Enzymatic post-treatment of ozonation: laccase-mediated removal of the by-products of acetaminophen ozonation

Abstract

Ozonation is a powerful technique to remove micropollutants from wastewater. As chemical oxidation of wastewater comes with the formation of varying, possibly persistent and toxic by-products, post-treatment of the ozonated effluent is routinely suggested. This study explored an enzymatic treatment of ozonation products using the laccase from Trametes versicolor. A high-performance liquid chromatography coupled with high-resolution mass spectrometry (HPLC-HRMS) analysis revealed that the major by-products were effectively degraded by the enzymatic post-treatment. The enzymatic removal of the by-products reduced the ecotoxicity of the ozonation effluent, as monitored by the inhibition of Aliivibrio fischeri. The ecotoxicity was more effectively reduced by enzymatic post-oxidation at pH 7 than at the activity maximum of the laccase at pH 5. A mechanistic HPLC-HRMS and UV/Vis spectroscopic analysis revealed that acidic conditions favored rapid conversion of the phenolic by-products to dead-end products in the absence of nucleophiles. In contrast, the polymerization to harmless insoluble polymers was favored at neutral conditions. Hence, coupling ozonation with laccase-catalyzed post-oxidation at neutral conditions, which are present in wastewater effluents, is suggested as a new resource-efficient method to remove persistent micropollutants while excluding the emission of potentially harmful by-products.

Keywords: Acetaminophen; Laccase; Mass spectrometry; Micropollutants; Organic trace contaminants; Ozonation; Polymerization; Quinone; Toxicity; Wastewater post-treatment.

Fig. 1

Kinetics of APAP ozonation: APAP degradation (A) and formation of transformation products and ecotoxicity as measured by the inhibitory effect on A. fischeri bioluminescence (B). The respective transformation products are indicated as squares (TP 168), triangles (TP 111), and circles (TP 200). Data points represent the mean normalized peak area of the replicate experiments ($\overline{x_{\mathrm{r}}}$). Error bars depict the standard deviation of the replicates (s_r). Each data point was measured at least in triplicate. The inhibitory effect of the APAP solution during the degradation by ozonation on A. fischeri after 15-min contact time is shown as bars. Data points represent the mean value ($\overline{x}$) and error bars the standard deviation (SD) of the inhibition assayed in duplicate

Fig. 2

Degradation of APAP without (unfilled diamonds) and with previous ozone treatment (filled diamonds) at pH 5 (A; orange) and pH 7 (B; blue) at 20 °C by laccase T. versicolor. Data points represent $\overline{x_{\mathrm{r}}}$ ± s_r. Each data point was determined at least in duplicate. APAP degradation after ozonation was analyzed in quadruplicate during the first 4 h

Fig. 3

Degradation of the transformation product TP 168 at pH 5 (A; orange) and pH 7 (B; blue) at 20 °C by laccase of T. versicolor. Data points represent $\overline{x_{\mathrm{r}}}$ ± s_r. Each data point was determined at least in quadruplicate during the first 4 h and at least in duplicate thereafter

Fig. 4

UV–Vis spectroscopic analysis of the degradation of pure TP 168 at 20 °C by laccase of T. versicolor: Time-dependent UV/Vis spectra during laccase treatment at pH 5 (A) and kinetic analysis of the formation of the product with an adsorption maximum of 450 nm at pH 5 (orange) and pH 7 (blue) (B)

Fig. 5

Formation of the secondary transformation product TP² 166 ([M + H]⁺ 166.0518, RT 5.7 min) via the degradation of pure TP 168 by laccase T. versicolor at pH 5 (orange) and pH 7 (blue). Data points represent $\overline{x_{\mathrm{r}}}$ ± s_r. Each data point was determined at least in triplicate

Fig. 6

Formation of multimer products [M + H]⁺ 392.09 (half-filled circles) and [M + H]⁺ 449.10 (circles) during the laccase treatment of pure TP 168 at pH 5 (orange) and pH 7 (blue). Data points represent $\overline{x_{\mathrm{r}}}$ ± s_r. Each data point was determined at least in duplicate

Fig. 7

Schematic representation of the suggested mechanism of laccase-catalyzed degradation of TP 168. First, the semiquinone radical of TP 168 (SQ^•) is formed which can either form oligomers or disproportionate to TP 168 and the quinone TP² 166

Fig. 8

Development of acute toxicity during treatment of ozonated APAP solution at pH 5 (orange bars) and pH 7 (blue bars) with laccase T. versicolor after 5-min contact time with A. fischeri. Data represent $\overline{x}$ ± SD. Each data point was determined in quadruplicate