Sporicidal and bactericidal efficacy of plasma-treated liquids based on reaction kinetics of peroxynitrous acid

Introduction Plasma-treated aqueous solutions have proven effective for the inactivation of bacteria and even dormant spores. However, reported efficacies vary considerably across setups and experimental conditions. Consequently, different reactive species formed during treatment and their specific reaction kinetics are considered to be responsible. Their individual contribution to microbial inactivation depends on a thorough understanding of the underlying chemical processes. We found that the buffer capacity of an aqueous solution strongly influences the concentrations of reactive species required for effective microbial inactivation. Conversely, the temporal evolution of reactions allows for the optimization of bactericidal and sporicidal efficacy. Methods Using time-resolved in situ UV spectrometry, formation and degradation processes of significant reactive oxygen and nitrogen species (RONS) were observed and analyzed during and after plasma treatment. Results The availability and concentration of peroxynitrous acid (ONOOH) proved crucial for the antimicrobial activity of the liquid. ONOOH generation depends on hydrogen peroxide (H 2 O 2 ) and nitrite (NO 2 – ), both supplied by the plasma exposure, and eventually decays to nitrate (NO 3 – ), which remains in solution. Experimental data showed that liquids with higher buffer capacity accumulated higher concentrations of H 2 O 2 and NO 2 – during plasma exposure, enabling continued ONOOH production even after partial buffer depletion. Concurrently, the solutions acidified progressively. Bacteria, either vegetative cells or dormant spores, were added to the solutions at different time points during the process, and inactivation was monitored in relation to RONS concentrations. The observed antimicrobial efficacy correlated directly with ONOOH concentration, which can be adjusted via the buffer capacity of the medium. This resulted in a 3.83-log 10 reduction of Bacillus atrophaeus spores within 90 min and a 5.78-log 10 reduction of Escherichia coli within 45 min. Discussion Simulations reproduced these experimental trends, confirming three distinct kinetic regimes: a pre-reaction window (before ONOOH formation), a main reaction window (dominated by ONOOH production), and a post-reaction window (defined by decomposition).