Application of response surface methodology to study the removal of estrogens in a laccase-mediated continuous membrane reactor

A three-level Box-Behnken factorial design combined with response surface methodology (RSM) was applied as a tool to study the laccase-catalyzed removal of three estrogenic compounds: estrone (E1), estradiol (E2), and ethinylestradiol (EE2), in a continuous enzymatic membrane reactor (EMR). Three main factors affecting the treatment efficiency were considered: enzyme activity, hydraulic residence time (HRT) and oxygenation rate. As expected, laccase activity and HRT showed large effects and, interestingly, the relevance of oxygen in improving the oxidation kinetics through raising the dissolved oxygen above saturation levels was demonstrated. When considering elimination rates as the response, optimal conditions were: 1,000 U/L of laccase, 1 h HRT and 60 mgO(2)/(L.h) of oxygenation rate, predicting 2.82-3.24 mg eliminated/(L.h), (71-81% of oxidation). These optimum conditions were successfully validated, and 75% of estrogenicity reduction was achieved. On the other hand, only 100 U/L were found as optimal to maximize the efficacy of the enzyme: E1 was oxidized by 0.06 mg/(L.h.U), although the removal of estrogenicity decreased to 60%. The methodology was also applied to maximize the reduction of estrogenic activity: the highest values assayed [1,000 U/L, HRT 4 h and 60 mgO(2)/(L.h)] provided 99% detoxification.