Evaluating the Effect of encapsulated Bacillus subtilis Formulations on Viability, Controlled Release, and Bacterial Persistence Under Laboratory Condition

Articles in Press

Document Type : Complete scientific research article

Authors

1 . M.Sc. Student, Dept. of soil science, Gorgan university of Agricultural Sciences and Natural Resources, Gorgan, Iran

2 Corresponding Author, Associate Prof., Dept. of Soil Science, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran

3 Prof, Dept. of Food Science and Technology, Gorgan university of Agricultural Sciences and Natural Resources, Gorgan, Iran.

Abstract

Background and Objectives:

The rapid growth of the global population and increasing demand for agricultural production, coupled with the degradation of soil quality and water resources, have created significant challenges in sustainable agriculture. Therefore, the application of plant growth-promoting bacteria (PGPB) has been considered as a supplement to chemical fertilizers and to reduce their consumption. These bacteria play crucial roles in enhancing plant growth, improving soil fertility, and mitigating environmental stresses. However, their survival under field conditions is often compromised by unfavorable environmental factors such as drought, salinity. This study aimed to evaluate the effectiveness of a bioformulation composed of sodium alginate, bentonite, starch, and nanosilica on capsule stability, viability of encapsulated PGPB and the controlled release of bacterium into the environment.

Materials and Methods:

To conduct this study, a bacterial suspension was first prepared using a nutrient broth medium. Capsules were then produced using the droplet method in a 2% calcium chloride solution using formulations containing sodium alginate, bentonite, starch, and nanosilica. Various physical and biological parameters were assessed, including capsule diameter, expansion rate, loading efficiency, bacterial viability, biodegradability, bacterial release rate, and survival rate of the bacterial cells.

Findings:

The results indicated that incorporating bentonite into the formulation enhanced the mechanical strength of the capsules and reduced their biodegradation rate. Capsules containing both bentonite and starch exhibited less diameter reduction after the drying process than those without these components, indicating greater structural stability. The expansion rate was higher in capsules containing bentonite, suggesting improved water absorption and the formation of a more stable gel matrix. The bacterial cell counts within the capsules were influenced by the formulation type. While higher initial viability was observed in formulations without bentonite (CK and A), a significant decline in the bacterial population was recorded over 90 days. In contrast, formulations containing bentonite and starch demonstrated a slower and more controlled bacterial release and maintained higher viable bacterial populations after 90 days than formulations without these additives and the non-encapsulated control (free cells).

Conclusion:

This study demonstrated that the use of a sodium alginate–bentonite–starch–nanosilica formulation is an effective strategy for enhancing the viability and persistence of plant growth-promoting bacteria under laboratory conditions. Bentonite contributed to improved mechanical strength, reduced biodegradability, and better-controlled bacterial release, while starch served as a protective agent enhancing bacterial survival. Therefore, this technology holds promise in the future as a suitable supplement to chemical fertilizers in agriculture, and play an important role in improving soil fertility and promoting plant growth.

Keywords

Main Subjects

References

  1.  Shabaan, M., Asghar, H. N., Zahir, Z. A., Zhang, X., Sardar, M. F., & Li, H. (2022). Salt-tolerant PGPR confer salt tolerance to maize through enhanced soil biological health, enzymatic activities, nutrient uptake and antioxidant defense.

    1. Saiz-Rubio, V., & Rovira-Más, F. (2020). From smart farming towards agriculture 5.0: A Review on Crop Data Management. Agronomy, 10(2), Article 2. doi.org/10.3390/agronomy10020207
    2. Long, X., Luo, Y., Sun, H., & Tian, G. (2018). Fertilizer using intensity and environmental efficiency for China’s agriculture sector from 1997 to 2014. Natural Hazards, 92(3), 1573–1591. doi.org/10.1007/s11069-018-3265-4
    3. Jalal, A., Filho, M. C. M. T., da Silva, E. C., da Silva Oliveira, C. E., Freitas, L. A., & do Nascimento, V. (2022). Plant Growth-Promoting Bacteria and Nitrogen Fixing Bacteria: Sustainability of Non-legume Crops. In D. K. Maheshwari, R. Dobhal, & S. Dheeman (Eds.), Nitrogen Fixing Bacteria: Sustainable Growth of Non-legumes (pp. 233–275). Springer Nature. doi.org/10.1007/978-981-19-4906-7_11
    4. Hashem, A., Tabassum, B., & Abd_Allah, E. F. (2019). Bacillus subtilis: A plant-growth promoting rhizobacterium that also impacts biotic stress. Saudi journal of biological sciences, 26(6), 1291-1297. doi.org/10.1016/j.sjbs.2019.05.004
    5. Szopa, D., Mielczarek, M., Skrzypczak, D., Izydorczyk, G., Mikula, K., Chojnacka, K., & Witek-Krowiak, A. (2022). Encapsulation efficiency and survival of plant growth-promoting microorganisms in an alginate-based matrix – A systematic review and protocol for a practical approach. Industrial Crops and Products, 181, 114846. doi.org/10.1016/j.indcrop.2022.114846
    6. Rojas-Sánchez, B., Guzmán-Guzmán, P., Morales-Cedeño, L. R., Orozco-Mosqueda, M. del C., Saucedo-Martínez, B. C., Sánchez-Yáñez, J. M., Fadiji, A. E., Babalola, O. O., Glick, B. R., & Santoyo, G. (2022). Bioencapsulation of microbial inoculants: mechanisms, formulation types and application techniques. Applied Biosciences, 1(2), Article 2. doi.org/10.3390/applbiosci1020013
    7. Balla, A., Silini, A., Cherif-Silini, H., Chenari Bouket, A., Alenezi, F. N., & Belbahri, L. (2022). Recent advances in encapsulation techniques of plant growth-promoting microorganisms and their prospects in the sustainable Aagriculture. Applied Sciences, 12(18), Article 18. doi.org/10.3390/app12189020

    9.Simó, G., Fernández‐Fernández, E., Vila‐Crespo, J., Ruipérez, V., & Rodríguez‐Nogales, J. M. (2017). Research progress in coating techniques of alginate gel polymer for cell encapsulation. Carbohydrate Polymers, 170, 1–14. doi.org/10.1016/j.carbpol.2017.04.013

    1. Desoky, E.-S. M., Mansour, E., El-Sobky, E.-S. E. A., Abdul-Hamid, M. I., Taha, T. F., Elakkad, H. A., Arnaout, S. M. A. I., Eid, R. S. M., El-Tarabily, K. A., & Yasin, M. A. T. (2021). Physio-biochemical and agronomic responses of faba beans to exogenously applied nano-silicon under drought stress conditions. Frontiers in Plant Science, 12. doi.org/10.3389/fpls.2021.637783
    2. de Moraes, A. C. P., & Lacava, P. T. (2022). Use of silicon and nano-silicon in agro-biotechnologies. In H. Etesami, A. H. Al Saeedi, H. El-Ramady, M. Fujita, M. Pessarakli, & M. Anwar Hossain (Eds.), Silicon and Nano-silicon in Environmental Stress Management and Crop Quality Improvement (pp. 55–65). Academic Press. doi.org/10.1016/B978-0-323-91225-9.00017-0
    3. Adam, W., Lukacs, Z., Kahle, C., Saha-Möller, C. R., & Schreier, P. (2001). Biocatalytic asymmetric hydroxylation of hydrocarbons by free and immobilized Bacillus megaterium cells. Journal of Molecular Catalysis B: Enzymatic, 11(4), 377–385. doi.org/10.1016/S1381-1177(00)00028-X
    4. Zhang, W., Zheng, L., Lang, D., Zhang, X., Ma, X., Li, X., & Zhang, X. (2023). Eco-friendly bio-encapsulation from sodium alginate-trehalose-kaolin and its performance evaluation in improving plant growth under salt or/and drought conditions. International Journal of Biological Macromolecules, 225, 123–134. doi.org/10.1016/j.ijbiomac.2022.12.009
    5. He, Y., Wu, Z., Tu, L., Han, Y., Zhang, G., & Li, C. (2015). Encapsulation and characterization of slow-release microbial fertilizer from the composites of bentonite and alginate. Applied Clay Science, 109–110, 68–75. doi.org/10.1016/j.clay.2015.02.001
    6. Li, X., Wu, Z., He, Y., Ye, B.-C., & Wang, J. (2017). Preparation and characterization of monodisperse microcapsules with alginate and bentonite via external gelation technique encapsulating Pseudomonas putida Rs-198. Journal of Biomaterials Science, Polymer Edition. doi: 10.1080/09205063.2017.1335075.
    7. Hafezi Ghehestani, M. M., Azari, A., Rahimi, A., Maddah-Hosseini, S., & Ahmadi-Lahijani, M. J. (2021). Bacterial siderophore improves nutrient uptake, leaf physiochemical characteristics, and grain yield of cumin (Cuminum cyminum L.) ecotypes. Journal of Plant Nutrition, 44(12), 1794–1806. doi.org/10.1080/01904167.2021.1884703
    8. Rani, U., & Kumar, V. (2019). Microbial bioformulations: present and future aspects. In R. Prasad, V. Kumar, M. Kumar, & D. Choudhary (Eds.), Nanobiotechnology in Bioformulations (pp. 243–258). Springer International Publishing. doi.org/10.1007/978-3-030-17061-5_10
    9. Brar, S. K., Verma, M., Tyagi, R. D., & Valéro, J. R. (2006). Recent advances in downstream processing and formulations of Bacillus thuringiensis based biopesticides. Process Biochemistry, 41(2), 323–342. doi.org/10.1016/j.procbio.2005.07.015
    10. Bashan, Y., de-Bashan, L. E., Prabhu, S. R., & Hernandez, J.-P. (2014). Advances in plant growth-promoting bacterial inoculant technology: Formulations and practical perspectives (1998–2013). Plant and Soil, 378(1), 1–33. doi.org/10.1007/s11104-013-1956-x
    11. Forni, C., Duca, D., & Glick, B. R. (2017). Mechanisms of plant response to salt and drought stress and their alteration by rhizobacteria. Plant and Soil, 410(1), 335–356. doi.org/10.1007/s11104-016-3007-x
    12. Chaudhary, T., Dixit, M., Gera, R., Shukla, A. K., Prakash, A., Gupta, G., & Shukla, P. (2020). Techniques for improving formulations of bioinoculants. 3 Biotech, 10(5), 199. doi.org/10.1007/s13205-020-02182-9
    13. Brahmaprakash, G. P., Sahu, P. K., Lavanya, G., Gupta, A., Nair, S. S., & Gangaraddi, V. (2020). Role of additives in improving efficiency of bioformulation for plant growth and development. In Frontiers in Soil and Environmental Microbiology. CRC Press.

    23.Wu, Z., Guo, L., Qin, S., & Li, C. (2012). Encapsulation of R. planticola Rs-2 from alginate-starch-bentonite and its controlled release and swelling behavior under simulated soil conditions. Journal of Industrial Microbiology and Biotechnology, 39(2), 317–327. doi.org/10.1007/s10295-011-1028-2

    1. Schoebitz, M., López, M. D., & Roldán, A. (2013). Bioencapsulation of microbial inoculants for better soil–plant fertilization. A review. Agronomy for Sustainable Development, 33(4), 751–765. doi.org/10.1007/s13593-013-0142-0
    2. Magan, N. (2001). Physiological approaches to improving the ecological fitness of fungal biocontrol agents. In Fungi as biocontrol agents: Progress, problems and potential (pp. 239–251). doi.org/10.1079/9780851993560.0239
    3. Schoebitz, M., Simonin, H., & Poncelet, D. (2012). Starch filler and osmoprotectants improve the survival of rhizobacteria in dried alginate beads. Journal of Microencapsulation. doi:10.3109/02652048.2012.665090
    4. Vassilev, N., Vassileva, M., Martos, V., Garcia del Moral, L. F., Kowalska, J., Tylkowski, B., & Malusá, E. (2020). Formulation of microbial inoculants by encapsulation in natural polysaccharides: Focus on beneficial properties of carrier additives and derivatives. Frontiers in Plant Science, 11. doi.org/10.3389/fpls.2020.00270
    5. Nayana, A. R., Joseph, B. J., Jose, A., & Radhakrishnan, E. K. (2020). Nanotechnological advances with PGPR applications. In S. Hayat, J. Pichtel, M. Faizan, & Q. Fariduddin (Eds.), Sustainable Agriculture Reviews 41: Nanotechnology for Plant Growth and Development (pp. 163–180). Springer International Publishing. doi.org/10.1007/978-3-030-33996-8_9
    6. Panichikkal, J., Prathap, G., Nair, R. A., & Krishnankutty, R. E. (2021). Evaluation of plant probiotic performance of Pseudomonas sp. encapsulated in alginate supplemented with salicylic acid and zinc oxide nanoparticles. International Journal of Biological Macromolecules, 166, 138–143. doi.org/10.1016/j.ijbiomac.2020.10.110
    7. Saberi-Rise, R., & Moradi-Pour, M. (2020). The effect of Bacillus subtilis Vru1 encapsulated in alginate – bentonite coating enriched with titanium nanoparticles against Rhizoctonia solani on bean. International Journal of Biological Macromolecules, 152, 1089–1097. doi.org/10.1016/j.ijbiomac.2019.10.197
Journal of Soil Management and Sustainable Production

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Available Online from 16 November 2025

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