## 4 Discussion
### Quantification of the Generated (Reactive) CAP Species
#### 4.1.2 Generation of ROS/RNS
Regarding the effect of the electrode configuration on the intracellular ROS concentration, it can be concluded that the DBD electrode configuration often resulted in significantly higher values in comparison to the SBD electrode. This can also be a consequence of the generally higher dissipated power values resulting in a more efficient generation of different CAP species [17]. In addition, when the SBD electrode configuration is applied, most reactive (short-living) species recombine before they reach the sample. Therefore, fewer species are able to diffuse into the biofilm matrix to eventually interact with the biofilm-associated cells [19; 43]. The effect of the discharge power on the intracellular ROS concentration was not very pronounced as this effect was influenced by the oxygen level and the applied electrode configuration. Finally, it was generally observed that the CAP treatment had a more pronounced effect on the intracellular ROS concentration of the biofilm-associated _Salmonella_ Typhimurium cells than for the corresponding _Listeria monocytogenes_ cells. This can be the result of the difference in biofilm matrix composition, allowing different ROS species to reach the biofilm-associated cells [41], and/or by the Gram type of the cells. It has been reported before that the composition of the cell wall of bacterial cells can have an effect on the ability of CAP species to penetrate into the cells. The thick peptidoglycan layer of Gram-positive cells (such as _Listeria monocytogenes_) still allows the penetration of ROS species, but the diffusion rate is lower as compared to Gram negative cells (such as _Salmonella_ Typhimurium) with a much thinner peptidoglycan layer [44]. The presence of nitrite and/or nitrate in the CAP treated water (Figure 3) indicates that RNS are generated in the gas phase at certain combinations of plasma characteristics since the generation of nitrite and nitrate is the result of nitric oxide (NO) that diffuses into the water [27]. This NO can be generated due to impurities from (i) the environment and/or (ii) the applied helium and oxygen gas. As mentioned before, no clear correlation was observed between the oxygen level of the gas flow and the generation of nitrite. It would be expected, however, that the highest concentrations would be obtained without the addition of oxygen since this resulted in the highest dissipated plasma power (Table 1) and because dissociation of N\({}_{2}\) requires a sufficiently high amount of energy [45]. For the influence of the electrode configuration and the discharge power, this correlation between the dissipated plasma power and the generation of nitrite can indeed be confirmed for most examined treatment combinations. For the influence of the plasma characteristics on the formation of nitrate, again higher concentrations were obtained at the highest dissipated plasma power, i.e., when no oxygen was added to the gas flow, when the DBD electrode was applied, and when the highest discharge power was used. However, similar as for the detection of H\({}_{2}\)O\({}_{2}\), a low concentration of nitrite/nitrate following 10 min of CAP treatment does not necessarily mean that no nitrite/nitrate (and other RNS species) was formed over the course of the treatment (0-10 min). Previous studies indicated that the nitrite concentration first gradually increases as function of the CAP treatment time to decrease again to a relatively low concentration [46; 47]. As mentioned before, the decrease in nitrite is, amongst others, the result of its reaction with hydrogen peroxide (at acidic conditions) to form nitrate and peroxnitrite [39; 40]. Therefore, the study of [PERSON] et al. [46] observed as well that the nitrate concentration increased as function of the CAP treatment time. Though, other pathways can be involved as well for the increase/decrease of nitrite/nitrate at a prolonged CAP treatment time.