(190ba) High-Throughput Screening of Alkaline Phosphatase Activity in Single Algal Cells Shows Heterogeneity Under Deviant Phosphorus Conditions

Authors: 
Vaithiyanathan, M., Louisiana State University
Pettigrew, J., Louisiana State University
Dugas, T., Louisiana State University
Kana, Y., Louisiana State University
Nguyen, A., St. Joseph's Academy
Melvin, A., Louisiana State University
Phytoplankton play an essential role in fixing carbon, nitrogen, and phosphorus levels which constitute freshwater and marine ecosystems. The unicellular algal species C. reinhardtii requires phosphorous (P) for growth and typically utilize dissolved orthophosphate. However, water stratification due to climate change and fertilizer influxes can lead to inconsistency in the availability of nutrients like nitrogen (N) and P which can strongly affect algal growth. High N and P levels are a contributing factor for harmful algal blooms (HABs), a natural overgrowth of algal species. As increased P levels have been linked to HABs, it is important to understand how algae utilize P and the link between available P and algal growth. Current approach to quantify P levels rely on bulk measurement of available P; however, these measurements fail to account for how much P is actually processed by algae. A more accurate metric is to measure the activity of the surface enzyme alkaline phosphatase (AP), which is produced when organic P is more plentiful and is responsible for converting organic P to inorganic P to be utilized by algae. Current methods to quantify AP activity are time consuming, require a lot of reagents, compromises ~30-40% of the collected cells, and cannot detect population-based enzyme activity from a single-cell level. The goal of this work was to develop a microfluidic device capable of isolating single algal cells and quantifying AP activity to understand population-based heterogeneity at the single cell level. The microfluidic device utilized sheath flow to direct and trap single C. reinhardtii cells in a 110-member trapping array. COMSOL simulations confirmed the fluid profile to maximize trapping efficiencies (~90%). To mimic real-time influxes of P in water systems, C. reinhardtii were subjected to a 48-h starvation-spiking cycle to induce an AP response. AP activity was visualized in individual cells using a commercially available AP stain and fluorescent microscopy. AP activity was correlated to fluorescence intensity to identify distinct subpopulations of cells with high, intermediate, and low AP activity. Using the device, it was found that the degree of heterogeneity was a function of the available P level, with higher P levels producing greater cellular heterogeneity. A similar degree of heterogeneity was observed in cells exposed to different heavy metals (Cu2+ and Zn2+) that have been shown to diminish AP activity. These findings were finally incorporated into a mathematical model depicting how diversity in the single cell response influences population-based studies in terms of intra/inter-taxonomic and geographical heterogeneity.