Alkaline phosphatase activity of marine bacteria studied with ELF 97 substrate: success and limits in the P-limited Mediterranean Sea

The fluorogenic substrate Enzyme-Labeled Fluorescence 97 (ELF-P) is hydrolyzed by the P-cleaving enzyme phosphatase, producing ELF 97 alcohol (ELFA), a fluorescent-insoluble product. This reaction is used for monitoring phosphatase activity at the single-cell level. Most frequently, ELF-P has been used to determine the P-limitation status of microphytoplankton, but rarely of heterotrophic bacteria. We incubated ELF-P on filters to monitor marine bacterial cultures and oligotrophic Mediterranean Sea samples. Results were compared to classical measurements of bulk alkaline phosphatase activity using the fluorogenic substrate 4-methylumbelliferyl phosphate (MUF-P). A high percentage of the cultured cells were labeled with ELFA (the ratio of ELFA spots to total DAPI counts in P-limited cultures ranged from 26 to 100%, depending on the strain). In contrast, this ratio never exceeded 0.01% in Mediterranean samples, even when P was demonstrated to be a significant limiting factor. This protocol is useful for application on cruises and with cultures, but was not sufficiently sensitive to detect P-stressed bacterial cells in oligotrophic marine environments.