Download A new analytical tool to look at phosphate limitation in mixed

American society for Limnology and Oceanography
February 2010
A new analytical tool to look at phosphate limitation in mixed
assemblages of phytoplankton
Melissa Blakely, Raphael M. Kudela
University of California Santa Cruz, Santa Cruz, CA
Alkaline phosphatase is used as a proxy for determining phosphate limitation in
marine phytoplankton. Phosphate limitation in natural communities of
phytoplankton has previously been determined using two analytical methods:
enzyme labeled fluorescence (ELF) and bulk alkaline-phosphatase activity enzyme
assays. Bulk alkaline phosphatase activity enzyme assays can be used to derive
patterns spatially and temporally for alkaline phosphatase activity (e.g. mapping
and time-series), but does not differentiate between species of marine
phytoplankton. ELF can be used in combination with traditional epifluorescence
microscopy to identify both the algal species and/or heterotrophic bacteria, but is
laborious and time consuming. This study developed a new more streamlined
method for assessing phosphate limitation using ELF-97®, a commercially available
alkaline phosphatase probe. Laboratory cultures of dinoflagellates were grown
under phosphate replete and phosphate deplete conditions and labeled with ELF97®. Cells grown under phosphate deplete conditions were assumed to be 100%
labeled with the alkaline phosphatase probe whereas cells grown under phosphate
replete conditions were assumed to be 0% labeled. Samples with varying
percentages of labeled cells were analyzed using a SpectraMax M2
spectrofluorometer plate reader at an excitation wavelength of 345 nm and an
emission wavelength of 525 nm. When corrected for biomass (using chlorophyll
fluorescence), results exhibited an exponential curve relating fluorescence to
percentage of cells labeled with ELF. The curve was reproducible with both pure
and mixed cultures of different dinoflagellates. The use of this technique to
determine percent abundance of labeled cells would greatly reduce laboratory time
by limiting the requirement for microscopy. Since we estimate biomass (bulk
fluorescence) simultaneously, this method has the potential to provide a relative
index of phosphorous limitation of the entire community. We will present results on
this new method for laboratory samples as well as preliminary results from natural
samples.