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

Abstract

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

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Acknowledgments

We gratefully acknowledge Waterschap Brabantse Delta (the Netherlands), Aquafin (Belgium), and Veolia Water Technologies Techno Center B.V. (Biothane, the Netherlands) for providing sludge samples and reactor operational data. We thank Johan Martens and Tom Bosserez for providing access to the Infinite M200 pro microplate reader and Koen Rummens for assistance in statistical analysis.

Funding

Pieter Van Gaelen was supported as doctoral candidate (aspirant) by the Research Foundation-Flanders (FWO–Vlaanderen; file number 28738).

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PVG conceived, designed, and conducted experiments, performed data analysis, and wrote the first draft of the manuscript. DS and IS supervised the project, reviewed the manuscript, and acquired funding. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Ilse Smets.

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The authors declare that they have no conflict of interest.

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Van Gaelen, P., Springael, D. & Smets, I. A high-throughput assay to quantify protein hydrolysis in aerobic and anaerobic wastewater treatment processes. Appl Microbiol Biotechnol 104, 8037–8048 (2020). https://doi.org/10.1007/s00253-020-10751-4

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Keywords

  • Protein
  • Hydrolysis
  • Enzyme activity
  • Fluorescence
  • Wastewater treatment