Spray-drying is the preferred method for producing dry microbial inoculants for agriculture, offering low cost and easy transport. But many beneficial Gram-negative bacteria—including nitrogen-fixing Klebsiella michiganensis M5al—struggle to survive the rapid dehydration and heat involved. Previous solutions focused on optimizing protective formulations, adding sugars or proteins to shield cells. This study took a different approach: instead of changing the environment, let the bacteria evolve to handle it.
The team serially passaged K. michiganensis M5al through a spray-drying encapsulation system called CLAMs (cross-linked alginate microcapsules). After each drying cycle, surviving cells were collected, regrown, and put through the process again—this time under slightly harsher conditions. Over 16 passages, the population’s survival improved steadily. Early rounds saw a loss of roughly 5 log 10 in viable cells; by the end, that loss had shrunk to less than 1.5 log 10 .
Paired side-by-side trials confirmed the gain was real and significant: evolved isolates consistently outperformed the parent strain by an average of 1.39 log 10 , even as outlet temperatures rose to 80 °C.
To uncover the genetic basis of this newfound toughness, the team sequenced the genomes of evolved populations and compared them to the starting strain using the breseq analysis pipeline. Among 55 candidate mutations, one stood out: a single-base deletion in the pepC gene encoding phosphoenolpyruvate carboxylase, appearing at 100% frequency in the population by the fifth passage. This mutation truncates the PepC protein in half, likely abolishing its enzymatic activity.
PepC catalyzes the irreversible conversion of phosphoenolpyruvate (PEP) to oxaloacetate, a key junction linking glycolysis to the TCA cycle. The researchers hypothesize that losing PepC function alters central carbon metabolism during desiccation—potentially slowing metabolic activity or shifting PEP toward the uptake of compatible solutes that stabilize cellular structures under stress.
The findings offer a practical framework for developing robust microbial products for agriculture, where shelf-stable, easy-to-apply powders are essential. Future studies will investigate the precise mechanism of PepC-related tolerance and explore whether similar adaptations emerge in other drying systems or formulations.
The work entitled “ Directed evolution of Gram-negative Klebsiella michiganensis M5al for desiccation tolerance and survivability of a spray-drying encapsulation process ” was published in Systems Microbiology and Biomanufacturing (published on June 8, 2026).
Systems Microbiology and Biomanufacturing
Experimental study
Not applicable
Directed evolution of Gram-negative Klebsiella michiganensis M5al for desiccation tolerance and survivability of a spray-drying encapsulation process
8-Jun-2026