Supervisor: Zdenko Machala Consultant: -- Title: Investigation of cold air plasma generation of aqueous reactive oxygen and nitrogen species with focus on their detection and related antibacterial effects Abstract: In this PhD thesis we investigate formation of reactive oxygen and nitrogen species (RONS) in cold air plasma of transient spark activated water (PAW) and buffered solutions by electrospray and their related antibacterial effect. Aqueous reactive species are typically formed due to chemical reactions occurring at the gas plasma-liquid interface. Antibacterial properties of PAW depend on its composition and presence of RONS. Therefore, we investigate their formation in the gas phase, their transfer into the aqueous solutions and particularly we focus on their specific detection in PAW.
DC transient spark discharge was generated in ambient air at atmospheric pressure in combination with water electrospray. The electrospray improved the inter-phase contact and enhanced the transfer of plasma reactive species into the droplets of electrosprayed solutions. The optical emission spectroscopy (OES) of the discharge with electrospray showed a significant number of emission lines: Fe, Fe+, Cr+, Ni+, second positive system of N2, O+, N+, O and N. We observed the cooling effect due to the water electrospray on the gas temperature of the discharge (700 K→ 400 K). Stable gaseous reactive species were identified by Fourier-transform infrared spectroscopy (FTIR), UV absorption and by electrochemical sensors. Transient spark produced mainly nitrogen oxides (NO + NO2) and in the presence of electrospray also other abundant species (H2O2, HNO2, HNO3, •OH, HO2•) were observed. Formation of O3 in other than negligible concentrations was not observed. Formation of aqueous RONS correlated with the production of gaseous reactive species. Detection of aqueous RONS in PAW showed that their formation and life-time depend on the pH. Based on the kinetic chemical analysis we proved formation of peroxynitrous acid, which was determined to be the main antibacterial agent in PAW formed by TS discharge. Highly reactive radicals •OH, HO2•, •NO and •NO2 formed in the PAW initiated the decomposition of the cell membranes via the process of lipoperoxidation and resulted in sublethal injury of the cells. We showed that antibacterial properties of PAW can be preserved by immediate deep freeze and storage at low temperatures (-70°C). Direct treatment of bacterial suspension in PAW resulted in the strong antibacterial effect (i.e. complete sterilization) and these bacteria showed no metabolic activity due to the synergetic effect of RONS induced chemistry, acidic pH and other plasma agents (e.g. UV emission or electric field).
Formation of peroxynitrite/peroxynitrous acid in PAW was proved also by their fluorescent detection. We investigate the suitability and specificity of 2,7-dischlorodihydrofluorescein diacetate (H2DCFDA) fluorescent dye in plasma activated solutions. We demonstrated that in plasma activated solutions without the presence of Cl- and thus prohibiting formation of hypochlorite anions (OCl-) or hypochlorous acid (HOCl), the H2DCFDA fluorescence signal can be attributed primarily to ONOO-/ONOOH formed primary via the reaction of NO2- and H2O2 in PAW. Simple UV-spectroscopic methods for the detection of aqueous NO2- and O3 in PAW were examined for their specificity and possible interferences. We confirmed the accuracy of the widely used Griess colorimetric assay for NO2- detection and we excluded the interference of H2O2 on the assay by using the enzyme catalase. By analysis of the phenol degradation products we confirmed that transient spark did not produce aqueous O3 and thus the Indigo blue assay for dissolved O3 detection is non-specific in PAW and strongly interference with the peroxynitrite chemistry in PAW.
MSc. study Supervisor: Zdenko Machala Consultant: -- Title: Mechanisms of the interaction of plasma with microbial cells and related chemical effects in water Abstract: In this Master thesis we investigated bio-decontamination effects of cold non-equilibrium plasma generated by transient spark discharge in air at atmospheric pressure and chemical changes that were induced by the plasma affecting the water bacterial suspensions. We used a model organism - Gram negative (G-) Escherichia coli bacterial cells that formed a suspension in non-buffered aqueous solutions – saline solution and water (solution of NaH2PO4.2 H2O) with different conductivities. In both cases, we observed 100% decontamination effect 5 hours after plasma treatments. In these solutions, we observed a significant decrease in pH ~ 3 and an increase in conductivity. These chemical changes are caused by the formation of peroxides, nitrites NO2ˉ and nitrates NO3ˉ in water. For comparison, we have also worked with bacterial suspensions prepared in the buffers. We used phosphate buffer (PB) Na2HPO4.2 H2O + KH2PO4 and the same PB in saline solution (PBS). The observed decontamination effect was ~ 2 log immediately after treatment and ~ 4 log 5 hours later. At the same time there was no significant decrease in pH, indicating that the rate of decontamination is associated with the acidification of solutions. Non-equilibrium plasma in the presence of water vapor or water is a rich source of reactive species derived from oxygen (ROS) and nitrogen (RNS), which are responsible for oxidative cell damage and subsequent decontamination. The extent of oxidative damage was evaluated by the concentration c(TBARS) - thiobarbituric acid reactive substances. We found an increased degree of oxidative damage of bacteria in non-buffered aqueous solutions, in which high decontamination was observed. We observed the presence of hydrogen peroxide H2O2 and peroxynitrite ONOO ˉ as reactive species. We detected their increased presence in non-buffered aqueous solutions. They are probably responsible for the oxidative damage and a high decontamination rate in these solutions.
Bc. study 2009 - 2010
Supervisor: Zdenko Machala Consultant: -- Title: Oxidative damage of Gram positive bacteria by non-thermal plasmas Abstract: