Plant growth-promoting rhizobacteria (PGPR) is a soil bacterium that positively impacts soil and crops. These microbes invade plant roots, promote plant growth, and improve crop yield production. Bacillus subtilis is a type of PGPR with a short shelf-life due to its structural and cellular components, with a non-producing resistance structure (spores). Therefore, optimum formulations must be developed to prolong the bacterial shelf-life by adding humic acid (HA) as an amendment that could benefit the microbes by providing shelter and carbon sources for bacteria. Thus, a study was undertaken to develop a biofertilizer formulation from locally isolated PGPR, using HA as an amendment. Four doses of HA (0, 0.01, 0.05, and 0.1%) were added to tryptic soy broth (TSB) media and inoculated with B. subtilis (UPMB10), Bacillus tequilensis (UPMRB9) and the combination of both strains. The shelf-life was recorded, and viable cells count and optical density were used to determine the bacterial population and growth trend at monthly intervals and endospores detection using the malachite green staining method. After 12 months of incubation, TSB amended with 0.1% HA recorded the highest bacterial population significantly with inoculation of UPMRB9, followed by mixed strains and UPMB10 at 1.8x10⁷ CFUmL^-1, 2.8x10⁷ CFUmL^-1 and 8.9x106 CFUmL^-1, respectively. Results showed that a higher concentration of HA has successfully prolonged the bacterial shelf-life with minimal cell loss. Thus, this study has shown that the optimum concentration of humic acid can extend the bacterial shelf-life and improve the quality of a biofertilizer.

• Al Hamad, B. M., Al Raish, S. M., Ramadan, G. A., Saeed, E. E., Alameri, S. S., Al Senaani, S. S., AbuQamar, S. F., & El-Tarabily, K. A. (2021). Effectiveness of augmentative biological control of Streptomyces griseorubens UAE2 depends on 1-aminocyclopropane-1-carboxylic acid deaminase activity against Neoscytalidium dimidiatum. Journal of Fungi, 7(11), Article 885. https://doi.org/10.3390/jof7110885

• Al Raish, S. M., Saeed, E. E., Alyafei, D. M., El-Tarabily, K. A., & AbuQamar, S. F. (2021). Evaluation of streptomycete actinobacterial isolates as biocontrol agents against royal poinciana stem canker disease caused by the fungal pathogen Neoscytalidium dimidiatum. Biological Control, 164, Article 104783. https://doi.org/10.1016/j.biocontrol.2021.104783

• Alblooshi, A. A., Purayil, G. P., Saeed, E. E., Ramadan, G. A., Tariq, S., Altaee, A. S., El-Tarabily, K. A., & AbuQamar, S. F. (2021). Biocontrol potential of endophytic actinobacteria against Fusarium solani, the causal agent of sudden decline syndrome on date palm in the UAE. Journal of Fungi, 8(1), Article 8. https://doi.org/10.3390/jof8010008

• Ali-Tan, K. Z., Radziah, O., Halimi, M. S., Rahim, K. B. A., Abdullah, M. Z., & Shamsuddin, Z. H. (2017). Growth and yield responses of rice cv. MR219 to rhizobial and plant growth-promoting rhizobacterial inoculations under different fertilizer-N rates. Bangladesh Journal of Botany, 46(1), 481-488.

• Arriel-Elias, M. T., Oliveira, M. I., Silva-Lobo, V. L., Filippi, M. C. C., Babana, A. H., Conceição, E. C., & Cortes, M. D. C. (2018). Shelf-life enhancement of plant growth promoting rhizobacteria using a simple formulation screening method. African Journal of Microbiology Research, 12(5), 115-126. https://doi.org/10.5897/AJMR2017.8787

• Awulachew, M. T. (2021). Food product shelf stability overview of sourdough-risen flatbread. Journal of Food Technology & Nutrition Sciences, 3(3), 1-3. https://doi.org/10.47363/JFTNS/2021(3)123

• Berninger, T., González López, Ó., Bejarano, A., Preininger, C., & Sessitsch, A. (2018). Maintenance and assessment of cell viability in formulation of non‐sporulating bacterial inoculants. Microbial Biotechnology, 11(2), 277-301. https://doi.org/10.1111/1751-7915.12880

• Bhakyaraj, R., Arunkumar, D., & Anitha, A. (Eds.). (2022). Synthetic Microbial Research-Challenge and Prospects. Darshan Publishers.

• Calvo, P., Zebelo, S., McNear, D., Kloepper, J., & Fadamiro, H. (2019). Plant growth-promoting rhizobacteria induce changes in Arabidopsis thaliana gene expression of nitrate and ammonium uptake genes. Journal of Plant Interactions, 14(1), 224-231. https://doi.org/10.1080/17429145.2019.1602887

• Cho, W. I., & Chung, M. S. (2020). Bacillus spores: A review of their properties and inactivation processing technologies. Food Science and Biotechnology, 29(11), 1447-1461. https://doi.org/10.1007/s10068-020-00809-4

• Dehsheikh, A. B., Sourestani, M. M., Zolfaghari, M., & Enayatizamir, N. (2020). Changes in soil microbial activity, essential oil quantity, and quality of Thai basil as response to biofertilizers and humic acid. Journal of Cleaner Production, 256, Article 120439. https://doi.org/10.1016/j.jclepro.2020.120439

• Durga, C. S. S., Ruben, N., Chand, M. S. R., Indira, M., & Venkatesh, C. (2021). Comprehensive microbiological studies on screening bacteria for self-healing concrete. Materialia, 15, Article 101051. https://doi.org/10.1016/j.mtla.2021.101051

• Ekin, Z. (2019). Integrated use of humic acid and plant growth promoting rhizobacteria to ensure higher potato productivity in sustainable agriculture. Sustainability, 11(12), Article 3417. https://doi.org/10.3390/su11123417

• El-Ghamry, A., Mosa, A. A., Alshaal, T., & El-Ramady, H. (2018). Nanofertilizers vs. biofertilizers: New insights. Environment, Biodiversity and Soil Security, 2(2018), 51-72. https://doi.org/10.21608/jenvbs.2018.3880.1029

• El-Tarabily, K. A., AlKhajeh, A. S., Ayyash, M. M., Alnuaimi, L. H., Sham, A., ElBaghdady, K. Z., Tariq, S., & AbuQamar, S. F. (2019). Growth promotion of Salicornia bigelovii by Micromonospora chalcea UAE1, an endophytic 1-aminocyclopropane-1-carboxylic acid deaminase-producing actinobacterial isolate. Frontiers in Microbiology, 10, Article 1694. https://doi.org/10.3389/fmicb.2019.01694

• El-Tarabily, K. A., ElBaghdady, K. Z., AlKhajeh, A. S., Ayyash, M. M., Aljneibi, R. S., El-Keblawy, A., & AbuQamar, S. F. (2020). Polyamine-producing actinobacteria enhance biomass production and seed yield in Salicornia bigelovii. Biology and Fertility of Soils, 56(4), 499-519. https://doi.org/10.1007/s00374-020-01450-3

• El-Tarabily, K. A., Ramadan, G. A., Elbadawi, A. A., Hassan, A. H., Tariq, S., Ghazal, E. W., Gamar, M. I. A., & AbuQamar, S. F. (2021). The marine endophytic polyamine-producing Streptomyces mutabilis UAE1 isolated from extreme niches in the Arabian Gulf promotes the performance of mangrove (Avicennia marina) seedlings under greenhouse conditions. Frontiers in Marine Science, 8, Article 710200. https://doi.org/10.3389/fmars.2021.710200

• Haruta, S., & Kanno, N. (2015). Survivability of microbes in natural environments and their ecological impacts. Microbes and Environments, 30(2), 123-125. https://doi.org/10.1264/jsme2.ME3002rh

• Hong, Z., Chen, W., Rong, X., Cai, P., Tan, W., & Huang, Q. (2015). Effects of humic acid on adhesion of Bacillus subtilis to phyllosilicates and goethite. Chemical Geology, 416, 19-27. https://doi.org/10.1016/j.chemgeo.2015.10.017

• Itelima, J. U., Bang, W. J., Onyimba, I. A., Sila, M. D., & Egbere, O. J. (2018). Bio-fertilizers as key player in enhancing soil fertility and crop productivity: A review. Direct Research Journal of Agriculture and Food Science, 6(3), 73-83.

• Kamil, F. H., Saeed, E. E., El-Tarabily, K. A., & AbuQamar, S. F. (2018). Biological control of mango dieback disease caused by Lasiodiplodia theobromae using streptomycete and non-streptomycete actinobacteria in the United Arab Emirates. Frontiers in Microbiology, 9, Article 829. https://doi.org/10.3389/fmicb.2018.00829

• Kapadia, C., Sayyed, R. Z., El Enshasy, H. A., Vaidya, H., Sharma, D., Patel, N., Abd malek, R., Syed, A., Elgorban, A.M., Ahmad, K., & Zuan, A. T. K. (2021). Halotolerant microbial consortia for sustainable mitigation of salinity stress, growth promotion, and mineral uptake in tomato plants and soil nutrient enrichment. Sustainability, 13(15), Article 8369. https://doi.org/10.3390/su13158369

• Lahlali, R., Ezrari, S., Radouane, N., Kenfaoui, J., Esmaeel, Q., El Hamss, H., Belabess, Z., & Barka, E. A. (2022). Biological control of plant pathogens: A global perspective. Microorganisms 2022, 10(3), Article 596. https://doi.org/10.3390/microorganisms10030596

• Li, X., Liu, H., Yang, W., Sheng, H., Wang, F., Harindintwali, J. D., Herath, H. M. S. K., & Zhang, Y. (2022). Humic acid enhanced pyrene degradation by Mycobacterium sp. NJS-1. Chemosphere, 288(Part 3), Article 132613. https://doi.org/10.1016/j.chemosphere.2021.132613

• Li, Y., Fang, F., Wei, J., Wu, X., Cui, R., Li, G., Zheng, F., & Tan, D. (2019). Humic acid fertilizer improved soil properties and soil microbial diversity of continuous cropping peanut: A three-year experiment. Scientific Reports, 9(1), Article 12014. https://doi.org/10.1038/s41598-019-48620-4

• Lindsay, D., Robertson, R., Fraser, R., Engstrom, S., & Jordan, K. (2021). Heat induced inactivation of microorganisms in milk and dairy products. International Dairy Journal, 121, Article 105096. https://doi.org/10.1016/j.idairyj.2021.105096

• Lipczynska-Kochany, E. (2018). Humic substances, their microbial interactions and effects on biological transformations of organic pollutants in water and soil: A review. Chemosphere, 202, 420-437. https://doi.org/10.1016/j.chemosphere.2018.03.104

• Liu, K., McInroy, J. A., Hu, C. H., & Kloepper, J. W. (2018). Mixtures of plant-growth-promoting rhizobacteria enhance biological control of multiple plant diseases and plant-growth promotion in the presence of pathogens. Plant Disease, 102(1), 67-72. https://doi.org/10.1094/PDIS-04-17-0478-RE

• Luu, J., Mott, C. M., Schreiber, O. R., Giovinco, H. M., Betchen, M., & Carabetta, V. J. (2022). Nε-Lysine acetylation of the histone-like protein HBsu regulates the process of sporulation and affects the resistance properties of Bacillus subtilis spores. Frontiers in Microbiology, 12, Article 782815. https://doi.org/10.3389/fmicb.2021.782815

• Mahalakshmi, S., Vijayapriya, M., & Pandeeswari, N. (2019). Studies on developing PGPR consortium with improved shelf life. Journal of Pharmacognosy Phytochemistry, 8(2S), 545-548.

• Mahapatra, S., Yadav, R., & Ramakrishna, W. (2022). Bacillus subtilis impact on plant growth, soil health and environment: Dr. Jekyll and Mr. Hyde. Journal of Applied Microbiology, 132(5), 3543-3562. https://doi.org/10.1111/jam.15480

• Mathew, B. T., Torky, Y., Amin, A., Mourad, A. H. I., Ayyash, M. M., El-Keblawy, A., Hilal-Alnaqbi, A., AbuQamar, S. F., & El-Tarabily, K. A. (2020). Halotolerant marine rhizosphere-competent actinobacteria promote Salicornia bigelovii growth and seed production using seawater irrigation. Frontiers in Microbiology, 11, Article 552. https://doi.org/10.3389/fmicb.2020.00552

• Mendoza-Labrador, J., Romero-Perdomo, F., Abril, J., Hernández, J. P., Uribe-Vélez, D., & Buitrago, R. B. (2021). Bacillus strains immobilized in alginate macrobeads enhance drought stress adaptation of guinea grass. Rhizosphere, 19, Article 100385. https://doi.org/10.1016/j.rhisph.2021.100385

• Meng, F., Huang, Q., Yuan, G., Cai, Y., & Han, F. X. (2021). The beneficial applications of humic substances in agriculture and soil environments. In New Trends in Removal of Heavy Metals from Industrial Wastewater (pp. 131-160). Elsevier. https://doi.org/10.1016/B978-0-12-822965-1.00007-6

• Morawska, L. P., Hernandez‐Valdes, J. A., & Kuipers, O. P. (2022). Diversity of bet‐hedging strategies in microbial communities-Recent cases and insights. WIREs Mechanisms of Disease, 14(2), Article e1544. https://doi.org/10.1002/wsbm.1544

• Mukherjee, S., Pandey, V., Parvez, A., Qi, X., & Hussain, T. (2022). Bacillus as a Versatile Tool for Crop Improvement and Agro-Industry. In M. T. Islam, M. Rahman & P. Pandey (Eds.), Bacilli in Agrobiotechnology (pp. 429-452). Springer. https://doi.org/10.1007/978-3-030-85465-2_19

• Nagpal, S., Kumawat, K. C., & Sharma, P. (2022). Insights into novel cell immobilized microbial inoculants. In New and Future Developments in Microbial Biotechnology and Bioengineering (pp. 289-318). Elsevier. https://doi.org/10.1016/B978-0-323-85577-8.00001-9

• Nardi, S., Schiavon, M., & Francioso, O. (2021). Chemical structure and biological activity of humic substances define their role as plant growth promoters. Molecules, 26(8), Article 2256. https://doi.org/10.3390/molecules26082256

• Pashang, R., Ronan, E., Kroukamp, O., Korber, D. R., Laursen, A. E., Wenk, J., & Wolfaardt, G. M. (2022). Huddling together to survive: Population density as a survival strategy of non-spore forming bacteria under nutrient starvation and desiccation at solid-air interfaces. Microbiological Research, 258, Article 126997. https://doi.org/10.1016/j.micres.2022.126997

• Połaska, M., Dekowska, A., & Sokolowska, B. (2021). Isolation and identification of guaiacol producing Alicyclobacillus fastidiosus strains from orchards in Poland. Acta Biochimica Polonica, 68(2), 301-307. https://doi.org/10.18388/abp.2020_5574

• Pota, G., Venezia, V., Vitiello, G., Di Donato, P., Mollo, V., Costantini, A., Avossa, J., Nuzzo, A., Piccolo, A., Silvestri, B., & Luciani, G. (2020). Tuning functional behavior of humic acids through interactions with stöber silica nanoparticles. Polymers, 12(4), Article 982. https://doi.org/10.3390/polym12040982

• Pukalchik, M., Kydralieva, K., Yakimenko, O., Fedoseeva, E., & Terekhova, V. (2019). Outlining the potential role of humic products in modifying biological properties of the soil-A review. Frontiers in Environmental Science, 7, Article 80. https://doi.org/10.3389/fenvs.2019.00080

• Rashad, M., Hafez, M., & Popov, A. I. (2022). Humic substances composition and properties as an environmentally sustainable system: A review and way forward to soil conservation. Journal of Plant Nutrition, 45(7), 1072-1122. https://doi.org/10.1080/01904167.2021.2005801

• Rattray, J. E., Chakraborty, A., Li, C., Elizondo, G., John, N., Wong, M., Radovic, J. R., Oldenburg, T. B. P., & Hubert, C. R. (2021). Sensitive quantification of dipicolinic acid from bacterial endospores in soils and sediments. Environmental Microbiology, 23(3), 1397-1406. https://doi.org/10.1111/1462-2920.15343

• Rizvi, A., Ahmed, B., Khan, M. S., El-Beltagi, H. S., Umar, S., & Lee, J. (2022). Bioprospecting plant growth promoting rhizobacteria for enhancing the biological properties and phytochemical composition of medicinally important crops. Molecules, 27(4), Article 1407. https://doi.org/10.3390/molecules27041407

• Saeed, E. E., Sham, A., Salmin, Z., Abdelmowla, Y., Iratni, R., El-Tarabily, K., & AbuQamar, S. (2017). Streptomyces globosus UAE1, a potential effective biocontrol agent for black scorch disease in date palm plantations. Frontiers in Microbiology, 8, Article 1455. https://doi.org/10.3389/fmicb.2017.01455

• Schaeffer, A. B., & Fulton, M. D. (1933). A simplified method of staining endospores. Science, 77(1990), 194-194.

• Shen, C., & Zhang, Y. (2017). Staining technology and bright-field microscope use. In Food microbiology laboratory for the food science student (pp. 9-14). Springer. https://doi.org/10.1007/978-3-319-58371-6_2

• Shultana, R., Kee Zuan, A. T., Yusop, M. R., & Saud, H. M. (2020). Characterization of salt-tolerant plant growth-promoting rhizobacteria and the effect on growth and yield of saline-affected rice. PLoS One, 15(9), Article e0238537. https://doi.org/10.1371/journal.pone.0238537

• Shultana, R., Kee Zuan, A. T., Yusop, M. R., Saud, H. M., & El-Shehawi, A. M. (2021). Bacillus tequilensis strain ‘UPMRB9’improves biochemical attributes and nutrient accumulation in different rice varieties under salinity stress. Plos One, 16(12), Article e0260869. https://doi.org/10.1371/journal.pone.0260869

• Silaban, S., Marika, D. B., & Simorangkir, M. (2020). Isolation and characterization of amylase-producing amylolytic bacteria from rice soil samples. Journal of Physics: Conference Series, 1485(1), Article 012006. https://doi.org/10.1088/1742-6596/1485/1/012006

• Sootahar, M. K., Zeng, X., Wang, Y., Su, S., Soothar, P., Bai, L., Kumar, M., Zhang, Y., Mustafa, A., & Ye, N. (2020). The short-term effects of mineral-and plant-derived fulvic acids on some selected soil properties: improvement in the growth, yield, and mineral nutritional status of wheat (Triticum aestivum L.) under soils of contrasting textures. Plants, 9(2), Article 205. https://doi.org/10.3390/plants9020205

• Stevenson, I. L., & Schnitzer, M. (1982). Transmission electron microscopy of extracted fulvic and humic ACIDS1. Soil Science, 133(3), 179-185.

• Sun, S., Abdellah, Y. A. Y., Miao, L., Wu, B., Ma, T., Wang, Y., Zang, H., Zhao, X., & Li, C. (2022). Impact of microbial inoculants combined with humic acid on the fate of estrogens during pig manure composting under low-temperature conditions. Journal of Hazardous Materials, 424(Part D), Article 127713. https://doi.org/10.1016/j.jhazmat.2021.127713

• Tapia, M. S., Alzamora, S. M., & Chirife, J. (2020). Effects of water activity (aw) on microbial stability: As a hurdle in food preservation. In G. V. Barbosa-Canovas, A. J. Fontana Jr., S. J. Schmidt & T. P. Labuza (Eds.), Water Activity in Foods: Fundamentals and Applications (pp. 323-355). Wiley. http://dx.doi.org/10.1002/9780470376454.ch10

• Tehri, N., Kumar, N., Raghu, H. V., & Vashishth, A. (2018). Biomarkers of bacterial spore germination. Annals of Microbiology, 68, 513-523. https://doi.org/10.1007/s13213-018-1361-z

• Tikhonov, V. V., Yakushev, A. V., Zavgorodnyaya, Y. A., Byzov, B. A., & Demin, V. V. (2010). Effects of humic acids on the growth of bacteria. Eurasian Soil Science, 43(3), 305-313. https://doi.org/10.1134/S1064229310030087

• Timmis, K., & Ramos, J. L. (2021). The soil crisis: The need to treat as a global health problem and the pivotal role of microbes in prophylaxis and therapy. Microbial Biotechnology, 14(3), 769-797. https://doi.org/10.1111/1751-7915.13771

• 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, Article 270. https://doi.org/10.3389/fpls.2020.00270

• Wrangham, J. B. (2019, March 24). Endospore production in response to microbiocides in the microbiologically influenced corrosion implicated genus clostridium. Paper presented at the CORROSION 2019, Nashville, Tennessee, USA.

• Yang, F., Tang, C., & Antonietti, M. (2021). Natural and artificial humic substances to manage minerals, ions, water, and soil microorganisms. Chemical Society Reviews, 50(10), 6221-6239. https://doi.org/10.1039/d0cs01363c

• Young, C. C., Rekha, P. D., Lai, W. A., & Arun, A. B. (2006). Encapsulation of plant growth‐promoting bacteria in alginate beads enriched with humic acid. Biotechnology and Bioengineering, 95(1), 76-83. https://doi.org/10.1002/bit.20957

• Zvinavashe, A. T., Mardad, I., Mhada, M., Kouisni, L., & Marelli, B. (2021). Engineering the plant microenvironment to facilitate plant-growth-promoting microbe association. Journal of Agricultural and Food Chemistry, 69(45), 13270-13285. https://doi.org/10.1021/acs.jafc.1c00138