Freezing the biotransformation potential of activated sludge in time: towards reproducible incubation experiments for micropollutant risk assessment

The omnipresence, persistence and possible ecotoxicological effects of micropollutants are of major concern for the functioning of ecosystems. In parallel to mitigating micropollutants emissions, designing more sustainable chemicals is consequently of crucial importance. The reliability of biodegradability screening tests in wastewater is nevertheless impeded by their low reproducibility, which arise from the variability of activated sludge (AS) microbial communities combined with the nonexistence of long-term preservation methods for this type of microbiome. To fill this gap, three conservation protocols were developed in the framework of this master thesis: cryopreservation at-80 °C using 5 % DMSO or 10 % glycerol as cryoprotectant and lyophilization with a combination of skimmed milk (12 %) and trehalose (7 %) as lyoprotectant. Their performance at preserving the biotransformation potential of 36 representative micropollutants was assessed after one week of storage in lab-scale batch assays of 72 h with fresh AS as reference. Due to its substantial stresses on viable biomass, lyophilization was the least representative method with more than 85 % of micropollutants being significantly more slowly or rapidly biodegraded than in fresh AS. Cryopreservation using either DMSO or glycerol was more effective with approximately 40 % of micropollutants exhibiting significantly different biotransformation kinetics than in the fresh matrix. Considering the apparent sensitivity of underrepresented microorganisms, the performance of cryopreservation may promisingly be improved by increasing AS biomass density prior to freezing for instance. Regarding the growth medium used for biotransformation assays, artificial wastewater did not appear representative of AS supernatant due to its elevated nutrients content and/or deficiency in extracellular enzymes.