Quantification of hydrolysis activity in a biological wastewater treatment context

This paper reviews currently available methods for hydrolysis activity monitoring of the most commonly encountered enzyme categories in biological wastewater treatment. While highlighting the relevant methods for protein, lipid, carbohydrate, organic phosphate, and ester hydrolysis, the discussion of their pros and cons is predominantly aimed at revealing the relevance of the to-be-hydrolyzed substrates that are used in the methods. These "substrates" should mimic the proteins, lipids, or other polymers that are present in the wastewater and are in the reviewed methods (i) real substrates (i.e., naturally present in the wastewater), (ii) chromogenic substrates, or (iii) fluorogenic substrates. We conclude that exploiting relevant substrates such as casein or starch, containing fluorophores, has the highest potential for meaningful high throughput hydrolysis quantification and that lipase activity monitoring is still cumbersome. Monitoring the hydrolysis activity in biological wastewater treatment systems is an underdeveloped area. With this review, which aims at providing a condensed and practice-oriented overview, we hope to facilitate the start or continuation of such monitoring. This monitoring will only grow in importance, given the transition from wastewater treatment plants towards water resource recovery facilities. KEY POINTS: • Colorimetric-based methods are vulnerable to sludge matrix interference. • Bonds in p-nitrophenol-based methods are not representative for the targeted substrates. • Direct methods with relevant/real substrates are preferred. • Fluorophore-containing (real) substrates enable high throughput screening.