Directed evolution of Gram-negative Klebsiella michiganensis M5al for desiccation tolerance and survivability of a spray-drying encapsulation process

Spray drying is a scalable method for producing dry microbial inoculants, but Gram-negative bacteria often exhibit poor survival under desiccation stress. We employed a directed-evolution approach to improve the survivability of diazotroph Klebsiella michiganensis M5al during spray-drying encapsulation in cross-linked alginate microcapsules (CLAMs). Wildtype K. michiganensis M5al was serially passaged through the CLAMs spray-drying process under incrementally increasing selection pressure. Viable cell density in powders improved over 1000-fold compared to baseline, reaching 3.07 × 10 10 CFU/g over the course of the directed evolution experiment. Paired trials confirmed a statistically significant increase in survival of evolved isolates relative to the parental strain (mean difference of 1.39 log 10 , P < 0.05). Whole-genome sequencing and analysis using breseq identified candidate mutations associated with desiccation tolerance, including a single point mutation in the phosphoenolpyruvate carboxylase gene ( pepC ), suggesting a potential link between central carbon metabolism and stress adaptation. These results demonstrate that genetic adaptation can rapidly enhance microbial fitness under industrial drying conditions without modifying formulation, providing a practical strategy for improving the manufacturability and deployment of Gram-negative microbial biostimulants and related products.