Monitoring the kinetics of biocatalytic removal of the endocrine disrupting compound 17α-ethinylestradiol from differently polluted wastewater bodies

Highlights

• 17α-Ethinylestradiol (EE2) was degraded by Horseradish peroxidase (HRP).

• Kinetics for EE2 degradation in artificial wastewater samples of different composition were monitored.

• Different variants of HRP displayed different activity under changing environmental conditions.

• EE2-degradation products differ from those described in literature.

• Outcomes pave the way for future wastewater treatment by immobilized HRP.

Abstract

Horseradish peroxidase (HRP)-mediated degradation of 17α-ethinylestradiol (EE2) in artificial wastewater samples was studied qualitatively (HPLC–MS) and quantitatively (HPLC-UV). Experiments were performed with two different HRP isoenzyme variants either of herbal origin (HRPc) or expressed by recombinant yeasts (C1A_6). Experiments were performed under different pH- and salinity conditions, with and without addition of H₂O₂ at different organic pollution levels mimicked by humic acids. The rapid degradation of the hormone by HRP was demonstrated, and characteristic peaks representing water-soluble degradation products (molar masses 97.2 and 195) were detected and tentatively classified as C5 and C10 compounds. Furthermore, it turned out that H₂O₂ addition has positive impact on EE2 degradation; especially repeated supplementation of small amounts of H₂O₂ resulted in complete EE2 degradation within a reasonable time frame for both isoenzyme variants. Increasing humic acid concentration displayed no major inhibition of HRPc’s biocatalytic activity, in contrast to a significant inhibition of variant C1A_6 at increasing humic acid concentrations. Detailed kinetic data were elaborated for the degradation rate of the target substrate EE2; these data shall be used in future to estimate the degradation performance of HRP under operational conditions during wastewater treatment, e.g., when used as enzyme immobilized on hollow fiber membrane modules.

Introduction

Based on the current progress of global industrialization and the growing need of mankind for food, agro-fertilizers, bulk-chemicals, diverse pharmaceuticals and other fine chemicals, increasing concentrations of various precarious chemicals have been detected in the environment in recent studies [1], [2], [3], [4], [5]. 17α-Ethinylestradiol (EE2), a widely utilized synthetic component in contraceptive pills, is a pre-example of anthropogenic pollutants which are to an increasing extent endangering aquatic environments. Because only an evanescent part of EE2 undergoes catabolic breakdown by the human metabolism, this compound enters the environment via renal excretion [6]. In addition, waste originating from pharma industry constitutes another source of disposed EE2, resulting in an accumulation of EE2 in sewage water plants [1], [7]. When consulting the pertinent literature, it is evident that already low concentrations of EE2 and related hormones in wastewater can have considerable effects on fertility of animals and humans by tremendously interfering with the usual balanced function of the endocrine system [6]. In several countries, the effect of EE2 on fish populations has been studied in details. Researchers found out that some male fish feminize if they are exposed over extended time periods to tiny estrogen concentrations in the ng/L range, as shown e.g., in the case of the zebrafish (Danio rerio) [8]. Studies carried out in Canada report that in the case of the fathead minnow (Pimephales promelas), already 5 ng/L EE2 severely impacts testicle development, causes feminization through vitellogenin mRNA and protein production, and results in intersex in males and disturbed oogenesis in females [9]. Hence, chronic exposure of fish to EE2 adversely impacts the sustainability of aquatic wild populations, thus resulting in a severe disturbance of the gender distribution of fish populations and consequently the ecological equilibrium. Accordingly, considerable economic concerns for aquaculture are provoked, inter alia by suspected extinction of different species [8], [9]. EE2′s chemical structure is shown in Fig. 1.

Literature reports the degradation of EE2 by chemical methods like ozonolysis [10], [11], UV photolysis [12], [13], by the action of microorganisms in activated sludge [14], [15], and by simple exposure to aquatic environments [16]. Reported degradation products are mainly water soluble organic acids like adipic, glutaric, succinic, malonic, or oxalic acid, hence, substances not exceptionally precarious for the ecosphere. A oxidative degradation mechanism, based on the action of ozone, which results in such organic acids is proposed in literature [11].

To an increasing extend, free or immobilized enzymes are applied for deactivation of hazardous compounds [17], [18], [19]. As an enzyme of growing interest, horseradish peroxidase (HRP, EC 1.11.1.7), a representative of the oxidase family, is well-known for its high potential for oxidative conversion of various eco-pollutants like endocrine disruption compounds and a broad range of additional precarious aromatic substances [20], [21], [22]. By analogy with the majority of peroxidases, HRP is characterized by a prosthetic heme moiety, and catalyzes the reduction of peroxides (e.g., H₂O₂) and parallel oxidation of predominantly organic compounds [reviewed in 19]. Only recently, HRP, covalently immobilized on calcium alginate supports, was successfully used in a packed bed bioreactor for detoxification and degradation of the synthetic dyes Red 120, Reactive Blue, and Reactive Orange 16 [19]. It is pivotal to emphasize that different variants of HRP isoenzymes show differently pronounced activity to target substrates and display different optima regarding the process conditions (temperature, pH-value, etc.) [23], [24]. Therefore, it was important to characterize available variants of HRP isoenzymes in order to identify the most suitable one for future application in wastewater treatment. The crystal structure of C-class HRP isoenzymes, as the variants investigated in this study, was for the first time provided in a study by Gajhede et al. [25]. This study gave also evidence that the ring system of three peripheral Phe residues (142, 68 and 179) is responsible for the interaction with aromatic residues like the one present in our target molecule EE2. Later, Shiro and associates discovered the pivotal role of exogenous Ca²⁺ ions in maintaining the protein structure of HRP in the environment of heme and to conserve the spin state of the heme iron, which is decisive for the enzymatic activity of HRP [26].

Aims of the study: Although the effect of pH-value and temperature on the removal of EE2 and related endocrine disrupting compounds by free HRP is reported [20], until now, no literature data are available neither for the detailed mechanism of the enzymatic break down of EE2 and the resulting degradation products, nor for kinetics of HRP-mediated oxidative EE2 breakdown under fluctuating environmental conditions. Convenient, inexpensive and fast methods to monitor the kinetics of EE2 degradation during the process are currently missing; therefore, fast and reliable analytical techniques were urgently needed. In the present study, we used an HPLC-UV system for fast and reproducible EE2 quantitation, enabling to establish degradation kinetics. In this context, we demonstrated the reproducible quantitation of EE2 in a range of 0.1–10 mg/L. In addition, the application of HPLC coupled with a mass spectroscopy (MS) device serving as detector was selected as a sophisticated state-of-the-art technique to separate and detect different substances both qualitatively and quantitatively [9]. These methods constitute strategies to track EE2 in polluted synthetic wastewater samples and could be implemented as alternative to other recent, cost-intensive and/or immature technologies for EE2 quantification, such as microfluidic immunoassay methodology based on anti-EE2 polyclonal antibodies immobilized on magnetic microspheres [27], or the error-prone, highly matrix-dependent and cumbersome determination of EE2 via fluorescence spectroscopy [23]. In the present study, artificial wastewater samples of different composition were subjected to the biocatalytic action of different variants of HRP (native herbal enzyme and recombinant enzyme produced in yeasts as host organism) displaying different performance in EE2 degradation.