Poly-β-hydroxybutyrate accumulation via a two-stage cultivation strategy increases the survival rate of Azospirillum argentinense Az39 during spray-drying using low-cost wall materials

The viability of microbial inoculants during spray drying remains a significant bottleneck for the large-scale agricultural application of these inoculants. To overcome this challenge, we developed a strategy that integrates a two-stage cultivation process, designed to induce poly-β-hydroxybutyrate (PHB) accumulation in the Azospirillum argentinense strain Az39 for enhanced cellular resilience, with optimization of spray-drying parameters. A central composite design was employed to optimize the drying parameters, including two low-cost agro-industrial co-products, maltodextrin and whey powder concentrations (g L−1), and the drying air inlet temperature (° C). PHB content was quantified by direct staining, and cell integrity was assessed by flow cytometry based on forward (FSC-H) and side (SSC-H) scatter signals. Cultures exposed to the two-stage protocol showed higher PHB accumulation, FSC-H, and SSC-H values, indicating improved size and internal complexity, which are crucial for maintaining integrity and enhancing resilience to desiccation stress during spray drying. Under optimized conditions, a desirability score of 87% was achieved, as multi-objective optimization, simultaneously achieving a survival rate of 99%, a drying yield of 81%, and water activity of 0.33 (104.2 °C drying air inlet temperature, 150 g L−1 maltodextrin, 76 g L−1 whey powder). For cell size and granularity, all pairwise comparisons were significant (adjusted p-values from 2.3 × 10−9 to < 2.2 × 10−16). Bland–Altman validation confirmed agreement between predicted and observed values. Our results show that combining PHB-inducing conditions with optimized encapsulation matrices greatly enhances bacterial survival, providing a robust and cost-effective solution for developing spray-dried Azospirillum-based inoculants.