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

Abstract

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 × 10¹⁰ 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 log₁₀, 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.

Keywords

Klebsiella michiganensis M5al / Spray drying / Encapsulation / Desiccation tolerance / Directed evolution

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Funding

College of Agricultural and Environmental Sciences, University of California, Davis(Henry A. Jastro Graduate Research Award)

UC Davis DNA Technologies Core(Seed Grant)

Jack Kent Cooke Foundation

Beatrice Oberly and S. Atwood McKeehan Fellowship

Graduate Studies, University of California, Davis

RIGHTS & PERMISSIONS

The Author(s)