Adaptive Laboratory Evolution of Probiotics toward Oxidative Stress Using a Microfluidic‐Based Platform

Abstract Adaptive laboratory evolution (ALE) can be used to make bacteria less susceptible to oxidative stress. An alternative to large batch scale ALE cultures is to use microfluidic platforms, which are often more economical and more efficient. Microfluidic ALE platforms have shown promise, but many have suffered from subpar cell passaging mechanisms and poor spatial definition. A new approach is presented using a microfluidic Evolution on a Chip (EVoc) design which progressively drives microbial cells from areas of lower H 2 O 2 concentration to areas of higher concentration. Prolonged exposure, up to 72 h, revealed the survival of adaptive strains of Lacticaseibacillus rhamnosus GG, a beneficial probiotic often included in food products. After performing ALE on this microfluidic platform, the bacteria persisted under high H 2 O 2 concentrations in repeated trials. After two progressive exposures, the ability of L. rhamnosus to grow in the presence of H 2 O 2 increased from 1 m m H 2 O 2 after a lag time of 31 h to 1 m m after 21 h, 2 m m after 28 h, and 3 m m after 42 h. The adaptive strains have different morphology, and gene expression compared to wild type, and genome sequencing revealed a potentially meaningful single nucleotide mutation in the protein omega‐amidase.