Lipid hydrolysis monitoring in wastewater treatment: proof-of-concept for a high throughput vegetable oil emulsion based assay

Abstract

Lipids are one of the main organic components in industrial and municipal wastewaters. Lipid hydrolysis is the first step in the biological conversion process and requires a close contact between lipid emulsion droplets and microbial hydrolytic enzymes. Adequate lipid hydrolysis monitoring is crucial to obtain mechanistic knowledge on lipid hydrolysis in response to changes in the process conditions and to improve the overall lipid conversion efficiency in aerobic and anaerobic wastewater treatment systems. We set out to develop a high-throughput lipid hydrolysis monitoring method based on vegetable oil model substrates and fluorescent quantification of product formation by exploiting the interaction with Rhodamine B. Olive oil and soybean oil emulsions were prepared with a high interfacial area and acceptable emulsion stability. The method was easy to apply and allowed to obtain detailed kinetic data over a time course of several hours for up to 16 samples in parallel. A proof-of-concept was obtained with a commercial enzyme, Amano lipase, but remains to be provided for wastewater treatment sludge samples. The findings of this study pave the way for further method development in lipid hydrolysis monitoring.

Rhodamine B‐based lipase activity assay with natural substrates—development and application

“…The emission wavelength was kept at 580 nm. In the original workflow by van Gaelen et al . (2021), RB was added to the emulsified substrate before partitioning the solution in wells. In the new workflow as described in 2.2, RB was added directly before the analysis.…”

“…The modifications to the original workflow by van Gaelen et al . (2021) led to an increase in sensitivity, reproducibility and comparability of the procedure. This was achieved by adjusting the excitation wavelength and the addition of the dye directly before measuring.…”

“…The assay was based on Zottig et al (2016) and van Gaelen et al (2021) and modified in order to optimise its sensitivity, repeatability and comparability. The first set-up corresponded to the procedure described by van Gaelen et al: A buffer solution (1.36 g potassium dihydrogenphosphate and 10 g gum arabic in 200 mL distilled water, pH 7.0) was used to dissolve lipases (10 mg/mL) and to emulsify the substrates (25 mg/mL).…”

“…(2016) and by van Gaelen et al . (2021). Rhodamine B can be used to detect released FA independently of the alcohol moiety.…”

“…For this purpose, the fluorescent dye Rhodamine B (RB) was chosen. It was first presented as a tool to detect lipase activity qualitatively in media in 1987 (Kouker & Jaeger, 1987) and first steps towards its use in quantitative assays were undertaken by Zottig et al (2016) and by van Gaelen et al (2021). Rhodamine B can be used to detect released FA independently of the alcohol moiety.…”