Green synthesis of ZnO nanoparticles using Microbacterium arborescens: a multifunctional approach in environment, health, and agriculture Open Access

ZnO nanoparticles (ZnO NPs) have gained immense attention due to their multipurpose applications in antimicrobial activity, adsorbing material, catalyst, gas sensors, luminescent materials, photocatalysts, and solar cells due to their non-toxicity, biocompatibility, and biosafety properties (Nguyen et al. 2024). Nano-ZnO is applied in the medical industry as well as in the food industries and, therefore, is recognized as safe by the Food & Drug Administration (Barage et al. 2022). In addition, ZnO NPs have optical attributes which makes them unable to be used in biomedical applications (Udayagiri et al. 2024).

They are commercially utilized in many industries to manufacture different products such as automobile and engineering (Ashfaq et al. 2022), the rubber and leather industry (Irshad et al. 2024), additives in cement production (Kumar et al. 2021), and protecting agents from the sunlight (Uribe-López et al. 2021). A variety of physical and chemical approaches have been used to synthesize ZnO NPs, including chemical reduction, hydrothermal reactor, microwave, microemulsion, physical vapour deposition, plasma, precipitation, sol–gel methods, ultrasonic irradiation are some of examples of these methods (Singh et al. 2021). These methods, however, are costly and require the use of toxic substances, as well as accumulation and limited nanoparticle stability (Gonçalves et al. 2021).

The biosynthesis of ZnO nanoparticles involves various natural materials including microorganisms, such as bacteria (Gangadhar et al. 2022), fungi (Shamim et al. 2019), plant extracts (Ukidave & Ingale 2022), and waste materials (Okpara et al. 2020) have acted as eco-friendly methods for the synthesis of ZnO NPs. The biosynthesis method involves secondary metabolites that trap the metallic ions and behave as a capping agent that allows to stabilize the ZnO NPs in terms of depletion, electrostatic, hydration, steric hindrance, and Van der Waals forces (Ajitha et al. 2016).

Microbial synthesis of ZnO NPs is influenced by the metabolic processes of microorganisms under moderate experimental conditions (Ajitha et al. 2016). The microbial synthesis approach is facile and does not use any hazardous chemicals. However, the challenges in microbial synthesis to obtain specific NPs. Therefore, optimization needs to be performed with experimental parameters and the different types of microbes (Mohd Yusof et al. 2019).

Therefore, Microbacterium arborescens, a robust microbe that was isolated from the Thar desert in India, is an unexplored resource to produce ZnO NPs. These microbes can generate bioactive metabolites to facilitate the biosynthesis of ZnO NPs with various physicochemical characteristics using different experimental conditions. ZnO NPs with improved quality and adjustable characteristics can be produced using this novel and sustainable biogenic process. The ZnO NPs were characterized using various techniques (spectroscopic and microscopic) such as UV, Fourier Transform Infrared Spectroscopy (FTIR), X-ray Diffraction (XRD) and SEM-EDX techniques for their bond energy, functional group, elemental composition, surface morphology, size, and crystalline structure. Removal of methylene blue (MB) dye using batch study experiments contributes to environmental pollution remediation. Their effective antimicrobial activity can inhibit multiple pathogens, addressing challenges in healthcare and agriculture. Furthermore, the ZnO NPs have enhanced seed germination, providing potential benefits for crop productivity in arid and nutrient-deficient regions. This multi-functionality of biogenic ZnO NPs is a flexible solution for addressing global challenges.

Nutrient agar, nutrient broth, zinc acetate, agar, ethanol, sodium hydroxide (NaOH), antibiotic assay agar, and MB were AR-grade chemicals procured from the SRL, Gujarat, India. Additionally, bacterial cultures, including Bacillus cereus, Bacillus megaterium, Staphylococcus aureus, and Serratia marcescens, were obtained from the GBRC, Gandhinagar, Gujarat, India. The green grams were purchased from the APMC (Agricultural Produce & Livestock Market Committee) Patan, India, 384265.

Microbacterium arborescens was isolated from Fagonia sp. Plant leaf from the Thar desert (28.189784 N, 73.411467 E), Rajasthan. The extracted bacterial colony M. arborescens was inoculated into a nutrient broth (N-broth). The N-broth was incubated for 24 h at 37 °C in an incubator shaker (RICO Orbital Incubator Shaker RS/OIS-240) at 150 rpm to confirm the optimal growth. After 24 h, the absorbance spectrum of the culture was taken at 600 nm with optical density (OD) > 1 to examine the sufficient growth. Afterwards, the culture was separated from the supernatant using centrifugation at 10,000 rpm for 10 mins. The supernatant, rich in bioactive metabolites, was further utilized for ZnO NP synthesis.

A precursor solution was prepared by making a 25 mM solution of zinc acetate dihydrate [Zn(CH₃COO)₂·2H₂O] in 80 mL of deionized water using a magnetic stirrer at 180 rpm. 20 mL of bacterial supernatant was added dropwise into the solution (till 5 pH) under continuous stirring for 2 h. In the end, white precipitates were formed at the bottom. The reaction mixture subject to centrifugation collects ZnO NPs. The particles were washed with deionized water, and then ethanol wash was given to remove impurities. The washed ZnO NPs were dried at 70 °C. Subsequently, the dried ZnO NPs were calcined at 300 °C for 2 h to complete dehydration. An additional experiment was performed using NaOH (up to 9 pH) with bacterial supernatant in the same manner to synthesise ZnO NPs. The obtained nanoparticles, including ZnO NPs- 300°-5 pH, ZnO NPs- 300°-9 pH, ZnO NPs- 30°-5 pH, and ZnO NPs- 30°-9 pH, were stored.

UV–visible spectrophotometric analysis of each ZnO NPs was conducted (LABMAN, LMPS-UV1900S) at wavelength extending from 200 to 800 nm, recorded at 1 nm intervals. FTIR analysis was conducted from 500 to 4,000 cm⁻¹ using an FTIR spectrophotometer (Perkin Elmer spectrum 65). The XRD (Rigaku MiniFlex 600) was used to calculate the degree of crystallinity of the developed film. The operation condition, such as Cu-Kα radiation, was 1.54059 Å, at 30 kV and 2 mA, and data were collected at 3°–80° 2 Ɵ values. The crystal size was obtained using the Debye–Scherrer equation. The surface micro-structural analysis of the ZnO NPs was conducted using SEM (FEI Apreo S) operated with high vacuum mode at a distance of 6.8 mm and energy at 20 kV. The elemental profile of ZnO NPs was estimated with an EDX spectrum. A Brunauer–Emmett–Teller (BET) analyzer and (Quantachrome-Autosorb iQ Station 2) were used to measure the specific surface area, pore volume, and pore size of the ZnO NPs. The sample was degassed at 200 °C for 2 h before the nitrogen adsorption measurements. The BET surface area was determined using multi-point adsorption data in the 0.05–0.3 P/Po relative pressure range. The Barrett–Joyner–Halenda (BJH) method calculates the pore diameter and volume. The Particle size and zeta potentials of the ZnO NPs sample were determined using a Particle size and Zeta Potential Analyzer (HORIBA SZ-100). It was measured mean value of zeta potential and electrophoretic mobility at 0.891 mPas dispersion medium viscosity, 3.4 V electrode voltage, and 25 °C temperatures.

The effect of different pH and dose of ZnO NPs were studied under experimental conditions. At the equilibrium, a kinetic study was conducted, to check the liner and non-liner fitting with various adsorption kinetic models, including pseudo-first-order (PFO), pseudo-second-order (PSO), intraparticle diffusion (IPD), and Elovich. The equation and parameters used for kinetic models are shown in Table S1.

ZnO NPs samples were examined for antimicrobial activity with four bacteria: S. marcescens, B. cereus, B. megaterium, and S. aureus, using a well diffusion method at different concentrations of ZnO NPs, such as 2, 4, and 6 mg mL⁻¹.

The peaks at 1,372, 1,474, and 1,481 cm⁻¹ correspond to the carboxyl group (Jay Chithra et al. 2015), while the carboxyl group was confirmed at 1,582 cm⁻¹ (Kalpana et al. 2018) due to the capping agent, which was eliminated after calcination at 300 °C and also at pH 9. Similarly, the adsorption peaks at 2,392, 2,384, 2,367 cm⁻¹, and 2,316 indicate the primary amine of stabilizing agents (Jayaseelan et al. 2012; Acharya et al. 2024), which disappeared after the calcination. A peak at 3,409 cm⁻¹ corresponds to the –OH group (Selvarajan & Mohanasrinivasan 2013) found reduced significantly with increasing pH and temperature. Biogenic-synthesized ZnO NPs are often additional peaks observed due to the biomolecules which interact with the NPs during the synthesis.

The XRD plots of ZnO NPs samples for different temperatures (30 and 300 °C) and pH (5 and 9) are presented in Figure 2(b). The XRD plot of all ZnO NPs samples indicates prominent peaks at various 2θ values with their corresponding basal plane values around 31.54^◦ [0 1 0], 34.16^◦ [0 0 2], 36.02^◦ [0 1 1], 47.28^◦ [0 1 2], 56.38^◦ [1 1 0], 62.64^◦ [0 1 3], and 67.82^◦ [1 1 2] (Priyadarshi et al. 2022). The spectrum of ZnO NPs corresponds to Joint Committee on Powder Diffraction Standards card No. 36-1451. Each of the broad peaks was a crystalline sharp peak at the same 2θ after calcination. These particular lattice plane reflections positively confirmed the hexagonal wurtzite structure of nano-sized ZnO crystals with a mean crystallite size of 134 nm for ZnO NPs 30° pH 5, 112 nm for ZnO NPs 30° pH 9, while after calcination, it becomes 15 nm for ZnO NPs 300° pH 5 and 12 nm ZnO NPs 300° pH 9 are in line with the previously described results (Verma et al. 2017). The effect of calcination temperature shows increases in the intensity of diffraction peaks that indicate the strengthening of ZnO NPs (Ashraf et al. 2015).

EDX of ZnO NPs elemental composition is shown in Figures 3(e)–3(h). The peaks at 1 and 8.6 keV confirmed the representation of the Zn element in ZnO NPs samples. The elemental composition shows the amount of Zn is increasing (∼ 10%) with rising pH and temperature. The peak at 0.2 keV represents Au, which is associated with the gold coating that was applied during the sample preparation phase for the EDX and SEM analyses. After the calcination, the amount of C and O elements is decreased due to the removal or purification of ZnO NPs.

The nitrogen adsorption-desorption isotherm, as shown in Figure S2 of ZnO NPs, represents a type IV isotherm with a hysteresis loop, a characteristic of mesoporous material. The adsorption curve shows a gradual increase in N₂ uptake at a lower relative pressure (P/Po), indicating the presence of micropores. At higher relative pressure, a straight-up increase in adsorption volume indicated the filling of mesopores. The BET surface area of the ZnO NPs was 31.179 m²/g, highlighting the material's high surface area. The BJH pore size distribution analysis indicated a mesoporous structure, with a dominant pore diameter of 3.814 nm and a total pore volume of 0.1542 cc/g, as shown in Figure S3. Additionally, the average pore size was 19.78 nm, further confirming the mesoporous nature of the synthesized ZnO NPs, aligning with the International Union of Pure and Applied Chemistry classification for mesoporous material (Khezami et al. 2018). The observed surface area and porosity contribute significantly to the adsorption efficiency of MB dye.

The dynamic light scattering (DLS) analysis of ZnO NPs revealed a Z-average hydrodynamic diameter of 254.7 nm, with a polydispersity index (PI) of 0.708, indicating a moderately broad size distribution. The results suggest significant agglomeration in aqueous dispersion. In contrast, the SEM analysis showed an average particle size of ∼46 nm at pH 9, 300 °C, which is much smaller than the DLS-measured size. DLS measures the hydrodynamic size of nanoparticles in suspension, where they form larger aggregates due to van der Waals forces and electrostatic interactions. Despite agglomeration in aqueous suspension, the observed moderate PI (PI = 0.708) suggests that the ZnO NPs maintain a relatively stable dispersion, which is crucial for their applications in adsorption and antimicrobial studies (Zakharova et al. 2019).

The zeta potential of ZnO NPs was −32.5 mV, confirming their high negative surface charge at pH 9. This negative charge arises due to the deprotonation of hydroxyl (–OH) groups on the ZnO surface, which enhances colloidal stability by electrostatic repulsion. A zeta potential value below −30 mV typically suggests that the nanoparticles are electrostatically stabilized and resistant to uncontrolled aggregation in the solution. The negative charge influences interactions with cationic species like MB dye and bacterial cell membranes, enhancing adsorption and antibacterial efficacy (Pervez et al. 2024).

The effect of pH on MB dye removal using ZnO NPs is illustrated in Figure 4(c). At pH 5, the MB removal was fairly low, approximately 24% after 100 min. This low efficiency could be ascribed to the acidic pH of the solution, due to the protonation of ZnO NPs, which limits their interaction with cationic MB molecules (Mulaw et al. 2024). At pH 7, the removal efficiency distinctly improved up to 69% within 120 min, which provides better conditions for electrostatic interactions between ZnO NPs and MB dye. The highest removal was observed at pH 9, around 72% removal. The alkaline conditions promote deprotonation of ZnO NPs enhancing the availability of negatively charged –OH, making stronger interactions with the cationic MB molecules (Mouni et al. 2018). The increased removal efficiency at 9 pH highlights the role of pH in optimizing the adsorption capacity.

The effect of ZnO NPs adsorbent dosage on MB dye removal efficiency is presented in Figure 4(d). The results indicate a dose-dependent improvement in MB removal efficiency, with higher ZnO NPs doses in mg exhibiting better dye removal. At a dosage of 25 mg, the removal was plateaued at 74% within 70 min.

Increasing the dosage to 50 mg resulted in a substantial improvement, succeeding around 81% dye removal. Further increasing the dose to 75 and 100 mg showed a slight improvement, achieving 87 and 90% removal within 70 min, respectively. The results suggest that after a certain concentration, the active adsorption sites become saturated, and further increases in the ZnO NPs do not significantly enhance the removal efficiency (Raval et al. 2022).

The IPD model shown in Figure 5(c) revealed a multi-stage adsorption process with a diffusion rate constant K_diff of 8.06 mg g min^−0.5 and an intercept C of 15.50, corresponding to boundary layer effects. However, the R² value of 0.8013 suggests that IPD is not the sole rate-controlling mechanism. The Elovich model presented in Figure 5(d) provided insights into the heterogeneity of the adsorption surface, with the desorption constant (β) of 0.0481 and the initial adsorption rate (α) of 17.76. An R² value of 0.9093 confirmed that this model adequately represents the chemisorption of MB molecules onto ZnO NPs (Wu et al. 2009; Zhou et al. 2014).

Overall, the PSO demonstrated the best fit, emphasizing chemisorption as the predominant mechanism, while the IPD and Elovich models highlighted the secondary processes, including diffusion and surface heterogeneity.

The IPD model shown in Figure 6(c) revealed a multi-step adsorption process, with a diffusion rate constant K_diff of 8.06 mg g⁻¹min^−0.5 and an intercept C of 15.50, attributed to boundary layer effects. However, the R² value of 0.801 suggested IPD as a contributing, but not exclusive, rate-controlling step (Li et al. 2024). The Elovich model, as shown in Figure 6(d), highlighted surface heterogeneity and chemisorption dynamics, with an initial adsorption rate α of 12.94 mg g⁻¹ min⁻¹ and a desorption constant β of 0.0435. The R² value of 0.907 indicated that this model effectively describes the non-uniform adsorption surface and chemical interactions. Among all models, the PSO model provided the best fit, underscoring chemisorption as the primary mechanism. However, the contributions of IPD and surface heterogeneity, as explained by the IPD and Elovich models, cannot be overlooked, reflecting the complexity of the adsorption process. The comparative maximum adsorption capacity for MB dye removal using various adsorbents is listed in Table S2. The synthesized ZnO NPs provide a balance between adsorption capacity, fast kinetics, and eco-friendly synthesis, making them a promising alternative to other conventional and nanocomposite adsorbents for MB removal.

The antimicrobial activities of ZnO NPs against gram-positive (S. aureus, B. cereus, and B. megaterium) and gram-negative (S. marcescens) microorganisms are depicted in (Table 3 and Figure S4). The observed zone of inhibition indicates a dose-dependent (increasing from 10 to 16 mm) inhibitory against all bacteria; however, S. aureus was lower than other bacteria. The zeta potential of ZnO NPs at pH 9 was −32 mV, indicating a strongly negative surface charge. This supports the proposed ionic interaction mechanism, where the electrostatic attraction between negatively charged ZnO NPs and the bacterial cell membrane influences antibacterial activity. The lower inhibition zone observed for S. aureus than gram-negative bacteria can be attributed to differences in cell wall composition. S. aureus, a gram-positive bacterium, has a thicker peptidoglycan layer that may reduce ZnO NPs' penetration and interaction with cellular components, thereby limiting antimicrobial efficacy. The negative zeta potential further supports ROS generation, which contributes to bacterial inactivation (Shaikh et al. 2023).

ZnO NPs generate various reactive oxygen species (ROS), including hydrogen peroxide, hydroxyl radicals, and superoxide radicals that play a vital role in Deoxyribonucleic acid (DNA) damage and cell death (Ma et al. 2024). A limitation of this study is that ROS production was inferred from the existing ZnO literature rather than directly measured. While the antimicrobial activity observed suggests ROS-mediated bacterial inactivation, experimental validation, using assays such as DCFH-DA fluorescence or electron spin resonance ,was not performed. Future studies should include direct ROS quantification to confirm the precise mechanism of bacterial inhibition and further elucidate the role of ZnO NPs in oxidative stress generation. The finding exhibits considerable antimicrobial activity of ZnO NPs through ionic interaction and ROS generation.

Table 4 and Figure S5 represent the results of seeding parameters. Seed germination % after 10–14 h shows a gradual increase (80–100%) in the number of germinated seeds compared to control (55–95%). Similar observations were found in previous studies (Ukidave & Ingale 2022; Mathin et al. 2023) that the ZnO NPs significantly (up to 100%) enhance the seed germination in green grams. The ZnO NPs show the enhancement in the process of seed germination. The study shows the gradual reduction (1.3–1.1 cm) in average root length and dry weight (0.780–0.838 g) of the seeds compared to the control (3.09 cm–0.723 g).

Higher concentrations, of ZnO NPs >640 mg L⁻¹ have been associated with phytotoxic effects, such as reduced growth metrics (Prajapati et al. 2024). The application of ZnO NPs, specifically at lower concentrations (Kalimuthu et al. 2023), improves seed yield, making them a promising biofertilizer.

The first documentation of biogenic fabrication of ZnO NPs using M. arborescens isolated from the Thar desert, India. The effect of experimental parameter variation on ZnO NPs was evaluated, using UV, FTIR, XRD ,and SEM-EDX analytic techniques. The finding indicates changes in band gap, increase in crystallinity, and size reduction by increasing up to pH 9 and calcination at 300 °C. The synthesis conditions, particularly pH 9 and calcination at 300 °C, significantly influenced the structural, optical, and surface properties of ZnO NPs, enhancing their performance in MB dye removal, antimicrobial activity, and seed germination. The adsorption kinetics revealed chemisorption as the dominant mechanism, while antimicrobial efficacy was attributed to ROS generation and surface charge interactions. In agricultural applications, ZnO NPs improved seed germination while maintaining minimal phytotoxicity. These findings emphasize the multifunctionality of biosynthesized ZnO NPs, bridging environmental remediation, antimicrobial applications, and agricultural advancements. Future work could explore scaling the synthesis process, investigating long-term impacts, and extending applications to other environmental and biological systems.

The authors acknowledge and extend their appreciation to the ongoing Research Funding Program (ORF-2025-739), King Saud University, Riyadh, Saudi Arabia. The authors wholeheartedly thank and appreciate the support given by the Department of Life Sciences, Hemchandracharya North Gujarat University for providing the lab facilities.

The authors acknowledge and extend their appreciation to the ongoing Research Funding Program (ORF-2025-739), King Saud University, Riyadh, Saudi Arabia.

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All the authors have given their consent to publish this article.

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The authors declare there is no conflict.