a b s t r a c tA procedure was deve

a b s t r a c t
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, bgalactosidase
and b-glucuronidase in conventional colorimetric enzyme assays, whereas heatdeactivated
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-OHCBZ
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.
© 2016 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND