Cell homogenization and subcellular fractionation in two phytoplanktonic algae: implications for the assessment of metal subcellular distributions
Limnol. Oceanogr. Methods 7:277-286 (2009) | DOI: 10.4319/lom.2009.7.277
ABSTRACT: Metal subcellular distribution in phytoplankton is of interest from both ecotoxicologic and trophic transfer perspectives. Differential centrifugation preceded by cell disruption is frequently used to separate metals in different intracellular compartments. Homogenization efficiency varies widely among species, however, and a quantitative assessment of this parameter is necessary. Moreover, fractions isolated by differential centrifugation remain operationally defined, and confirmation of the nature of these fractions is thus needed. In the present study, homogenization efficiencies for two chlorophytes were evaluated for different methods (sonicator, beadbeater, rotor-stator homogenizer). For the most promising approach (sonication), homogenization efficiency was optimized, using a particle counter, 14C uptake, and growth experiments. The separation efficiency of a subcellular fractionation protocol was also optimized and applied to algae that had been exposed to environmentally relevant concentrations of Cd (0.7 nM Cd2+). The homogenization efficiency could be reliably estimated with the particle counter. Contrasting homogenization efficiencies were obtained for the two test species; virtually all C. reinhardtii cells were easily broken, whereas a large proportion of P. subcapitata cells remained intact (73.2 ± 2.8%). Failure to consider the specific homogenization efficiency for P. subcapitata cells would lead to a greater than threefold underestimate of Cd quotas in the organelle and cytosol fractions.