Spectrally Resolved Cell Imaging for Enhanced Production of ε-Caprolactone via an Enzyme Cascade Reaction in E. coli Immobilized Within Barium–Calcium Alginate Beads Using JetCutter

Jet-cutting—the most powerful immobilization technique—was utilized for the entrapment of recombinant E. coli cells expressing a cascade of enzymes, including alcohol dehydrogenase, enoate reductase, and cyclohexanone monooxygenase, within mechanically reinforced barium–calcium alginate beads. Cost-effective alginate beads with entrapped cells were applied in a model process for the production of the industrially relevant ε-caprolactone under bioreactor-controlled conditions, enabling parallel repeated biotransformations. Immobilization resulted in a reduced rate of cell deactivation over four biotransformation cycles, leading to overall ε-caprolactone yield increases of 36% using 0.55 mm beads and 22% using 0.9 mm beads compared to the use of free cells. Additionally, the model bioprocess was employed to investigate the metabolic adaptation of cells to immobilization and repeated biotransformations using viability assays and spectrally resolved confocal microscopy. These measurements, conducted for the first time throughout the entire cellular life cycle, clearly demonstrated that the cells retained high viability during cultivation, immobilization, and repeated use in biotransformations. Moreover, based on characteristic spectral shifts, advanced analysis via spectrally resolved confocal microscopy revealed distinct mechanisms of metabolic adaptation in entrapped cells versus free cells during repeated cascade reactions in parallel bioreactors.