Study of the synergistic effect of singlet oxygen with other plasma-generated ROS in fungi inactivation during water disinfection.

Cold atmospheric plasma (CAP) possesses the ability of high-efficiency disinfection. It is reported that mixtures of reactive oxygen species (ROS) including OH, O, O and HO generated from CAP have better antimicrobial ability than mimicked solution of mixture of single ROS type, but the reason is not clear. In this study, CAP was applied to treat yeasts in water in order to investigate the fungal inactivation efficiency and mechanism. The results showed that plasma treatment for 5 min could result in >2-log reduction of yeast cells, and application of varied ROS scavengers could significantly increase the yeast survival rate, indicating that OH and O played the pivotal role in yeast inactivation. Moreover, the synergistic effect of O with other plasma-generated ROS was revealed. O could diffuse into cells and induce the depolarization of mitochondrial membrane potential (MMP), and different levels of MMP depolarization determined different cell death modes. Mild damage of mitochondria during short-term plasma treatment could lead to apoptosis. For long-term plasma treatment, the cell membrane could be severely damaged by the plasma-generated OH, so a large amount of O could induce more depolarization of MMP, leading to increase of intracellular O and Fe which subsequently caused cell inactivation. O could also induce protein aggregation and increase of RIP1/RIP3 necrosome, leading to necroptosis. With participation of O, endogenous OH could also be generated via Fenton and Haber-Weiss reactions during plasma treatment, which potentiated necroptosis. Adding l-His could mitigate membrane damage, inhibit the drop of MMP and the formation of necrosome, and thus prevent the happening of necroptosis. These findings may deepen the understanding of plasma sterilization mechanisms and provide guidance for microbial killing in the environment.