Development of Yeast-Based Measurement Methods for Detecting Bioavailable Nutrients

Preventing eutrophication of inland freshwater ecosystems requires quantifying the excess nutrient (i.e., phosphorus (P) and nitrogen (N)) content of the streams and rivers that feed them. Identification of these at-risk waterways will help prevent harmful algae blooms that make drinking water unsafe. Here we present a novel method for measuring the more relevant bioavailable P (BAP). Where typical methods for measuring P assess soluble reactive P (SRP) or total P (TP) and require expensive analytical techniques that produce hazardous waste, this assay utilizes the growth of familiar baker’s yeast, avoids production of hazardous waste, and reduces cost relative to measurements of SRP and TP. The yeast BAP (yBAP) assay takes advantage of the observation that yeast density at saturating growth increases linearly with provided P. We show that this relationship can be used to measure P in freshwater in concentration ranges relevant to eutrophication. In addition, we measured yBAP in water containing a known amount of fertilizer and in samples from agricultural waterways. We observed that the majority of yBAP values were between those obtained from standard SRP and TP measurements, demonstrating that the assay is compatible with real-world settings. To expand the yBAP assay, we developed two additional derivatives for use with turbid samples that utilize the fact that growing cultures produce the gas carbon dioxide. The first derivative directly measures the released carbon dioxide by measuring pressure build-up in a closed culture chamber. The second derivative indirectly measures carbon dioxide release by measuring pH in the headspace (space above the yeast culture in a closed system) as indicated by the color of a gel containing cresol red suspended above the liquid culture. In either case, the amount of P in a sample can be determined by comparing the observed result to standard curves constructed from solutions of known P concentration. These new variants of the assay allow for the detection of yBAP in turbid (sediment-laden) or soil samples due to their readouts not requiring measurement through the sample matrix itself but rather above it. The final portion of this thesis focuses on applying the same principles of growth to measure the excess nutrient nitrogen, a large component of eutrophication of saltwater systems, using the yeast strain Blastobotrys adeninivorans, which is capable of metabolizing more complex forms of N. In either system the cost-effective and nonhazardous nature of yeast-based assays suggests that they could have utility in a range of settings, offering added insight to identify water systems at risk of eutrophication.