## 4 Discussion
### Effect of the Generated (Reactive) CAP Species on the Biofilms
#### 4.2.2 Membrane Integrity  
As mentioned before, CAP treatment resulted in DNA leakage (Figure 7) for both biofilm forming species and for almost all examined combinations of plasma characteristics. As a consequence, it can be concluded that CAP treatment has an effect on the membrane of the biofilm-associated cells. Based on previously discussed results (Section 4.1), this can be attributed to the generation of (intracellular) ROS, the drop in pH, and the formation of RNS. In literature, it has been reported that ROS can interact with the peptidoglycan of the cell wall and with the lipopolysaccharide and phospholipids present in the (outer) cell membranes [28]. The lipids can be oxidized and C-C, C-O, and C-N bonds within the peptidoglycan structure can be destroyed due to the presence of ROS such as OH and O [14]. The drop in pH, on the other hand, can result in cell leakage due to the neutralization of the net negative charge of the bacterial cell wall [60]. The leakage of intracellular DNA was, however, often higher when no oxygen was added to the gas flow. This can originate from the higher dissipated plasma power and the lower quenching rate of the reactive species. However, it can indicate that other reactive species such as RNS can be involved as well. This is in agreement with the results observed for the nitrite and nitrate concentrations, i.e., higher total concentrations were often measured following 10 min of CAP treatment when no oxygen was added to the gas flow, which might be similar for all other (unmeasured) RNS species. However, these results should be interpreted with caution as the nitrite/nitrate concentration can change as a function of the CAP treatment time. Regarding the influence of the electrode configuration, higher absorbance values were often observed when the DBD electrode was applied. This higher membrane damage can originate from (i) the higher amount of (intracellular) ROS, (ii) the higher total amount of nitrite and nitrate, and (iii) the lower pH value. Moreover, it indicates too that charged particles can contribute to the observed membrane damage since these charged species can only reach the biofilm samples when a direct treatment is applied [61]. Finally, for the influence of the power discharge, more membrane damage can be obtained at the highest value, which is in agreement with previous studies investigating the influence of the plasma power on CAP's inactivation efficacy (e.g., [28]). This increase in membrane damage (Figure 7) can be correlated to a higher concentration of nitrite + nitrate (Figure 3) and a high drop in pH (Figure 4). However, there is no clear correlation between the increased membrane damage and the generation of (intracellular) ROS (Figure 5). Compared to the _Salmonella_ Typhimurium biofilms, a slightly higher increase in absorbance was observed for the _Listeria monocytogenes_ biofilms, which indicates that the latter cells are more vulnerable to membrane damage. In literature, the opposite trend has been observed. The study of [PERSON] et al. [14] reported that the CAP efficacy was directly correlated to the thickness of the bacterial cell wall. Gram positive bacteria, with a thick peptidoglycan layer, showed the highest resistance both for planktonic cells and biofilms. Similar, the study of [PERSON] et al. [28] reported that Gram-negative (planktonic) cells were mainly inactivated due to cell leakage and low-level DNA damage, while Gram-positive (planktonic) cells were killed due to intracellular damage and little envelope damage. The contradictory results observed within the presented study can originate from a difference in (i) applied treatment characteristics and/or (ii) (biofilm forming) bacterial species. The latter is of high importance since the composition of the biofilm matrix, and the ability of the CAP species to penetrate into the biofilm, depends on the biofilm forming species [62]. Moreover, the research of [PERSON] et al.