Preserving the Biotransformation Potential of Activated Sludge in Time: Toward Reproducible Incubation Experiments for Persistence Assessment

Biotransformation assays conducted in activated sludge (AS) from wastewater treatment plants (WWTPs) offer various benefits, most notably the high microbial density and comparably high bioavailability of the chemicals, enabling short experimental times of 72 h. Moreover, rate constants determined in AS experiments have shown the potential to be used as predictors for half-lives in other environmental compartments, such as soil. Therefore, biotransformation experiments with AS could serve as a valuable basis for developing standardized, high-throughput persistence tests used for screening purposes, e.g., in a benign-by-design framework, if reproducibility of experimental outcomes can be ensured. Here, we tested protocols for the preservation of AS microbial communities using lyophilization or cryopreservation. Their preservation performance was evaluated for 36 representative micropollutants (MPs) in 72 h, lab-scale batch experiments, with fresh AS as a reference. Cryopreservation, using either DMSO or glycerol as a protective agent, preserved the biotransformation potential for most of the MPs (~65%), showing significant deviations in biotransformation kinetics almost exclusively for amine-containing substances. Lyophilization, however, performed worse with over ~89% of MPs exhibiting significantly decreased or enhanced biotransformation compared to fresh AS. We further demonstrate nonsignificant impacts of storage time and the possibility of using artificial instead of preserved native supernatant. Major shifts in community composition based on 16S rRNA gene sequencing results aligned with biotransformation outcomes. Overall, the results suggest that our optimized cryopreservation protocol holds promise to preserve the biotransformation potential of AS and, upon further refinement and testing, might effectively support long-term reproducibility in persistence assessment.