Enhance the inactivation of fungi by the sequential use of cold atmospheric plasma and plasma-activated water: Synergistic effect and mechanism study

Cold atmospheric plasma (CAP) has been widely applied for microbe inactivation. Previous studies revealed that the inactivation efficiency could be improved with the sequential use of plasma-activated water (PAW) after the direct CAP treatment, while the mechanism requires further exploration. In this work, we employed a CAP jet device and investigated the inactivation effect of CAP combined with PAW treatment on two kinds of fungi (Saccharomyces cerevisiae and Aspergillus flavus), and particularly, we examined the synergistic effect by scrutinizing the roles of varied reactive species. Our experimental results showed that short-term CAP treatment combined with the continued long-term (e.g., 120 min) PAW treatment led to significant reduction of Saccharomyces cerevisiae/Aspergillus flavus, with the disinfection effect equivalent to the single CAP approach with much longer treatment time. The comparative analysis revealed that the inactivation of fungi was induced by different reactive oxygen and nitrogen species (RONS) during the combination of CAP and PAW treatments, which had different influences on the fungal morphology, membrane permeability, intracellular RONS and energy metabolism. In the first stage of CAP treatment, the plasma produced ·OH and 1O2, could directly destroy the structure of fungal wall and membrane, leading to cell death. While in the subsequent PAW treatment, the reactive nitrogen species (RNS) could enter cell and influence metabolic activities, leading inactivation of more fungi. The synergistic effect was attributed to the increasing of intracellular 1O2 and ONOO– induced membrane damage, causing the irreparable oxidative damage to mitochondria and energy metabolic system. This mechanistic study may thus provide a new guidance for the optimal application of low-temperature plasma technology in the application of fungal disinfection with high efficiency.