From Cell Physiology to Process Design: Spray-Drying-Based Production of a Stable and Functional Ensifer meliloti Bioinoculant

The formulation of rhizobial bioinoculants remains a critical bottleneck for the large-scale deployment of biological nitrogen fixation in sustainable agriculture, mainly due to limitations in the stability and viability of conventional liquid products. In this study, a spray-drying-based process was developed and optimized to produce a stable and functional bioinoculant using Ensifer meliloti Rm8530, an EPS II–producing strain with enhanced stress tolerance. Strain robustness was evaluated through thermal and osmotic stress assays, together with growth performance across relevant temperature and pH ranges. Six carrier-based formulations combining polysaccharides and proteins were then tested under controlled spray-drying conditions. Process performance was assessed in terms of powder recovery, residual moisture, bacterial survival, yield, and storage stability over 16 weeks. The morphological integrity of spray-dried particles and rehydrated cells was analyzed by scanning electron microscopy. The biological functionality of selected formulations was subsequently validated in planta using alfalfa as a host model. Among the formulations tested, a mixed alginate–gum Arabic matrix showed the best overall balance between process efficiency, post-drying viability, long-term stability, and symbiotic performance. Spray-dried cells retained the ability to induce nodulation and support early plant responses under the conditions evaluated. These results demonstrate that spray drying, combined with appropriate strain selection and formulation design, constitutes a viable and scalable platform for producing stable, functional rhizobial bioinoculants.