Enhancing Microalgae Cell Inactivation Through Hydrodynamic Cavitation: Insights from Flow Cytometry Analysis

Abstract

Hydrodynamic cavitation (HC) has recently emerged as an effective method for disrupting microalgae cells with lower energy consumption compared to traditional mechanical methods like bead milling and ultrasonication. However, the efficiency and energy utilization of HC can vary depending on the microalgae species and operating conditions. To assess the efficacy of HC and quantify the extent of cellular damage at the cell level, we designed a bench-top cavitation system to investigate the time-dependent responses of the microalgae Dunaliella viridis to multiple HC passes. We evaluated cell disruption efficiency by monitoring cell concentration using a cell counter and analyzing cell size distribution and whole cell counts via flow cytometry. Additionally, we assessed cell viability, metabolic activity, and reactive oxygen species (ROS) levels using the fluorescent probes fluorescein diacetate (FDA), erythrosine B (EB), and 2′,7′-dichlorofluorescein diacetate (DCFDA). We further analyzed cell chlorophyll autofluorescence and the kinetics of overall cell inactivation. Our results demonstrated that HC effectively disrupted and inactivated cells, approximating pseudo-first-order kinetics. However, the cell inactivation rate and energy utilization efficiency declined rapidly in the early stages, likely due to the accumulation of cell debris. A P-factor model incorporating two first-order rate constants was then developed to better predict the cell inactivation kinetics and further demonstrated that cavitation number alone was insufficient to characterize the dynamic change in the inactivation rate during cavitation. To maintain high inactivation efficiency, it is recommended to keep the cell debris fraction below 10-20%. HC was found to inactivate cells by rupturing cell membranes, leading to the rapid release of intracellular contents such as esterase and chlorophyll. HC did not affect intracellular esterase activity or chlorophyll content in cells with intact membranes, but the endogenous ROS levels in viable cells were reduced. The mechanisms of cell damage were discussed in detail. For bioproduct harvesting, cell membrane integrity is suggested as a key physiological endpoint for optimizing HC treatment protocols.