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.

Enzyme response of activated sludge to a mixture of emerging contaminants in continuous exposure

“…The enzyme-containing fractions from AS samples were obtained by sonication method. In brief, ice cooled samples of 10 mL sludge (~0.5 g TSS L -1 ) were individually homogenized using an ultrasonic cell disintegrator Sonics-VibraCell (BioBlock Scientific, France) with a power density of 1 W/mL for 5 (net) min in intervals of 5 s with 5 s breaks to avoid sample heating [37]. The crude cell lysate was subsequently purified through 0.45 μm and 0.20 μm syringe filters (Whatman 25 mm GD/X polyethersulfone membrane with glass microfiber prefilter).…”

Cometabolic Enzymatic Transformation of Organic Micropollutants under Methanogenic Conditions

“…[16][17][18][19][20][21] The action of enzymes on OMPs is poorly investigated, and most studies have focused on oxygenases. [21][22][23] A recent study by Krah et al 24 tested the activity of a cocktail of extracted enzymes from activated sludge towards OMPs. By measuring several TPs and using several enzymatic inhibitors, they concluded that amide hydrolases could be involved in the biotransformation of acetaminophen, acetyl sulfamethoxazole, atenolol and bezafibrate, that oxidoreductases transform 10-OH-carbamazepine and that erythromycin is affected by hydrolases acting on ester bonds.…”

Enzymes as Enhancers for the Biodegradation of Synthetic Polymers in Wastewater

“…Indeed, our results suggest that extracellular hydrolases from typical wastewater microorganisms can hydrolyze polyesters under realistic WWTP conditions. The assumption is further supported by the fact that hydrolases such as esterases, lipases, and proteases are already being utilized successfully to enhance the bioremediation of effluents, and they are, furthermore, used for sludge pretreatment and flocculation reduction in wastewater . Enzymes could be applied in WWTPs both in free form and as immobilized enzymes, and both applications have their advantages and disadvantages.…”

“…[18][19][20][21][22][23] Another approach to regulate and to enhancem icrobial hydrolytic activities in WWTPs is to add enzymef ormulations.I tw as shown in several studies that extracellular enzymes such as esterases, lipases, and proteasesc ould be utilized to enhancethe bioremediation of effluents and for sludge pretreatment and flocculation reductioni nw astewater. [24][25][26][27][28][29][30][31] Microbial lipases have been proven to be effective in reducing fat ando il contents in effluents dischargedb yt he dairy industry,s laughter houses, restaurants, hospitals, and health resorts. [25,32,33] Esterases have provent o play an important role in the biodegradation of many synthetic chemicals by hydrolyzing short-chain esters.…”