Daily Monitoring at a Full-Scale Wastewater Treatment Plant Reveals Temporally Variable Micropollutant Biotransformations

Abstract

Despite decades of micropollutant (MP) monitoring at wastewater treatment plants (WWTPs), we lack a fundamental understanding of the time-varying metabolic processes driving MP biotransformations. To address this knowledge gap, we collected 24-h composite samples from the influent and effluent of the conventional activated sludge (CAS) process at a WWTP over 14 consecutive days. We used liquid chromatography and highresolution mass spectrometry to (i) quantify 184 MPs in the influent and effluent of the CAS process; (ii) characterize temporal dynamics of MP removal and biotransformation rate constants; and (iii) discover biotransformations linked to temporally variable MP biotransformation rate constants. We measured 120 MPs in at least one sample and 66 MPs in every sample. There were 24 MPs exhibiting temporally variable removal throughout the sampling campaign. We used hierarchical clustering analysis to reveal four temporal trends in biotransformation rate constants and found MPs with specific structural features co-located in the four clusters. We screened our HRMS acquisitions for evidence of specific biotransformations linked to structural features among the 24 MPs. Our analyses reveal that alcohol oxidations, monohydroxylations at secondary or tertiary aliphatic carbons, dihydroxylations of vicunsubstituted rings, and monohydroxylations at unsubstituted rings are biotransformations that exhibit variability on daily timescales.

Differential biotransformation of micropollutants in conventional activated sludge and up-flow anaerobic sludge blanket processes

“…We selected 175 MPs for this study (the original mixture contained 184 MPs as previously described, 42 but nine MPs could not be measured due to analytical issues related to their assigned isotope-labelled internal standard). The selected MPs are commonly observed in WWTPs and consist of pharmaceuticals, industrial chemicals, pesticides, human metabolites, and food additives.…”

“…The phenomenon of negative removal has been reported before for some of these MPs 53,54 and is attributed to formation resulting from back-transformation of human metabolites or the MP being a TP itself [56][57][58] or variable biotransformation within a biological process. 42 The remaining 15 MPs exhibited significantly higher removal in the CAS and are presumed to be better removed in the CAS based on this metric (t-test, p < 0.05).…”

“…Upon arrival in the laboratory, subsamples were collected to measure total suspended solids (TSS). 42,43 The wastewater samples were then filtered with glass-fiber filters (grade GF/F, diameter 4.7 cm, pore size 0.7 μm, VWR) to remove suspended solids and subsequently with cellulose acetate filters (diameter 4.7 cm, pore size 0.45 μm, VWR) to generate sample filtrate for solid-phase extraction (SPE). CAS_inf and UASB_inf samples were diluted by a factor of ten in nanopure water.…”

“…More details on our approach to biotransformation product screening is provided elsewhere. 42,50 We identified eight TPs for six of the 14 parent MPs in at least one of the batch experiments. All TPs were identified with a confidence of level 3.…”

Substrate promiscuity of xenobiotic-transforming hydrolases from stream biofilms impacted by treated wastewater

“…Most biofilm studies have been conducted using marker gene sequencing to profile taxonomic composition and community shifts that correlate with TrOC biotransformations in treated wastewater (Carles et al, 2022(Carles et al, , 2021Mansfeldt et al, 2020;Tien et al, 2013). Metagenomics and metatranscriptomics analyses have recently deepened the resolution of community analysis to identify functional shifts in TrOC-exposed engineered wetland biomats, yet the molecular mechanisms of TrOC biotransformations in freshwater biofilms or biomats remain poorly studied relative to other systems such as enriched soil consortia (Vasileiadis et al, 2022) or activated sludge (Rich and Helbling, 2023;Vasileiadis et al, 2022). Studies in activated sludge have additionally correlated microbial taxa, functional genes, and pollutant biotransformations using meta'omic datasets (Achermann et al, 2020;Athanasakoglou and Fenner, 2022;Kennes-Veiga et al, 2022;Stadler et al, 2018).…”