Photometric Determination of Ammonium and Phosphate in Seawater Medium Using a Microplate Reader

Abstract

To more efficiently process the large sample numbers for quantitative determination of ammonium (NH₄⁺) and phosphate (orthophosphate, PO₄^3-) generated during comprehensive growth experiments with the marine Roseobacter group member Phaeobacter inhibens DSM 17395, specific colorimetric assays employing a microplate reader (MPR) were established. The NH₄⁺ assay is based on the reaction of NH₄⁺ with hypochlorite and salicylate, yielding a limit of detection of 14 µM, a limit of quantitation of 36 µM, and a linear range for quantitative determination up to 200 µM. The PO₄^3-assay is based on the complex formation of PO₄^3- with ammonium molybdate in the presence of ascorbate and zinc acetate, yielding a limit of detection of 13 µM, a limit of quantitation of 50 µM, and a linear range for quantitative determination up to 1 mM. Both MPR-based assays allowed for fast (significantly lower than 1 h) analysis of 21 samples plus standards for calibration (all measured in triplicates) and showed only low variation across a large collection of biological samples.