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

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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 H2O2 concentration to areas of higher concentration. Prolonged exposure, up to 72h, 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 H2O2 concentrations in repeated trials. After two progressive exposures, the ability of L. rhamnosus to grow in the presence of H2O2 increased from 1mm H2O2 after a lag time of 31h to 1mm after 21h, 2mm after 28h, and 3mm after 42h. 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.