Customized microscale approach for optimizing two-phase bio-oxidations of alkanes with high reproducibility

Numerous challenges remain to achieve industrially competitive space-time yields for bio-oxidations. The ability to rapidly screen bioconversion reactions for characterization and optimization is of major importance in bioprocess development and biocatalyst selection; studies at conventional lab scale are time consuming and labor intensive with low experimental throughput. The direct ω-oxyfunctionalization of aliphatic alkanes in a regio- and chemoselective manner is efficiently catalyzed by monooxygenases such as the AlkBGT enzyme complex from Pseudomonas putida under mild conditions. However, the adoption of microscale tools for these highly volatile substrates has been hindered by excessive evaporation and material incompatibility.

This study developed and validated a robust high-throughput microwell platform for whole-cell two-liquid phase bio-oxidations of highly volatile n-alkanes. Using microwell plates machined from polytetrafluoroethylene and a sealing clamp, highly reproducible results were achieved with no significant variability such as edge effects determined. A design of experiment approach using a response surface methodology was adopted to systematically characterize the system and identify non-limiting conditions for a whole cell bioconversion of dodecane. Using resting E. coli cells to control cell concentration and reducing the fill volume it is possible to operate in non-limiting conditions with respect to oxygen and glucose whilst achieving relevant total product yields (combining 1-dodecanol, dodecanal and dodecanoic acid) of up to 1.5 mmol g _DCW^-1 .

Overall, the developed microwell plate greatly improves experimental throughput, accelerating the screening procedures specifically for biocatalytic processes in non-conventional media. Its simplicity, robustness and standardization ensure high reliability of results.