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

AMB Express, Feb 2018

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.

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

Hahn et al. AMB Expr Enhanced laccase-mediated transformation of diclofenac and flufenamic acid in the presence of bisphenol A and testing of an enzymatic membrane reactor Veronika Hahn 0 2 Mareike Meister 0 2 Stephan Hussy 1 Arno Cordes 4 Günther Enderle 1 Akuma Saningong 3 Frieder Schauer 0 0 Institute of Microbiology, Ernst-Moritz-Arndt-University Greifswald , Friedrich-Ludwig-Jahn-Str. 15, 17487 Greifswald , Germany 1 Atec Automatisierungstechnik GmbH , Emmi-Noether-Str. 6, 89231 Neu-Ulm , Germany 2 , 73479 Ellwangen , Germany 3 EurA Consult AG , Max-Eyth-Str 4 ASA Spezialenzyme GmbH , Am Exer 19 C, 38302 Wolfenbüttel , Germany 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. Quinone; Bioremediation; Detoxification; Pharmaceuticals; Biodegradation; Vibrio fischeri Introduction The daily use of anti-inflammatory drugs such as diclofenac (DCF) or flufenamic acid (FA) in creams or tablets, as well as of plastic materials which release bisphenol A (BPA), contributes to pollution with these environmentally dangerous compounds. BPA is used in plastic industries; consequently the release from different sources is conceivable such as packages for food and drink, varnishes, paintings and glues (Barnabe et al. 2009) . The main entries for pharmaceuticals such as DCF or FA in ground and surface water are sludge or effluents of wastewater treatment plants (WWTP) where the pollutants are insufficiently degraded. Another source is dung and manure of farm animals which were treated with these substances (Ternes 1998; Deblonde et al. 2011; Samaras et al. 2013; Carmona et al. 2014) . Only 17–69% of DCF is removed in sewage treatment plants (Ternes 1998; Zwiener et al. 2000; Heberer 2002a; Heberer and Feldmann 2005; Deblonde et  al. 2011; Samaras et al. 2013) . FA is also insufficiently removed in WWTP. Moreover, Gracia-Lor et al. (2012 ) and Carmona et al. (2014) described for DCF and FA higher concentrations in the effluent than in the corresponding influent of spanish WWTP. The authors proposed a release as result of a cleavage of transformation products, deconjugation of metabolites and desorption from organic material. DCF, FA and BPA were found repeatedly in surface water. Ternes (1998) reported for DCF a median of 0.15  µg/l and a maximum of 1.20  µg/l for German river and stream water. In Berlin, up to 0.5–1  µg/l and 0.38  µg/l were found in the Teltowkanal and in ground water, respectively (Heberer 2002b) . FA was detected in river and tap water with average concentrations of 21 and 16 ng/l, respectively (Carmona et al. 2014). BPA was also found in surface and ground water (Loos et al. 2009, 2010) . In small streams of the German region called Hessisches Ried BPA concentrations of up to 1.92 µg/l were detected (Quednow and Püttmann 2008). DCF, FA as well as BPA may cause toxic effects. In rainbow trout DCF accumulates in the bile to a factor of approximately 580, and additionally the tested environmentally relevant concentrations caused necrosis in the kidney (Mehinto et  al. 2010) . The endocrine disrupting compound BPA leads to the induction of feminization in aquatic organisms, such as frogs (Levy et  al. 2004; Bhandari et  al. 2015) . Because of the potential risk for human health, DCF was included in a watch list of emerging pollutants by the EU (Commi (...truncated)


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Veronika Hahn, Mareike Meister, Stephan Hussy, Arno Cordes, Günther Enderle, Akuma Saningong, Frieder Schauer. Enhanced laccase-mediated transformation of diclofenac and flufenamic acid in the presence of bisphenol A and testing of an enzymatic membrane reactor, AMB Express, 2018, pp. 28, Volume 8, Issue 1, DOI: 10.1186/s13568-018-0546-y