title: Micropollutant degradation via extracted native enzymes from activated sludge.
abstract: A procedure was developed to assess the biodegradation of micropollutants in cell-free lysates produced from activated sludge of a municipal wastewater treatment plant (WWTP). This proof-of-principle provides the basis for further investigations of micropollutant biodegradation via native enzymes in a solution of reduced complexity, facilitating downstream protein analysis. Differently produced lysates, containing a variety of native enzymes, showed significant enzymatic activities of acid phosphatase, β-galactosidase and β-glucuronidase in conventional colorimetric enzyme assays, whereas heat-deactivated controls did not. To determine the enzymatic activity towards micropollutants, 20 compounds were spiked to the cell-free lysates under aerobic conditions and were monitored via LC-ESI-MS/MS. The micropollutants were selected to span a wide range of different biodegradabilities in conventional activated sludge treatment via distinct primary degradation reactions. Of the 20 spiked micropollutants, 18 could be degraded by intact sludge under assay conditions, while six showed reproducible degradation in the lysates compared to the heat-deactivated negative controls: acetaminophen, N-acetyl-sulfamethoxazole (acetyl-SMX), atenolol, bezafibrate, erythromycin and 10,11-dihydro-10-hydroxycarbamazepine (10-OH-CBZ). The primary biotransformation of the first four compounds can be attributed to amide hydrolysis. However, the observed biotransformations in the lysates were differently influenced by experimental parameters such as sludge pre-treatment and the addition of ammonium sulfate or peptidase inhibitors, suggesting that different hydrolase enzymes were involved in the primary degradation, among them possibly peptidases. Furthermore, the transformation of 10-OH-CBZ to 9-CA-ADIN was caused by a biologically-mediated oxidation, which indicates that in addition to hydrolases further enzyme classes (probably oxidoreductases) are present in the native lysates. Although the full variety of indigenous enzymatic activity of the activated sludge source material could not be restored, experimental modifications, e.g. different lysate filtration, significantly enhanced specific enzyme activities (e.g. >96% removal of the antibiotic erythromycin). Therefore, the approach presented in this study provides the experimental basis for a further elucidation of the enzymatic processes underlying wastewater treatment on the level of native proteins.
authors.affiliation: Federal Institute of Hydrology (BfG), D-56068 Koblenz, Am Mainzer Tor 1, Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Krah
authors.affiliation: Federal Institute of Hydrology (BfG), D-56068 Koblenz, Am Mainzer Tor 1, Germany.
authors.forename: Ann-Kathrin
authors.initials: AK
authors.lastname: Ghattas
authors.affiliation: Federal Institute of Hydrology (BfG), D-56068 Koblenz, Am Mainzer Tor 1, Germany.
authors.forename: Arne
authors.initials: A
authors.lastname: Wick
authors.affiliation: Federal Institute of Hydrology (BfG), D-56068 Koblenz, Am Mainzer Tor 1, Germany.
authors.forename: Kathrin
authors.initials: K
authors.lastname: Bröder
authors.affiliation: Federal Institute of Hydrology (BfG), D-56068 Koblenz, Am Mainzer Tor 1, Germany. Electronic address: ternes@bafg.de.
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Ternes
mesh_terms: D012722:Sewage; D053719:Tandem Mass Spectrometry; D062065:Wastewater; D014874:Water Pollutants, Chemical; D001616:beta-Galactosidase
chemical_list: D012722:Sewage; D062065:Wastewater; D014874:Water Pollutants, Chemical; D001616:beta-Galactosidase
keywords: Activated sludge; Enzyme assay; Micropollutant biodegradation; Native enzymes; Protein extraction