Application of a three-phase fluidized bed electrode for an electro-enzymatic batch process with the peroxygenase AaeUPO

Electrification of biological syntheses offers a sustainable route to decarbonize chemical production. This requires innovative reactor concepts that combine efficient electrochemical reaction steps with selective biocatalysts. Unspecific peroxygenases (UPOs) are attractive because they hydroxylate non-activated Csingle bondH bonds and need only hydrogen peroxide (H₂O₂) as co-substrate. However, excess H₂O₂ rapidly deactivates UPOs. Electrochemical reactors must therefore generate H₂O₂ in-situ at low, uniform levels while maintaining strong mixing and mass transfer — a capability conventional electrochemical cells struggle to provide. Here, we present the first three-phase electrochemical fluidized bed reactor (gas–liquid–solid) for combined electro-enzymatic synthesis. This reactor utilizes conductive graphite particles as a fluidized electrode on which oxygen reduction generates H₂O₂ throughout the particle bed, while air sparging resupplies O₂ and drives mixing, enhancing mass transfer. Using the unspecific peroxygenase from Agrocybe aegerita (AaeUPO) as a model enzyme, we successfully optimized the electrochemical H₂O₂ generation rate to maintain non-inhibitory H2O2 concentrations below 0.2 mM and achieved a high current efficiency of 54.5 ± 12.5% for enzymatic product formation. ... mehrMoreover, the batch process demonstrated scalability, as increasing the enzyme concentration enabled a Y$_{HEBA/EBA}$ of ~100% at even higher H₂O₂ generation rates, resulting in a space-time yield (STY) of 38.83 ± 0.32 g/(L·d) for the hydroxylation of 4-ethylbenzoic acid (EBA) to 4-(1-hydroxyethyl)benzoic acid (HEBA). These results establish the electrochemical fluidized bed reactor as a highly efficient platform for electro-enzymatic synthesis, particularly for processes requiring in-situ co-substrate generation.