Study of Serndipita indica and Sinorhizobium meliloti on concentration and kinetics of zinc release in alfalfa rhizosphere contaminated with zinc oxide nanoparticles

Background and Objectives: The application of nano-fertilizers, such as zinc oxide nanoparticles (ZnO-NPs), is an emerging strategy to improve micronutrient efficiency in sustainable agriculture. However, the overuse of ZnO-NPs, particularly in calcareous soils with low zinc availability, risks soil contamination and subsequent toxicity to plants and soil biota. The bioavailability of zinc from these nanoparticles is governed by complex interactions within the rhizosphere, involving the soil matrix, plant roots, and plant growth-promoting microorganisms (PGPMs). While PGPMs like fungi and bacteria can alter metal bioavailability, their combined effects and the resulting release dynamics are not fully understood. Therefore, this study aimed to investigate the effect of inoculating the fungus Serendipita indica and the bacterium Sinorhizobium meliloti, both individually and in combination, on the release kinetics of zinc in the rhizosphere of alfalfa (Medicago sativa L.) grown in soil contaminated with different levels of ZnO-NPs.




Materials and Methods: This study was conducted as a factorial experiment based on a completely randomized design with three replications on alfalfa (Medicago sativa L.) cv. Hamedani in a greenhouse. The soil used was a loamy-clay collected from the 0-30 cm layer of Chitgaran station in Shiraz, Iran, with a pH of 8.3, 0.71% organic matter, and 42.5% calcium carbonate equivalent. The soil was autoclaved at 121°C for 25 minutes before use. Treatments included three levels (0, 400, and 800 mg kg⁻¹ soil) of ZnO-NPs (average diameter of 10 nm, sourced from Pishgaman Nano Mavadd-e Iran Co.) and four levels of microbial inoculation (a non-inoculated control, S. indica alone, S. meliloti alone, and co-inoculation of the fungus and bacterium). Soils were incubated for three months at field capacity to allow for equilibration reactions. The S. indica inoculum was prepared by collecting spores from a 4-week-old culture, with the final concentration adjusted to 5×10⁵ spores mL⁻¹. The S. meliloti strain, selected for its high N-fixation and PGP traits (including possession of nfe, putA, and acdS genes), was cultured for 48 hours, and the inoculum was adjusted to 5×10⁷ cells mL⁻¹. In order to find the best model to describe the zinc release pattern, time-dependent release data were fitted to nine kinetic equations (zero-, first-, second-, third-order, parabolic diffusion, power function, simplified Elovich, pseudo-first-order, and pseudo-second-order). For each experimental treatment, the best kinetic equation was selected based on the highest coefficient of determination (R²) and the lowest standard error (SE). Zinc concentrations were measured using an atomic absorption spectrophotometer (Shimadzu AA 670). Statistical analysis was performed using SAS 9.1, and means were compared using the LSD test at P≤0.05.
Results: The results showed that co-inoculation of the fungus and bacterium at 400 and 800 mg Zn kg⁻¹ had the lowest rhizosphere pH value, resulting in a decrease of 15.25% and 6.81%, respectively, compared to the zero-Zn level. Also, at the 800 mg Zn kg⁻¹ level, inoculation with the fungus alone and co-inoculation with the bacterium were equally effective, showing the highest ability to release zinc from the rhizosphere soil; these treatments led to an increase of 27.79% and 26.42%, respectively, in the amount of cumulative zinc released compared to the uninoculated condition. The study of the zinc release pattern under the influence of different zinc levels and microbial inoculation showed that the process of zinc release in all treatments followed a two-stage kinetic process that starts with a fast release step and then reaches equilibrium after a slow step. In the initial rapid phase, zinc release corresponds to mobile forms with low bond energy, and in the second stage, to forms with less mobility. This was confirmed by the observation that approximately 68-76% of the total desorbed zinc was released within the first two hours of extraction. Analysis of the kinetic models showed that the pseudo-second-order (R² > 0.995) and power function (R² > 0.94) equations provided the best fit for the zinc release data in all experimental treatments, while the simplified Elovich equation was also suitable only at the control (zero Zn) level. The superiority of the pseudo-second-order model was further validated by the positive and significant correlation between its calculated equilibrium zinc concentration (qe) and the plant-available zinc concentrations (extracted by DTPA in 2 hours), as well as the zinc concentrations in the root and shoot tissues of the alfalfa plants.
 




Conclusion: The present study demonstrated that microbial inoculation, particularly co-inoculation with S. indica and S. meliloti, is an effective strategy for acidifying the rhizosphere and increasing the release of zinc from soil contaminated with ZnO-NPs. The highest efficacy was observed at high contamination levels, where fungal inoculation (alone or combined) enhanced cumulative zinc release by approximately 27%. The kinetics of zinc release in the calcareous soil followed a two-stage process, dominated by an initial rapid release of weakly bound zinc forms. Among the nine models tested, the pseudo-second-order kinetic model proved to be the most robust descriptor of the zinc release process across all treatments, which was confirmed by its strong correlation with plant-available zinc. These findings highlight the potential of using synergistic microbial partnerships to manipulate the bioavailability of nanoparticle-derived contaminants in the rhizosphere and underscore the importance of kinetic modeling in predicting nutrient and contaminant release patterns in complex soil systems.