Abstract
Two-phase biocatalysis in batch reactions often suffers from inefficient mass transfer, inconsistent reaction conditions, and enzyme inactivation issues. Microfluidics offer uniform and controlled environments ensuring better reproducibility and enable efficient, parallel processing of many small-scale reactions, making biocatalysis more scalable. In particular, the use of microfluidic droplets can increase the interfacial area between the two phases and can therefore also increase reaction rates. For these reasons, slug flow has been extensively used in two-phase biocatalysis in recent years. However, microdroplet flow has been largely neglected for this application despite its great potential. In this work, we performed biphasic biocatalysis in microfluidic droplets, both in microdroplets and slugs, as well as in batch, and systematically investigated the effect of various reaction parameters on the outcome of the reaction. We show that microdroplet flow outperforms the more commonly used batch and slug flow configurations for most reaction conditions by providing shorter substrate diffusion paths and larger interfacial area for the reaction. The potential trade-off between maximized mass transfer and possibly higher enzyme inactivation rates in small droplets with large surface-to-volume ratios was also investigated for the first time, and a pipeline was established to allow evaluation in other reactions. Finally, the effect of surfactant necessary for microdroplet stabilization was also investigated in all reaction setups for the first time, and it was shown that a properly selected surfactant can have a positive effect at low concentrations by creating more stable emulsions and smaller droplets, thus increasing the interfacial area between the two phases.