A density gradient centrifugation method for rapid separation of nanoTiO₂ and TiO₂ aggregates from microalgal cells...

In natural environment, the microorganisms are exposed to complex mixtures of contaminants, including manufactured nanoparticles and their aggregates. Evaluation of the toxicant accumulation in biota exposed to such cocktails is a challenging task because the microorganisms need to be separated from nanomaterial aggregates often of a comparable size. We propose a method for separation of TiO₂ aggregates from green microalga Chlamydomonas reinhardtii and subsequent determination of cellular Hg concentration in algae exposed to mixture of Hg with nanoTiO₂, known also to adsorb Hg. The method is based on differences in specific weight of algae and TiO₂ aggregates, using medium speed centrifugation on a step gradient of sucrose. The efficiency of the separation method was tested with nanoTiO₂ of three different primary sizes at four concentrations: 2, 20, 100 and 200 mg L^-1. The method gives a possibility to separate nanoTiO₂ and their aggregates from the algae with a mean recovery of 83.3% of algal cells, thus allowing a reliable determination of Hg accumulation by microalgae when co-exposed to Hg and nanoTiO₂. • A rapid and reliable method to separate algal cells and nanoparticle aggregates of comparable size. • A method to measure the cellular amount of Hg in green alga co-exposed to Hg and nanoTiO₂.