Directed Evolution of Gram-Negative Klebsiella michiganensis M5al for Desiccation Tolerance and Survivability of a Spray Dry 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 dry 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 (greater than three log ₁₀ ) compared to baseline, reaching 3.07 x 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. This work demonstrates that iterative selection under industrial drying conditions can rapidly enhance microbial fitness and provide genomic insights for metabolic and formulation strategies. The approach offers a framework for improving the manufacturability of Gram-negative biostimulants and other microbial products.