Enzymatic degradability of diclofenac ozonation products: A mechanistic analysis.

The treatment of waterborne micropollutants, such as diclofenac, presents a significant challenge to wastewater treatment plants due to their incomplete removal by conventional methods. Ozonation is an effective technique for the degradation of micropollutants. However, incomplete oxidation can lead to the formation of ecotoxic by-products that require a subsequent post-treatment step. In this study, we analyze the susceptibility of micropollutant ozonation products to enzymatic digestion with laccase from Trametes versicolor to evaluate the potential of enzymatic treatment as a post-ozonation step. The omnipresent micropollutant diclofenac is used as an example, and the enzymatic degradation kinetics of all 14 detected ozonation products are analyzed by high-performance liquid chromatography coupled with high-resolution mass spectrometry (HPLC-HRMS) and tandem mass spectrometry (MS2). The analysis shows that most of the ozonation products are responsive to chemo-enzymatic treatment but show considerable variation in enzymatic degradation kinetics and efficiencies. Mechanistic investigation of representative transformation products reveals that the hydroxylated aromatic nature of the ozonation products matches the substrate spectrum, facilitating their rapid recognition as substrates by laccase. However, after initiation by laccase, the subsequent chemical pathway of the enzymatically formed radicals determines the global degradability observed in the enzymatic process. Substrates capable of forming stable molecular oxidation products inhibit complete detoxification by oligomerization. This emphasizes that it is not the enzymatic uptake of the substrates but the channelling of the reaction of the substrate radicals towards the oligomerization of the substrate radicals that is the key step in the further development of an enzymatic treatment step for wastewater applications.

Enhanced laccase-mediated transformation of diclofenac and flufenamic acid in the presence of bisphenol A and testing of an enzymatic membrane reactor.

The inadequate removal of pharmaceuticals and other micropollutants in municipal wastewater treatment plants, as evidenced by their detection of these substances in the aquatic environment has led to the need for sustainable remediation strategies. Laccases possess a number of advantages including a broad substrate spectrum. To identify promoting or inhibitory effects of reaction partners in the remediation processes we tested not only single compounds-as has been described in most studies-but also mixtures of pollutants. The reaction of diclofenac (DCF) and flufenamic acid (FA), mediated by Trametes versicolor laccase resulted in the formation of products, which were more hydrophilic than the respective reactant (reactant concentration of 0.1 mM; laccase activity 0.5 U/ml). Analyses (HPLC, LC/MS) showed that the product 1a and 1b for DCF and FA, respectively, to be a para-benzoquinone imine derivative. The formation of 1a was enhanced by the addition of bisphenol A (BPA). After 6 days 97% more product was formed in the mixture of DCF and BPA compared with DCF tested alone. Product 1a was also detected in experiments with micropollutant-supplemented secondary effluent. Within 24 h 67% and 100% of DCF and BPA were transformed, respectively (25 U/ml). Experiments with a membrane reactor (volume 10 l; phosphate buffer, pH 7) were in good agreement with the results of the laboratory scale experiments (50 ml). EC50-values were also determined. The data support the use of laccases for the removal or detoxification of recalcitrant pollutants. Thus, the enzyme laccase may be a component of an additional environmentally friendly process for the treatment stage of wastewater remediation.