Does Culture Age Affect the Viability of Plant Growth-Promoting Bacteria, Including in Interaction with Environmental Factors? A Case Study on Two Wild Strains from the Durum Wheat Rhizosphere

Plant growth-promoting bacteria (PGPB) could exert positive effects on plant growth and productivity; however, little is known about the effects of variables during the production of PGPB biomass and how they could affect the performances of these microorganisms. This study investigated the effects of pH, temperature, and culture age on the growth of promising PGPBstrains, Bacillus sp. 36M and Stenotrophomonas sp. 20P, isolated from the rhizosphere of durum wheat. A fractional factorial 2k design was applied to evaluate bacterial growth under varying conditions (pH 5.0–7.5; 15–35 °C; 24–72 h precultures). Multifactorial ANOVA revealed that all independent variables and their interactions significantly affected cell concentration (p < 0.05). Bacillus sp. 36M exhibited optimal growth when inoculated from 24 h cultures and incubated under moderate conditions (15 °C, pH 7.5), whereas Stenotrophomonas sp. 20P showed higher viability with 72 h cultures. These results demonstrate that the inoculum physiological state is a critical determinant of PGPB stress tolerance and should be specifically optimized for each strain. The greater technological robustness of Stenotrophomonas sp. 20P suggests its suitability for biofertilizer formulations requiring extended shelf-life or application under variable environmental conditions. In conclusion, this work provides a quantitative framework for tailoring PGPB production protocols to maximize field performance in sustainable agriculture.