Measurement Of Phosphatase Activity In Activated Sludge Using Fluorogenic Substrates

Van Ommen Kloeke and Geesey41 measured PO4ase activity in activated sludge samples using the soluble, artificial, fluorogenic enzyme substrate methylumbelliferyl phosphate (MUFP) (Figure 14.1). MUFP has been used to monitor phosphomonoes-terase activity in lakes.42,43 MUF substrates are likely hydrolyzed by PO4ases either at or near the surface of intact cells or by PO4ases released into the environment by cells.31 After an appropriate incubation period, the reaction mixture was centrifuged to remove interfering particulate material and the supernatant fraction assayed for fluorescence intensity using a spectrofluorometer at an excitation wavelength of 360 nm and emission wavelength at 430 nm. This substrate yielded quantitative PO4ase activity in various activated sludge fractions at relatively high sensitivity. Using freshly collected activated sludge samples from the wastewater treatment plant in Bozeman, MT, which contained 2.9 ± 0.3 g l-1 total solids and 7 to 14 mg l-1 Pi (Table 14.2), it was determined that the particulate fraction (operationally defined as that which sed-imented by centrifugation at 27,000 x g for 15 min) exhibited linear PO4ase activity at pH 7.0 of 7.9 ¡¡moll-1 h-1, or 91% of the total activity in "as collected" samples at MUFP final concentrations of 100 ¡M over a 3 to 4-h period (Figure 14.2). At a final MUFP concentration of 500 ¡M, the PO4ase activity was approximately twice that observed at the 100 ¡M final MUFP concentration, and the activity associated with the particulate fraction was slightly greater than that associated with the "as collected" sample. A Lineweaver Burke plot of reaction rates over a range of added

4-MUF-PO4

Soluble

Blue fluorescence

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