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

ABSTRACT The viability of microbial inoculants during spray-drying (SD) remains a significant bottleneck for large-scale agricultural application. To overcome this challenge, we developed a strategy that integrates a two-stage cultivation process to induce poly-β-hydroxybutyrate (PHB) accumulation in Azospirillum argentinense Az39 for enhanced cellular resilience with optimization of SD parameters. A central composite design was employed to optimize the drying parameters, including two low-cost co-products, maltodextrin and whey powder concentrations (g L -1 ), and the drying air inlet temperature (°C). PHB content was quantified by direct staining, while flow cytometry based on forward (FSC-H) and side (SSC-H) scatter signals revealed significant treatment-related differences in cell size and granularity, with all pairwise comparisons being significant (adjusted p-values from 2.3 × 10 -9 to < 2.2 × 10 -16 ). Cultures exposed to the two-stage protocol showed higher PHB accumulation, FSC-H, and SSC-H values, indicating greater size and internal complexity associated with enhanced resilience to desiccation stress during SD. Under optimized conditions, a desirability score of 87% was achieved through multi-objective optimization, simultaneously obtaining a survival rate of 99%, a drying yield of 81%, a water activity of 0.33, and a final viable count of approximately 13.5 log 10 CFU g -1 (104.2 °C drying air inlet temperature, 150 g L -1 maltodextrin, and 76 g L -1 whey powder). Bland-Altman analysis supported the agreement between target and observed responses under the selected validation conditions. 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.