Detection of Intracellular Reactive Oxygen Species by Flow Cytometry in Pichia pastoris Fermentation

In Pichia pastoris fermentation, methanol was oxidized into carbon oxide, producing a byproduct, H2O2. This byproduct was one of the partially reduced forms of molecular oxygen known as reactive oxygen species (ROS). ROS are known to highly damage cellular constituents. Flow cytometry (FCM) is an excellent method that permits the rapid, optical analysis of individual cells and has many advantages over conventional cytometry. However, its use in detecting intracellular ROS levels during Pichia fermentation has rarely been reported. In this study, two fluorescent dyes 2′, 7′-dichlorofluorescin diacetate (DCFH-DA) and propidium iodide (PI) were used to detect ROS by the means of FCM. The effect of intracellular ROS on Pichia pastoris cells during fermentation was studied by comparing DCFH-DA/PI double-stained cells and PI single-stained cells. In this study, the loss of cell viability during fermentation was correlated with the accumulation of ROS. A small amount of ROS was accumulated intracellularly at the glycerol batch and the fed-batch phase, and cell viability reached almost 100 %. At the early methanol fed-batch phase, there was intracellular ROS accumulation but 98.5 % of cells were still viable. At the later methanol fed-batch phase, 94.0 % of cells accumulated high ROS. As a result, some cells lost their viability because of the damage to the ROS. Among 29.1 % of the total dead cells, 25.4 % of dead cells accumulated high ROS.