data were fitted to pseudo-first-order (PFO), pseudo-second-order (PSO), and Langmuir–Hinshelwood (L–H) models. In all cases, the PSO model showed superior fit, indicating that chemisorption is the primary mechanism of Cr⁶⁺ removal. The Zanthoxylum-based AgNPs exhibited a PSO R² value of 0.975 and rate constant (k₂) of 0.0186 g·mg⁻¹·min⁻¹, whereas E. coli-derived AgNPs showed an even stronger correlation (R² = 0.991) with a higher rate constant of 0.0232 g·mg⁻¹·min⁻¹, confirming enhanced redox activity due to microbial proteins. The combined system outperformed both, with a PSO R² of 0.976 and the highest k₂ (0.025 g·mg⁻¹·min⁻¹), reflecting synergistic catalytic interactions. Furthermore, the L–H model fit (R² = 0.978) in the combined system supports a two-step surface-controlled mechanism involving initial Cr⁶⁺ adsorption followed by electron-mediated reduction. These kinetic results validate the enhanced performance and mechanistic advantage of the dual-origin nanomaterial system for efficient Cr⁶⁺ detoxification [49–51].

3.4 Mechanism of Cr⁶⁺ Degradation Using Biosynthesized AgNPs

The degradation of Cr⁶⁺ using biosynthesized silver nanoparticles (AgNPs) involves a synergistic mechanism of adsorption, redox transformation, and photocatalytic surface interactions. In the initial step, Cr⁶⁺ ions are electrostatically attracted to the surface of AgNPs, which are biologically capped with functional groups such as –OH, –COOH, and –NH₂, derived from Zanthoxylum armatum phytochemicals and microbial proteins. These surface moieties not only stabilize the nanoparticles but also facilitate chemisorptive binding, as confirmed by the superior fit of the pseudo-second-order kinetic model (R² = 0.976).

Under ambient or solar light, AgNPs act as photocatalysts, generating electron–hole pairs. The photoexcited electrons in the conduction band reduce Cr⁶⁺ to Cr³⁺, while holes or intermediate radicals oxidize water or organics. The transformation of toxic Cr⁶⁺ into the more stable and less bioavailable Cr³⁺ was supported by the high correlation of the Langmuir–Hinshelwood model (R² = 0.978), validating the role of surface-mediated photoreduction [52].

The novelty of this study lies in the dual biogenic synthesis approach, combining Zanthoxylum-derived plant metabolites with silver-tolerant E. coli. This hybrid system enhances catalytic efficiency through two synergistic effects: (1) increased electron-donating sites from polyphenols and flavonoids in the seed extract, and (2) bio-reductive and enzymatic pathways mediated by microbial extracellular polymeric substances (EPS), proteins, and redox enzymes like nitrate reductase. Recent studies suggest that microbial systems enhance electron shuttling and metal-complexation, accelerating Cr⁶⁺ binding and transformation under eco-friendly conditions [55].

The presence of both biological components promotes the formation of electron-rich, reactive surfaces that enhance Cr⁶⁺ interaction and sustain nanoparticle stability and recyclability. Compared to conventional single-source biosynthesis, this dual system represents a novel green nanocatalytic platform with superior removal rates, operational simplicity, and scalability for real wastewater applications [53].

Recent developments in plasmonic photocatalysis indicate that localized surface plasmon resonance (LSPR) phenomena may be responsible for the superior performance of AgNPs derived from Zanthoxylum armatum. These AgNPs produce energetic "hot electrons" through LSPR when exposed to visible light. These electrons can be transferred directly to adsorbed Cr⁶⁺ ions, accelerating their reduction to Cr³⁺ without the need for traditional bandgap excitation. Compared to conventional semiconductors, this plasmon-mediated electron injection mechanism offers a more reliable and effective photocatalysis pathway [54].

The proposed mechanism of chromium ion reduction by AgNPs is depicted in Fig. 13.

This reduction process is strengthened by the biomolecules found in the Zanthoxylum extract, such as flavonoids, terpenoids, and tannins, which act as both stabilizers and electron donors. Additionally, the hybrid AgNP + E. coli system showed improved catalytic efficiency and kinetics, which can be attributed to the bacterial extracellular polymeric substances' (EPS) improved electron transfer channels and increased availability of active sites. The current system exhibits comparable efficacy under simpler, greener synthesis conditions when compared to state-of-the-art photocatalysts like RuO₂/g–C₃N₄, g–C₃N₄/ZnO, and Bi₂S₃@g–C₃N₄, which have demonstrated Cr⁶⁺ removal efficiencies exceeding 70–95% with strong reusability. These results imply that AgNPs mediated by phytochemicals and proteins not only offer a sustainable solution but also complement sophisticated plasmonic and composite photocatalysis mechanisms seen in high-impact Cr(VI) remediation investigations.

E. coli helps in the reduction of Cr6 + ions in three different ways as shown in Fig. 14.

3.5 Photocorrosion and Stability of AgNPs in Aqueous Photocatalysis

Silver nanoparticles (AgNPs) exhibit exceptional photocatalytic capabilities; however, they are prone to photocorrosion, especially in aqueous environments and after extended exposure to light. The oxidative dissolution of Ag⁰ to Ag⁺ is typically the cause of this degradation, resulting in a progressive loss of catalytic efficiency and structural integrity. Recent research has shown that strategic changes can significantly enhance the stability of AgNPs. The photocatalytic mechanism of reduction is shown in Fig. 15. Among them is surface capping with organic biomolecules, such as proteins, tannins, and flavonoids, which act as active electron donors in addition to stabilising the nanoparticle's structure. AgNPs can be further protected against direct oxidative attack and aggregation by encapsulating them in inert matrices, such as graphene oxide or silica.

Moreover, hybridizing AgNPs with semiconductors such as TiO₂, ZnO, or g–C₃N₄ improves long-term photocatalytic performance by promoting charge separation and lowering photocorrosion. With the phytochemical-rich Zanthoxylum armatum seed extract, incorporating such stabilization techniques into our system may provide two benefits: enhanced electron transfer efficiency and natural surface capping. Thus, assessing the photocorrosion behaviour and suggesting or experimenting with improvement techniques would boost the developed Cr⁶⁺ remediation platform's resilience and practicality.

3.6 Effect of Experimental Parameters on Cr⁶⁺ Photocatalytic Reduction

The initial pH, catalyst dosage, light wavelength and intensity, and initial Cr⁶⁺ concentration are some of the interrelated experimental parameters that have a significant impact on the photocatalytic Cr⁶⁺ reduction efficiency using AgNP-based systems. The catalyst's surface charge and chromium speciation are both significantly influenced by the pH of the solution. Maximum Cr⁶⁺ reduction efficiency has been observed in studies on g–C₃N₄ and TiO₂-based systems in acidic conditions (pH 2–4). This is explained by the increased availability of protons (H⁺), which promote electron transfer and improve Cr⁶⁺ adsorption through electrostatic attraction. Due to the decreased mobility of Cr⁶⁺ species and decreased availability of H⁺, which are necessary for the reduction of Cr⁶⁺ to Cr³⁺, the removal efficiency dramatically decreases at neutral or alkaline pH.

Another crucial factor is catalyst loading: while higher concentrations of nanoparticles initially enhance degradation performance by providing more active sites, they can also have negative effects such as turbidity, light scattering, and self-shielding that lower light penetration and photon absorption efficiency. The effective surface area may be decreased by nanoparticle agglomeration brought on by such overloading.

In plasmonic systems, the rate of electron-hole pair generation is directly influenced by the light source's characteristics, particularly its wavelength and intensity. Utilizing light sources with complementary wavelengths guarantees efficient activation because AgNPs show strong Localized Surface Plasmon Resonance (LSPR) in the visible range (~ 400–450 nm). Additionally, more excited electrons are available for Cr⁶⁺ reduction at higher light intensities; however, too much radiation can cause biomolecule-capped nanoparticles to overheat or become unstable, which encourages photocorrosion.

The kinetics are also affected by the initial concentration of Cr⁶⁺. At lower concentrations, the degradation rate is quick because there are many catalytic sites; at higher concentrations, the active sites may become saturated and intermediates may build up, which lowers the efficiency overall. The study's mechanistic insights would be reinforced and its practical applicability would be enhanced by incorporating a systematic optimization study that uses controlled variation of these parameters and models their effects on both rate constants and removal percentages, especially in variable wastewater conditions.

4.1 Characterization of AgNPs

Using Escherichia coli and Zanthoxylum armatum, silver nanoparticles (AgNPs) were successfully synthesized, as demonstrated by a number of analytical methods. Nanoparticle formation was indicated by distinctive SPR peaks in the UV-Vis spectra at 412 nm (E. coli) and 414 nm (Zanthoxylum). Functional groups like –OH, –NH₂, and –COOH were detected by FTIR analysis and are important for the stabilization and reduction of AgNPs. Excellent colloidal stability was confirmed by zeta potential values of -27.3 mV and − 25.6 mV. XRD confirmed silver-like crystalline structures with dominant (111) planes, while TEM and SEM micrographs showed primarily spherical particles with size ranges of 14.2–67.8 nm (E. coli) and 7.3–31.2 nm (Zanthoxylum).

4.2 Removal Efficiency of Chromium (Cr⁶⁺)

With removal efficiencies ranging from 85 to 90%, both kinds of AgNPs dramatically decreased the concentrations of Cr⁶⁺ in both synthetic and actual wastewater samples. Because proteinaceous surface capping improves metal ion binding and electron transfer, AgNPs produced using E. coli demonstrated a marginally higher efficiency. The reduction at the nanoparticle surface and the Cr⁶⁺ interaction was confirmed by the shifts in important functional group regions in the treated samples' FTIR spectra.

4.3 The AgNP Photocatalyst's Reusability and Recyclability

A nanocatalyst's performance stability over time is essential for its practicality in treating wastewater in the real world. Recent research on advanced nanocomposites, including RuO₂@g–C₃N₄ systems and ZnO–NiO/Ag hybrids, has shown excellent cyclic stability, consistently achieving over 90% Cr⁶⁺ reduction efficiency across 4–5 consecutive reuse cycles without a discernible loss in activity. These results highlight how crucial it is to assess catalyst robustness in real-world scenarios. Silver nanoparticles made from Zanthoxylum armatum seed extract, both by itself and in conjunction with E. coli biomass, offer a promising biogenic and environmentally benign platform within the framework of our system.

4.3 Reusability and Recyclability

A nanocatalyst's performance stability over time is essential for its practicality in treating wastewater in the real world. Recent research on advanced nanocomposites, including RuO₂@g–C₃N₄ systems and ZnO–NiO/Ag hybrids, has shown excellent cyclic stability, consistently achieving over 90% Cr⁶⁺ reduction efficiency across 4 consecutive reuse cycles without a discernible loss in activity. The assessment is illustrated in Fig. 16. These results underscore the importance of evaluating catalyst robustness in real-world scenarios. Silver nanoparticles made from Zanthoxylum armatum seed extract, both by itself and in conjunction with E. coli biomass, offer a promising biogenic and environmentally benign platform within the framework of our system.

Recyclability tests, in which the catalyst is recovered after the reaction (for example, by centrifugation or filtration), cleaned, dried, and then used again under the same circumstances, are necessary to verify their practical use. Any deactivation trends brought on by photocorrosion, surface fouling, or agglomeration can be identified by monitoring the degradation efficiency and structural integrity (using UV-Vis, FTIR, or TEM) over cycles. The system's suitability for scalable and economical Cr⁶⁺ remediation in wastewater treatment applications would be strongly supported by demonstrating sustained catalytic activity over multiple runs.

4.4 COMPARATIVE STUDY

The performance of the biosynthesised silver nanoparticles created in this study was placed in context by comparing them to recent Ag-based hybrid nanocomposites that have been published in the literature. Key examples of such systems are compiled in Table 1, along with information on their synthesis methods, target pollutants, mechanistic insights, and benefits for application. These systems demonstrate the adaptability of AgNPs in environmental remediation, encompassing carbon-supported hybrids, membrane-integrated formats, biopolymer matrices, and ternary metal oxides. By providing eco-friendly synthesis, high Cr⁶⁺ removal efficiency, and synergistic catalytic behaviour under real-world wastewater conditions, the green-synthesised hybrid (Zanthoxylum + E. coli) system proposed in this study demonstrates its competitive edge.

Table 1
Comparative overview of advanced Ag-based nanocomposites for environmental remediation applications.
Composite System	Synthesis Method	Target Pollutants	Mechanistic Insight	Key Features	Reference
Ag/ZnWO₄–TiO₂ ternary system	Sol–gel + AgNPs	Rhodamine B, Cd(II), MB	TiO₂ enables charge transport; Ag enhances separation; ZnWO₄ adds photostability	High photostability; ternary synergy	[56]
Ag–Bi₂WO₆/GO composite	In-situ AgNPs on GO	Congo Red, Cr(VI), pharmaceuticals	GO disperses AgNPs; improved charge mobility and recyclability	Graphene-supported; high conductivity	[57]
Ag₂WO₄–ZnO heterojunction	Solvothermal + Ag doping	Organic dyes, Pb(II), bacteria	S-scheme mechanism; Ag⁺ induces ROS and antibacterial action	Broad-spectrum action; visible light catalysis	[58]
Bi₂WO₆/AgNP–Chitosan hydrogel	Biopolymer-AgNP hybrid	Cr(VI), textile dyes	Biopolymer slows Ag⁺ release; Bi₂WO₆ drives oxidation reactions	Biocompatible; recyclable; sustained release	[59]
AgNP–CoWO₄–Biochar composite	Pyrolysis + in-situ AgNPs	Heavy metals, MB, phenol	Biochar adsorbs; AgNPs reduce; CoWO₄ oxidizes pollutants	Multimodal detoxification; carbon-supported hybrid	[60]
Ag–FeWO₄ on cellulose acetate membrane	Electrospinning + AgNP embedding	Mixed dyes (textile effluent)	Combines membrane filtration and Ag-based ROS generation	Real effluent-ready; reusability tested	[61]
Hybrid AgNPs (Zanthoxylum + E. coli)	Green synthesis (plant + microbial route)	Cr(VI), synthetic dyes	Initial adsorption by proteins/phytochemicals; surface-mediated redox reduction; Langmuir–Hinshelwood fit	Synergistic catalysis; high Cr(VI) removal; low-cost green route	[62]

This study uses a dual-origin biosynthetic system to effectively remediate toxic hexavalent chromium (Cr⁶⁺) from aqueous environments in a novel, environmentally friendly, and scalable manner. Silver nanoparticles (AgNPs) were produced using environmentally friendly methods using chromium-resistant Escherichia coli and seed extract from Zanthoxylum armatum, both of which produced unique reducing and stabilizing biomolecules. By efficiently utilizing the complementary benefits of microbial enzymes and plant phytochemicals, this hybrid approach improves the catalytic activity and functionality of nanoparticles.

Comprehensive characterization validated the biosynthesized AgNPs' high crystallinity, homogeneous morphology, and surface functionalization; these findings were corroborated by FE-SEM, HR-TEM, and UV-Vis analyses. Because of improved electron transfer, more active surface sites, and increased catalytic activity brought on by microbial-nanoparticle synergy, the integrated AgNP + E. coli system significantly improved Cr⁶⁺ reduction efficiency, outperforming its parts.

The reduction process was best described by the pseudo-second-order (PSO) and Langmuir–Hinshelwood (L–H) models, according to kinetic modelling, suggesting a surface-mediated adsorption–reduction mechanism dominated by chemisorption. In addition to FTIR spectra confirming strong interactions between Cr⁶⁺ ions and functional groups (–OH, –NH₂, –COOH) present on the nanoparticle surfaces, the L–H model's strong correlation supports the role of surface-bound intermediates in facilitating reduction. This dual-origin nanotechnology offers high efficiency, low toxicity, cost-effectiveness, and scalability, making it a ground-breaking and environmentally friendly substitute for traditional Cr⁶⁺ removal techniques. The study offers a hybrid remediation platform that is both environmentally safe and practically deployable for treating industrial wastewater by fusing green synthesis and microbial bioremediation. This invention is in line with the objectives of global sustainability and shows great promise for practical use in reducing heavy metal pollution, which will guarantee safer water supplies and better animal health.

Acknowledgements

The authors gratefully acknowledge the Sophisticated Analytical Instrument Facility (SAIF) lab, Chandigarh, for characterising the silver nanoparticles. We would also like to thank Dr. Arvind Bhatt for allowing us to use the Biotechnology lab at Himachal Pradesh University (HPU), Shimla.

Conflict of interest

The authors have no conflict of interest related to this publication.

Funding:

This research did not receive a specific grant from any funding agency in the public, commercial, or not–for–profit sectors.

AUTHOR CONTRIBUTION

Author b,Dr. Nidhi Gupta guided the whole process and methodology of heavy metal removal. Author b helped choose the heavy metals and the process selected for the work.
Author a has carried out the lab work and obtained it graphically. Author a has written the paper.

DATA AVAILABILITY STATEMENT

All other relevant data generated and analysed during this study, which include experimental, spectroscopic, crystallographic and computational data, are included in this article.

RESEARCH ETHICS

This research is conducted with the highest standards of scientific integrity and ethical responsibility. We adhere to all relevant ethical guidelines and regulations, including those established by professional organisations and regulatory bodies.

CONSENT TO PARTICIPATE

All participants involved in this study provided informed consent prior to inclusion. The research was conducted in accordance with ethical standards, and participation was entirely voluntary.

CONSENT TO PUBLISH

The authors affirm that participants provided explicit consent for their anonymized data, images, or results to be published in this article.

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Hybrid Green-Synthesised Silver Nanoparticles from Microbial and Seed Extracts for Efficient Heavy Metal Detoxification

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Abstract

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1. INTRODUCTION

2. MATERIALS AND METHODS

3. MODELLING KINETICS

3.5 Kinetic Modelling of Degradation of Chromium (Cr⁶⁺)

3.1 PFO

3.2 PSO

3.3 Langmuir–Hinshelwood (L–H)

3.6 Kinetic Modelling of Cr⁶⁺ Degradation

3.4 Mechanism of Cr⁶⁺ Degradation Using Biosynthesized AgNPs

3.5 Photocorrosion and Stability of AgNPs in Aqueous Photocatalysis

3.6 Effect of Experimental Parameters on Cr⁶⁺ Photocatalytic Reduction

4. RESULTS AND DISCUSSION

4.1 Characterization of AgNPs

4.2 Removal Efficiency of Chromium (Cr⁶⁺)

4.3 The AgNP Photocatalyst's Reusability and Recyclability

4.3 Reusability and Recyclability

4.4 COMPARATIVE STUDY

5. CONCLUSION

Declarations

References

Supplementary Files

Status:

Version 1