Automated high resolution ectoenzyme measurements: instrument development and deployment in three trophic regimes

Brian M. Gaas,James W. Ammerman

Limnol. Oceanogr. Methods 5:463-473 (2007) | DOI: 10.4319/lom.2007.5.463

ABSTRACT: A commercial flow-injection analysis (FIA) unit was adapted for rapid, automated measurements of ectoenzyme activity. Seawater was injected with a small amount of the substrate 6,8-difluoro-4-methylumbelliferyl phosphate or L-leucine-7-amido-4-methylcoumarin, the hydrolysis products of which were measured fluorometrically in a flow cell. The FIA system was run using sequential injection stopped-flow analysis, allowing activities to be calculated directly from the rate of substrate hydrolysis. The FIA unit sampled autonomously, with the exception of running standard curves and creating new killed controls. The FIA unit was deployed in three environmentally distinct locations – the hypereutrophic Louisiana shelf (July 2004), the oligotrophic Sargasso Sea (February–March 2005), and the eutrophic Hudson River estuary (April 2005). Both phosphatase (Louisiana shelf and Sargasso Sea cruises) and peptidase (Hudson estuary cruise) activities were detectable by the FIA unit. Good correlations were observed between activities measured by the FIA unit and a fluorescence microplate reader. Incubation times ranged from 5–20 min with sampling rates of one sample every 11–26 min. Differences in incubation time, mixing coil length, and detector settings altered the dilution of standards and substrates but did not affect the detection limit of fluorescence. High resolution contour maps and time-series measurements of ectoenzyme activities in eddies, localized upwellings and downwellings, blooms, and river plumes may be used to understand and predict biogeochemical changes in these and other aquatic environments.