Measuring the influence of nutrients and river water on the photosynthetic efficiency of <i>Didymosphenia geminata</i> using pulse amplitude modulated fluorometry
<p>Over the past three decades the freshwater diatom <i>Didymosphenia geminata</i> (Lyngbye) M. Schmidt has expanded its range globally. In some rivers <i>D. geminata</i> has become invasive, forming expansive and thick polysaccharide-dominated mats. Techniques to maintain and study <i>D. geminata</i> in the laboratory are limited. In this study, a 96-well plate format assay using pulse amplitude modulated (PAM) fluorometry was developed to study <i>D. geminata</i> under controlled conditions. The PAM assay and morphological assessments were used to investigate the addition of sodium nitrate (NaNO<sub>3</sub>) to a previously developed <i>D. geminata-</i>specific growth medium (Didymo Medium; DM). Addition of low concentrations (ca. 0.003–0.018 µM) enhanced cell survival and health. Central-composite design (CCD) experiments coupled with response surface methodology were then used to investigate optimal concentrations of six key chemicals in DM (magnesium sulphate, calcium chloride, mono-potassium phosphate, sodium metasilicate pentahydrate, ferric sodium ethylenediaminetetraacetic acid (EDTA) and NaNO<sub>3</sub>). An optimised DM recipe is provided. The PAM assay was also used to analyse the influence of maintaining <i>D. geminata</i> cells in river waters sourced from locations with and without the diatom. A NaNO<sub>3</sub> spiking experiment was undertaken using water from one location. The maximum quantum yields of cells maintained in all river waters remained relatively constant and higher than those maintained in DM or Milli-Q water for the seven day test period. The results of the NaNO<sub>3</sub> river water spiking experiment provided contrasting results to the culture medium trials, with minimal impact on photosynthetic efficiency. These data, coupled with the results of the CCD experiment, suggest complex interactions among nutrients that have varying effects on <i>D. geminata</i> cell health. Together with microscopical observations, the 96-well plate PAM assay provides a useful tool for improving knowledge of <i>D. geminata</i> biology and growth requirements.</p
