ena-STUDY-EAWAG-08-08-2017-12:13:19:282-1883
The influence of sludge retention time on the  expressed functional diversity of and biotransformation rates of micropollutants performed by a community originating from wastewater activated sludge
Reactor operation and SRT experiments:Six fully automated sequencing batch reactors (6 x 12 L) were operated at solids retention times of 1, 3, 5, 7, 10 and 15 days with local wastewater. At two different time points (experiment_1 and experiment_2) during reactor operation, a selection of micropollutants was spiked into the reactors (final concentration: 6 µg/L), and, using liquid chromatography coupled to high resolution mass spectrometry, chemical concentrations were determined and pseudo first-order biotransformation rate constants calculated. Additionally, activated sludge samples were collected for nucleic acid extraction and sequencing.Protocol RNA extraction (adapted from Johnson et al. 2014):Activated sludge from the six reactors was collected 5 hours after start of the experiments (addition of micropollutants), immediately frozen using liquid nitrogen and stored at -80 °C until further analysis (after not more than 16 months of storage). RNA from 4 mL of AS was extracted using phenol chloroform and precipitated as described elsewhere (Johnson, D.R., et al., Appl Environ Microbiol, 2005. 71(11): p. 7145-51.). Following the manufacturer's instructions, the RNA was purified using RNA pro clean up kit (Mo Bio) and remaining DNA was removed using TURBO DNase (Thermo Fisher Scientific). After extraction and after DNase treatment, the obtained RNA was analyzed using Qubit (Thermo Fisher Scientific) and Bioanalyzer (Agilent).The data provided have been quality checked using FastQC, trimmed with Trimmomatic, and filtered of rRNA reads using SortMeRNA.
Reactor operation and SRT experiments:Six fully automated sequencing batch reactors (6 x 12 L) were operated at solids retention times of 1, 3, 5, 7, 10 and 15 days with local wastewater. At two different time points (experiment_1 and experiment_2) during reactor operation, a selection of micropollutants was spiked into the reactors (final concentration: 6 µg/L), and, using liquid chromatography coupled to high resolution mass spectrometry, chemical concentrations were determined and pseudo first-order biotransformation rate constants calculated. Additionally, activated sludge samples were collected for nucleic acid extraction and sequencing.Protocol RNA extraction (adapted from Johnson et al. 2014):Activated sludge from the six reactors was collected 5 hours after start of the experiments (addition of micropollutants), immediately frozen using liquid nitrogen and stored at -80 °C until further analysis (after not more than 16 months of storage). RNA from 4 mL of AS was extracted using phenol chloroform and precipitated as described elsewhere (Johnson, D.R., et al., Appl Environ Microbiol, 2005. 71(11): p. 7145-51.). Following the manufacturer's instructions, the RNA was purified using RNA pro clean up kit (Mo Bio) and remaining DNA was removed using TURBO DNase (Thermo Fisher Scientific). After extraction and after DNase treatment, the obtained RNA was analyzed using Qubit (Thermo Fisher Scientific) and Bioanalyzer (Agilent).The data provided have been quality checked using FastQC, trimmed with Trimmomatic, and filtered of rRNA reads using SortMeRNA.