Motility-based viability assay for screening acid-tolerant microalgae

Motility of self-propelled microorganisms is a clear indicator of survival under acidic stress, enabling identification of acid-tolerant strains. Microalgae hold promise for carbon capture and storage (CCS) owing to their photosynthetic ability to convert carbon dioxide into biomass. However, in sequential chemical-biological CCS, chemical absorption occurs before microalgal inoculation; consequently, the resulting CO₂-rich, low-pH solution can markedly reduce cell survival during biological conversion, indicating the need for acid-tolerant strains to improve process efficiency. Here, this study presents a rapid, label-free viability assay that identifies acid-tolerant microalgal strains by tracking their motility under acidic stress. Mutant microalgae were cultured under acidic stress, and their motility was assessed using the proposed computer vision algorithm. This algorithm selectively identifies live motile cells while excluding non-motile cells and debris commonly found in culture media, eliminating the need for fluorescence staining. Mutant groups exhibiting higher fractions of motile microalgae at early cultivation stages showed greater proliferation during subsequent cultivation, validating early motility as a predictor of acid tolerance. By determining which groups exhibit the highest motile fractions, this method identifies the most acid-tolerant strains within one hour, unlike conventional methods that require several days. Furthermore, the proposed stochastic-kinetics model quantified acid tolerance through the cell motility-loss rate, providing a measure of enhanced resistance to acidic stress compared with the wild type. Therefore, this assay provides a practical solution for screening acid-tolerant microalgal strains and quantitatively evaluating their resistance to acidic stress. This work also highlights broader applicability to label-free cytotoxicity evaluations across diverse motile microorganisms.