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Algal growth depends strongly on phosphorus (P) as a key nutrient, underscoring the significance of monitoring P levels. Algal species display a sensitive response to fluctuations in P availability, notably through the expression of alkaline phosphatase (AP) when challenged with P-depletion. As such, alkaline phosphatase activity (APA) serves as a valuable metric for P availability, offering insights into how algae utilize and fix available P resources. However, current APA quantification methods lack single cell resolution, while also being time- and reagent consuming. Microfluidics offers a promising cost-effective solution to these limitations, providing a platform for precise single-cell analysis. In this study, a trap-based microfluidic device was integrated with a commercially available AP live stain to study the single cell APA response of a model algae strain, Chlamydomonas reinhardtii, when exposed to different exogenous P levels. A three-step culture-starve-spike process was used to induce APA in cells cultured under two different basal P levels (1 and 21 mM). When challenged with different spiked P levels (ranging from 0.1–41 mM), C. reinhardtii cells demonstrated a highly heterogeneous APA response. Two-way ANOVA confirmed that this response is influenced by both spiked and basal P levels. Utilizing an unsupervised machine learning approach (HDBSCAN), distinct subpopulations of C. reinhardtii cells were identified exhibiting varying levels of APA at the single-cell level. These subpopulations encompass significant groups of individual cells with either notably high or low APA, contributing to the overall behavior of the cohorts. Considerable intrapopulation differences in APA were observed across cohorts with similar average behavior. For instance, while some cohorts exhibited a concentrated distribution around the overall average APA, others displayed subpopulations dispersed across a wider range of APA levels. This underscores the potential bias introduced by analyzing a small number of cells in bulk, which may skew results by overrepresenting extreme behavioral subpopulations. The findings if this study highlight the need for analytical approaches that account for single cell heterogeneity in APA and demonstrate the utility of microfluidics as a well-suited means for such investigations. This study illuminates the complexities of APA regulation at the single cell level, providing crucial insights that advance our understanding of algal phosphorus metabolism and environmental responses. [ABSTRACT FROM AUTHOR]