A total of 10 bacteria (SL1–SL10) were isolated from wastewater treatment, slug (site), and slag (site) Fe-enriched sites from Hisar (Haryana, India) using a serial dilution method (Rana et al., 2011). Soil samples were diluted up to 10^–8 with sterilized water, and 100 µL from final dilutions was spread on Luria Bertani (LB) agar plates, followed by overnight incubation at 28 ± 2°C. The bacteria were isolated on the basis of distinguished colony shape, pigmentation, and other morphological features. Furthermore, six plant growth-promoting isolates (RP24, TMG3, WF, WH, WD, and AW5) were obtained from the Microbial Genomics Laboratory (115A), Department of Microbiology, College of Basic Sciences and Humanities, Chaudhary Charan Singh Haryana Agricultural University, Hisar, Haryana (India). The bacteria were grown on an LB agar medium and stored at 4°C for further use.

The isolates were evaluated for their Fe and Zn tolerance on nutrient agar media supplemented with different concentrations of ferric chloride (FeCl₃) and zinc sulfate (ZnSO₄) (3 mM, 6 mM, 9 mM, and 12 mM). The iron and zinc solutions were added to the growth media via syringe filtration before being poured onto a Petri plate. The bacteria were grown on the prepared media at 28 ± 2°C, and colony growth diameter was observed after 48 h of incubation.

The effects of synthesized Fe and Zn NPs, individually as well as in combination with PGPR (AW5), were tested on wheat. Wheat seeds were primed with the suspension of NPs and bacterial culture before sowing. The optimized doses of Fe and Zn NPs, which showed enhanced effects on the growth bacteria and PGP traits, were selected to evaluate their effect on growth, yield, and nutritional content of wheat under pot house conditions. A total of 18 treatments were designed, and the recommended dose of fertilizer (RDF) N120P60K60 was added in all the treatments, except the absolute control following two split doses of nitrogen as follows: T1: Absolute control; T2: RDF control; T3: RDF + PGPR; T4: RDF + PGPR + FeNPs (20 ppm); T5: RDF + PGPR + FeNPs (100 ppm); T6: RDF + PGPR + ZnNPs (20 ppm); T7: RDF + PGPR + ZnNPs (100 ppm); T8: RDF + PGPR + ZnNPs and FeNPs (20 ppm); T9: RDF + PGPR + ZnNPs and FeNPs (100 ppm); T10: RDF + PGPR + Foliar treatment (FeCl₃), T11: RDF + PGPR + Foliar treatment (ZnSO₄); T12: RDF + PGPR + ZnSO₄ and FeCl₃, T13: RDF + PGPR + Foliar treatment (FeNPs-20 ppm); T14: RDF + PGPR + Foliar treatment (FeNPs-100 ppm); T15: RDF + PGPR + Foliar treatment (ZnNPs-20 ppm); T16: RDF + PGPR + Foliar treatment (ZnNPs-100 ppm); T17: RDF + PGPR + Foliar treatment (ZnNPs and FeNPs-20 ppm); T18: RDF + PGPR + Foliar treatment (ZnNPs and FeNPs-100 ppm).

Surface sterilization of wheat seeds was performed with sodium hypochlorite (4%), followed by washing with sterilized distilled water (DW) and drying. The sterilized seeds were primed with FeNPs (20 ppm, 100 ppm) or ZnNPs (20 ppm, 100 ppm) or a combination of both nanoparticles (20 ppm; 100 ppm), followed by PGPR or combination (PGPR and NPs) for a 1-h treatment. The nanoparticle stock (1,000 ppm) was prepared by dispersing it in double-distilled water by sonication. Working concentrations (20 ppm and 100 ppm) were prepared by dilution of stock solution and employed for seed priming and foliar application. A foliar spray was performed on the crop at the three-leaf stage and the milking head stage.

The soil mix (5 g) with solution A (3% TTC (2,3,5 triphenyl tetrazolium chloride) and distilled water 1:2) was used to determine the dehydrogenase activity. Samples were incubated at 37°C for 24 h (in dark) after vigorous mixing. After incubation, methanol (10 mL) was added to the reaction and filtered (Whatman No. 1 paper). The absorbance of the filtrate was recorded at 485 nm using a spectrophotometer with methanol as a blank (Thalmann, 1968). The potassium phosphate buffer (5 mL, pH 7.6, 60 mM) was mixed with soil (1 g), followed by the addition of fluorescein diacetate (FDA) solution (10 µL) and incubation (37°C, 1 h). After incubation, acetone (200 μL, 5% v/v) was added to stop the reaction and filtered. The absorbance was recorded at 485 nm using a spectrophotometer method (Green et al., 2006).

After harvesting the crop, different parameters like auxin (Manna et al., 2024) and chlorophyll content (Wellburn, 1994) were analyzed. Fresh plant tissues were weighed 0.1 gm and crushed after freezing in deep at −20°C for 2 h. An aliquot of ethanol (∼2 mL) was added to the powdered sample before thawing, followed by thorough grinding. The homogenate (1 mL) was transferred to an Eppendorf tube and centrifuged at 10,000 × g for 2 min at 4°C. The supernatant (100 μL) was mixed with 900 μL of the reagent (mixture of water: concentrated sulfuric acid: 0.5 M FeCl₃ (25:15:0.75) and incubated at room temperature for 30 min. Absorbance was recorded at 540 nm, and the auxin concentration was calculated using a standard curve (Manna et al., 2024). The chlorophyll content was analyzed following the modified method of Wellburn (1994). Fresh tissue (0.1 g) was crushed using a chilled mortar and pestle, followed by the addition of methanol (5 mL). The homogenate was incubated at 65°C for 30 min in the dark. The mixture was centrifuged at 10,000 × g for 10 min, and the supernatant was analyzed using a spectrophotometer at 666 nm and 653 nm wavelengths. Total chlorophyll content, chlorophyll a, and chlorophyll b were calculated using the following equations: Chlorophyll a (µg/g) = (15.65 × A666–7.34 × A653) × 50; chlorophyll b (µg/g) = (27.05 × A653–11.21 × A666) × 50 (Wellburn, 1994).

The pots were filled with water overnight before harvesting the plants with their roots intact. The plant roots were washed to remove excess soil and dried before measuring biomass and shoot and root length (cm). Plant biomass (g) was measured with spikes, followed by grain harvesting and grain weight (100 seeds per treatment). All experiments were performed in triplicate, and the average was reported.

The plant samples were prepared using a standard protocol. In brief, plant straw and grain fine powder (0.5 g) were added with diacid (HNO₃+HClO₄, 4:1, 10 mL) and heated until a clear, colorless solution (reduced up to 3–4 mL) was obtained. The solution was cooled, filtered, and transferred to a volumetric flask, followed by analysis (Huang et al., 2004). In the case of P, the analysis digested plant material (1–5 mL) was taken in a volumetric flask, followed by the addition of 2,4 di-nitrophenol indicator (2–3 drops). Ammonia solution was added until a yellow color appeared. Furthermore, HCl (6 N) was drop-wise added up to a colorless appearance, followed by the addition of the vanadomolybdate solution (5 mL) and diluted up to 25 mL with distilled water, mixed well, and analyzed using a spectrophotometer (440 nm wavelength). The soil sample (2.0 g) was taken in a glass bottle (100 mL), to which was added a pinch of activated charcoal (Darco G-60). Sodium bicarbonate (NaHCO₃) solution (40 mL of 0.5 M) was added to the soil sample, agitated for 30 min, and filtered for analysis of nutrients (Alban and Kellogg, 1959).

Proximate analysis of wheat grain was performed using standard laboratory protocols. Moisture content in wheat grain samples (5 g, dried for 15 min, 100°C) was estimated. The moisture content was calculated using the following formula: Moisture (%) = (W2/W1) × 100, where W1 is the initial weight of the sample (g), and W2 is the weight loss (g) after drying. To determine the ash content, a grain sample (5.0 g) was placed in a silica crucible and heated in a muffle furnace (550°C, 15 min). The crucible was then removed, cooled in a desiccator for 1 hour, and weighed. The sample was heated on a hot plate until smoking ceased and thoroughly charred until it became white. The ash content was calculated using the formula: Ash (%) = (W2/W1) × 100, where W1 and W2 are the weight of the sample (g) and the loss of weight (g) in the sample after drying, respectively.

To determine the crude fat content, a finely ground wheat grain sample (5.0 g) was placed in an extraction thimble. The thimble was placed in an extractor and connected to a weighed flask containing petroleum ether (100 mL). The extractor was connected to a reflux condenser, and the sample was extracted under reflux for 2 h. After extraction, the petroleum ether extract was evaporated to dryness, and 2 mL of acetone was added. Air was gently blown into the flask to remove the last traces of the solvent. The flask containing the fat residue was dried in a hot air oven (100°C, 10–15 min), then cooled in a desiccator, and weighed. The crude fat content was calculated using the formula: Crude Fat (%) = ((W3 − W2)/ W1) × 100, where W1 is the weight of the sample before drying (g), W2 is the weight of the empty flask (g), and W3 is the weight of the flask with the fat residue (g).

The impact of amorphous Fe and Zn NPs on the growth of AW5 in a liquid medium was dependent on the concentration (1 ppm, 5 ppm, 10 ppm, 20 ppm, 30 ppm, 40 ppm, and 50 ppm). The maximum growth of AW5 was observed at 20 ppm for Fe (2.3 × 10⁸ CFU/mL) and Zn (2.1 × 10⁸ CFU/mL), with an increase in bacterial growth (up to 19%) versus control without NPs (Figures 5A,B). The effect of standard and annealed Fe and Zn NPs on the growth of bacteria was compared with their amorphous form in a liquid medium at an optimized concentration (20 ppm) of NPs. The amorphous bacterial nanoparticles (BNPs) significantly increased bacterial count with the amendment of Fe (12%–18%) and Zn (15%–17%) versus standard NPs and crystalline (annealed) BNPs, respectively. However, the standard (S) and annealed (A) nanoparticles did not show any significant effect on bacterial growth enhancement (Figure 6). The positive impact of amorphous Fe and Zn nanoparticles on bacterial growth showed their better compatibility and employability in sustainable agriculture.

The effect of amorphous FeNPs and ZnNPs on the PGP traits of AW5 was checked with optimized doses of NPs (10 ppm, 20 ppm, and 30 ppm) supplemented with a liquid medium. The NP treatment enhanced IAA production significantly (up to 39%) with ZnNPs (20 ppm) versus control (without NPs). However, IAA production in the case of FeNP treatment was not significant. Phosphate and zinc solubilization were increased in the case of FeNPs (60%) and ZnNPs (35%) with a 20 ppm dose. Moreover, siderophore production significantly improved by up to 10% with FeNPs (20 ppm) (Figure 7).

The enhanced growth and PGP traits of AW5 in response to NPs indicate the improved dynamics of nutrients leading to the enhancement of biochemical activity in bacteria for soil health and plant growth promotion. In this study, we have observed for the very first time the effect of bacterial Fe and Zn NPs on the PGP traits of rhizobacteria. It is pertinent to mention that the studies have generally reported the toxicity of NPs towards bacteria. For example, Chatterjee et al. (2011) reported a declined effect of different concentrations (50–200 μg/mL) of chemically synthesized iron oxide NPs on the growth of Escherichia coli at 6-h incubation due to reactive oxygen species (ROS) superoxide, hydroxide radical, and singlet oxygen (Chatterjee et al., 2011). Chemically synthesized FeNPs (10–100 ppm) showed a negative effect on the Serratia growth and reduced production of biosurfactants, while a positive effect of bacterial NPs on the growth of PGPR and activity thereof was noticed in our study within the same concentration range. Similar to our study, Kaur et al. (2024) reported an enhanced effect of titanium NPs (20 ppm) on the PGP traits of Rhizobium, namely, IAA, siderophore production, and phosphate solubilization.

The effect of NPs on soil enzymes (dehydrogenase and fluorescein diacetate) was studied. The FDA activity ranged from 11.43 to 20.4 µg fluorescein/g soil/h. Foliar applications of ZnNPs (100 ppm) with RDF and PGPR showed a maximum increase (64%) in T16 versus the RDF control (T2). It was up to a 53% increase compared to other treatments containing NPs (Figure 8A). The dehydrogenase activity ranges from 1.49 µgTPF/g soil/d to 2.2 µgTPF/g soil/d. The maximum increase (53%) was observed in T7 (seed-primed with RDF + PGPR+ ZnNPs, 100 ppm) versus the RDF control (T2). Moreover, dehydrogenase activity was also increased in other NP treatments up to 42% (Figure 8B). Soil microbial enzymatic activities of dehydrogenase and FDA were enhanced due to the interaction of NPs with bacteria that promoted the growth of microorganisms, leading to the mobilization of nutrients and the breakdown of soil organic matter for maintaining soil fertility and nutrient recycling. The findings suggest that FeNPs and ZnNPs may act as co-factors for soil microbial enzymes, enhancing their metabolic activity, organic matter decomposition, and nutrient dynamics, which ultimately contribute to improving soil health and plant growth (Ameen et al., 2021; Ahmed et al., 2023).

The effect of Fe and Zn NPs individually and in combination was observed on plant biochemical parameters, namely, chlorophyll and auxin (Figure 9). The chlorophyll content (µg/g) in leaf tissue ranged from 90.6 µg/g to 136 µg/g (Chl A) and from 42.2 µg/g to 87.4 µg/g (Chl B). The maximum increase was observed in treatment T9 (PGPR + ZnNPs + FeNPs, 100 ppm). Chl A and Chl B were increased by 51% and 107%, respectively, in T9 versus the RDF control (T2). The auxin content (µg/gm) ranged from 5.14 µg/gm to 8.16 µg/gm with a maximum increase in T15 (seed priming with PGPR + foliar application of ZnNPs-20 ppm). Auxin amount was increased by 59% in T15 versus the RDF control (T2), followed by T9 and T6 (up to 50%).

In a study performed by Seyed Sharifi et al., 2020, foliar application of FeNPs, ZnNPs (1,500 ppm), and their combination improved chlorophyll and auxin content in the plant via inducing the photosynthetic and plant metabolic pathways and significantly increased grain yield (22%, 11%, and 40%, respectively). A similar enhancing effect was recorded in our study using Fe and Zn NPs with increased chlorophyll levels and plant growth hormone (auxins). FeNP- and ZnNP-induced molecular pathways participate in electron transport chains, which especially involve photosystems I and II, thus improving ATP synthesis and overall energy efficiency for better plant growth and enhancement (Fallah et al., 2024; Qu et al., 2024). Auxins play an important role in plant growth, influencing cell elongation and division, and their enhancement is likely to contribute to the observed improvements in plant biomass and other growth traits. NPs generally showed a positive effect; the specific outcomes are dependent on the combination and concentration of NPs. Similarly, a significant enhancement in photosynthetic parameters (chlorophyll A, 45%, and B, 117%) was observed as a result of seed priming with FeNPs and ZnNPs (Rizwan et al., 2019). A dose-dependent effect of ZnNPs was reported via improving chlorophyll A (55%) and chlorophyll B (133%) at 50 ppm; a higher concentration (100 ppm) did not show a significant effect (Amooaghaie et al., 2017). The results are in line with our study, where an enhanced effect in plants treated with a 20 ppm dose of NPs versus 100 ppm was observed.

Wheat plants were uprooted after maturity, and physiological parameters (root and shoot length, plant biomass) were recorded (Figure 10). The shoot length ranged from 64.4 cm to 74.9 cm, with a maximum of 74.9 cm for treatment T4 (PGPR + FeNPs, 20 ppm) applied through seed priming, followed by T13 and T18. Shoot length was increased up to 8% in combination with NPs and PGPR versus the RDF control (T2). The root length ranged from 7.6 cm to 12.7 cm, with the maximum for T12, followed by T9, T10, and T11. There was an increase in root length (19%) in NP-treated seeds versus the RDF control (T2). The maximum plant biomass (14.9 g) was recorded in treatment T18 with an increase of 56% versus control (T2), followed by T10. The maximum 100 grains weight (6.4 g) was recorded in treatment T17 (seed priming with PGPR + foliar spray of Fe and Zn NPs (20 ppm), followed by T18 (6.02 g) versus the RDF control (T2). The grain weight was increased by 34% (T17) and 25% (T16).

The results of the present study, in the context of plant growth parameters, are similar to previous findings that demonstrated significant enhancement in key growth parameters with NP application. The interaction between bacteria and plant roots with NPs depends upon their characteristics, such as amorphous or crystallinity nature. The stability of crystalline NPs impacts the microbial community, whereas amorphous NPs discharge ions to modify bacterial functions and enzymatic processes. A similar effect with different doses of FeNPs and ZnNPs on plant growth attributes was reported. For example, seed priming of FeNPs and ZnNPs (20 ppm and 100 ppm) enhanced plant height by 37% and 35%, respectively, and other parameters such as shoot biomass (53%) and grain weight (74%) in all treatments containing NPs compared to the control (Rizwan et al., 2019). In another study, soil application with ZnNPs (20 ppm and 50 ppm) increased biomass (63%) and grain yield (56%) (Du et al., 2019). Similarly, ZnNPs and zinc oxide (ZnO) increased wheat shoot fresh weight (up to 21% and 28%) with 100 ppm and 50 ppm, respectively. The application of bulk ZnNPs (50 ppm and 200 ppm) also increased root and shoot weight (Amooaghaie et al., 2017).

The NP-induced micronutrient profiling of wheat and soil was studied (Figure 11). Iron (mg/kg) in wheat grains ranged from 40.4 mg/kg to 54.5 mg/kg (Figure 11A). The maximum increase of 27% was observed in the case of T17 (ZnNPs-20 ppm + FeNPs, 20 ppm foliar application + RDF and PGPR), followed by T13 (17%) versus the RDF control (T2). The iron content (ppm) in wheat straw ranged from 119 mg/kg to 276 mg/kg. The maximum increase of 82% was observed in T6 (seed priming-ZnNPs, 20 ppm, + RDF + PGPR), followed by T17 (63%) versus the RDF control (T2, Figure 11C). There was no significant increase in iron content in NP-treated soil versus the RDF control (T2, Figure 11E). The zinc content (mg/kg) in grain ranged from 15.8 mg/kg to 26.9 mg/kg (Figure 11B). The foliar application of NPs increased zinc content up to 53% (T16) versus the RDF control (T2). There was no significant increase of zinc in straw (Figure 11D). The zinc content (mg/kg) in soil ranged from 1.25 mg/kg to 2.32 mg/kg with a maximum rise of 48% in T14, followed by T3 (40%) and T9 (30%) versus the RDF control (T2, Figure 11F).

The manganese content in wheat grain increased up to 38% (T4 seed treatment) and 23% (T18 Foliar application) versus the RDF control (T2, Supplementary Figure S4B). Moreover, in the case of straw, an increase in Mn content was 47% (T4) and 55% (T6) applied via NP seed treatment and up to 15% (T17) through foliar application (Supplementary Figure S4D). NP treatments did not show any significant effect on soil manganese content versus the RDF control (T2, Supplementary Figure S4F). Copper content was increased up to 17% (T16) in wheat grains without any significant difference among the treatments (Supplementary Figure S4A). Moreover, Cu in soil (Supplementary Figure S4E) increased up to 83% (T14), followed by 46% (T3).

The combination of bacteria and NP could biofortify wheat crops to address the global challenge of hidden hunger. The biofortification of micronutrients (Fe, Cu, Zn, and Mn) and protein content in wheat under field conditions was reported in our previous study (Rana et al., 2012). Similarly, seed priming with FeNPs and ZnNPs enhanced the Zn and Fe content in plants and grains in other studies. Seed priming with Fe (10 ppm) and Zn (20 ppm) nanoparticles increased Fe and Zn in roots (28%), followed by shoots (18%) and grains (29%) versus the control (Rizwan et al., 2019). A distinct effect of bulk ZnO and ZnO NPs was reported on the increment of micronutrient content in wheat grain, straw, and soil (Amooaghaie et al., 2017).

The phosphorus content in grains was significantly increased up to 25% (T16) and 7% (T4) when applied via foliar treatment and seed treatment, respectively, versus the RDF control (T2, Supplementary Figure S5A). In the case of straw, the P content increased to 32% (T8 and T16) when applied through seed and foliar treatment of NPs versus the RDF control (T2, Supplementary Figure S5C). The soil P content was increased up to 47% (T9) and 40% (T14) through seed and foliar treatment, respectively, versus the RDF control (T2, Supplementary Figure S5E). The K content in grains was significantly increased up to 54% (T8) and 53% (T7) via seed treatment of NPs versus the RDF control (T2, Supplementary Figure S5B). In straw, the K content was increased up to 17% (T8 seed treatment) and 12% (T13 foliar spray, Supplementary Figure S5D). The soil K content was increased up to 12% (T9) and 16% (T14) through seed and foliar treatment of NPs, respectively, versus the RDF control (T2, Supplementary Figure S5F). FeNPs and ZnNPs enhanced translocation of metal ions through transport proteins combined with vascular sequestration processes, resulting in increased accumulation of nutrients in edible parts of the plant (Khan et al., 2021; Koç and Karayiğit, 2021; Sánchez-Palacios et al., 2023).

Seed carbohydrate and fat content in combination of PGPR and NPs increased up to 16% (T16) and 33% (T3), respectively, versus the RDF control (T2, Supplementary Figures S5A–C). The ash content in wheat grain increased up to 65% (T18-foliar spray) versus the RDF control (T2, Supplementary Figure S6B).

These results demonstrate the efficiency of NPs with PGPR to enhance nutrient content and metabolic pathways in bacteria for their better interaction with plants, leading to the biofortification of wheat in a sustainable manner. Wheat crop growth and yield were increased with the application of NPs (20 ppm and 100 ppm) through foliar application and seed priming. Moreover, wheat biofortification (nutrient content in wheat straw and grains) was achieved with NP treatment in combination with PGPR and correlated with all parameters and other studies (Figure 12; Supplementary Table S2). To explore the feasibility of the technology under field application, these NPs can be used with PGPR as a nanobiofertilizer through the seed treatment method. PGPR performed well in the root region, and their interaction with NPs bolsters plant growth hormones and the activity of bacterial soil enzymes that led to the mobilization of nutrients in the soil, making them available to the plant and biofortifying the crop.