A high-throughput assay to quantify protein hydrolysis in aerobic and anaerobic wastewater treatment processes

Proteins, an important fraction of the organic matter in wastewater, typically enter a treatment facility as high molecular weight components. These components are degraded by extracellular protein hydrolytic enzymes, denoted as proteases. Adequate protein hydrolysis monitoring is crucial, since protein hydrolysis is often a rate-limiting step in wastewater treatment. However, current monitoring tools lack a high sample throughput and reliable quantification. Here, we present an improved assay for high-throughput protein hydrolysis rate measurements in wastewater treatment applications. A BODIPY FL casein model substrate was implemented in a microplate format for continuous fluorescent quantification. Case studies on a conventional and a high-rate aerobic municipal wastewater treatment plant and a lab-scale, two-stage, anaerobic reactor provided proof-of-concept. The assay presented in this study can help to obtain monitoring-based process insights, which will in turn allow improving biological performance of wastewater treatment installations in the future. KEY POINTS: • Protein hydrolysis is a crucial step in biological wastewater treatment. • Quantification of the protein hydrolysis rate enables in-depth process knowledge. • BODIPY FL casein is a suitable model substrate for a protein hydrolysis assay. • High sample throughput was obtained with fluorescent hydrolysis quantification. Graphical abstract.