Freshwater Phytoplankton Populations Detected Using High Pressure Liquid Chromatography (HPLC) of Taxon-Specific Pigments

Abstract

Phytoplankton are key primary producers in aquatic ecosystems, and the principle food source for primary consumers. Individual phytoplankton species respond to different physical, chemical and biological parameters, so monitoring taxonomic composition of the phytoplankton community is a means to monitor changes in environmental conditions. Phytoplankton community changes have frequently been monitored by estimating biomass (using chlorophyll a, measured fluorometrically), and taxonomic data obtained from cell counts. While such methods are useful, they are time-consuming. I hypothesized that high pressure liquid chromatography (HPLC) methods, which have been frequently used in marine systems, would allow separation and identification of key pigments. These pigments could then be used to allocate chlorophyll a to particular taxa using an optimization algorithm (CHEMTAX), providing simultaneous biomass estimates and taxonomic information. The present study tested the applicability of HPLC/CHEMTAX methods in Lake Michigan phytoplankton communities. During Summer 2008, six synoptic cruises were conducted aboard the R/V Neeskay, sampling communities at nearshore and offshore sites and carrying out grazing and nutrient enrichment experiments. HPLC/CHEMTAX methods were compared with conventional methods over the seasonal cycle from June to August. Estimates of chlorophyll a biomass measured with the HPLC method agreed well with conventional fluorometric measurements of extracted pigments. Chlorophyll a reached maxima (averaging 5.0 μg L-1 nearshore and 1.5 μg L-1 offshore) in late June/early July, with the exception of the offshore metalimnion where chlorophyll a peaked (3.0 μg L-1) in late July. Nutrient enrichment experiments demonstrated that the algal communities were phosphorus-limited, while grazing experiments showed declines in phytoplankton biomass with increasing grazer abundance. Taxonomic groups were consistently misidentified by HPLC/CHEMTAX, relative to microscope methods; confusion between diatoms and chrysophytes was particularly serious. Specific improvement to the HPLC method that might help overcome the problems include faster sample processing techniques to prevent pigment degradation, eluting fewer critical pigments to improve resolution, and use of flow cytometric measurements in parallel. Although the HPLC method is faster and more efficient, it seems unlikely that microscopy to verify the specific taxa within the phytoplankton community can be avoided.