Rapid biotransformation and inactivation of subtilisin and α-amylase in wastewater treatment plants Article Type: Research Article

Evanthia Bournaka , Michael Thorsen, Bettina Margrethe Jensen, Torben Madsen

This study quantified the removal and biotransformation of the industrial enzymes
subtilisin and α-amylase in wastewater treatment plants (WWTPs) and activated
sludge systems. Measurements of subtilisin and α-amylase at an industrial WWTP
showed high inlet concentrations and concentrations below quantification limits in the
outlet, corresponding to removals of more than 99% of intact enzymes. Lower
concentrations of the enzymes were found in the inlet to a municipal WWTP at which
the concentrations in the outlet were below the quantification limits. Complementary
laboratory simulation experiments with activated sludge demonstrated extremely rapid
biotransformation of subtilisin and α-amylase under aerobic conditions. More than 70%
of the intact enzyme protein was lost within 0.25 min, and more than 90% within 5 min,
followed by slower continuous transformation to levels below quantification limits within
48 h. The transformation followed first order kinetics with Disappearance Time 50
(DT50) values of less than 10 seconds for both enzymes. In contrast, only minor losses
were observed in abiotic sludge, indicating that microbial degradation is the primary
removal mechanism. Overall, the results show that subtilisin and α-amylase are rapidly
transformed during wastewater treatment, supporting the assumption of very low
environmental concentrations and providing robust data for environmental exposure
modelling.