## 2 Materials and Methods
### Quantification of the Generated (Reactive) CAP Species
#### 2.4.2 Chemical Composition and pH of CAP Treated Water  
In order to simulate the chemical reactions that can occur between the generated CAP species and the intracellular fluid and/or the water inside the biofilm matrix, a small amount of water (i.e., 500 \(\upmu\)L) was CAP treated using the different combinations of plasma characteristics. The chemical composition and the pH of the water were examined following 10 min of CAP treatment.  
For the chemical composition, the concentration of three (relatively stable) CAP species was examined, i.e., hydrogen peroxide (H\({}_{2}\)O\({}_{2}\)), nitrite, and nitrate. For the H\({}_{2}\)O\({}_{2}\) concentration, the method of [PERSON] et al. [27] was applied. Here, 100 \(\upmu\)L of the CAP treated water was added to a single well of a 96-well microtiter plate (polystyrene, F-bottom, clear, Greiner Bio-One, Vilvoorde, Belgium) containing 10 \(\upmu\)L of TiOSO\({}_{4}\) (Carl Roth GmbH+Co, Karlsruhe, Germany). Following 10 min of incubation (at room temperature and in the dark), a microplate reader (VersaMax tuneable microtiter plate reader, Molecular Devices, Wokingham, UK) was used to measure the absorbance at 405 nm. In addition, a standard curve was developed in order to convert absorbance values into H\({}_{2}\)O\({}_{2}\) concentrations. For the development of this calibration curve, a 30% (_v/v_) H\({}_{2}\)O\({}_{2}\) standard solution (Sigma-Aldrich, St. Louis, MO, US) was diluted to obtain solutions with different known concentrations, i.e., 0, 2 \(\times\) 10\({}^{-4}\)%, 3 \(\times\) 10\({}^{-4}\)%, 5 \(\times\) 10\({}^{-4}\)%, 1 \(\times\) 10\({}^{-3}\)%, 2 \(\times\) 10\({}^{-3}\)%, 3 \(\times\) 10\({}^{-3}\)%, and 5 \(\times\) 10\({}^{-3}\)% (1% = 0.426 M). Three independent replicates (\(n=3\)) were used to determine (i) the equation of the absorbance as function of the H\({}_{2}\)O\({}_{2}\) concentration and (ii) the H\({}_{2}\)O\({}_{2}\) concentration of the CAP treated water using  
\begin{table}
\begin{tabular}{c c c c} \hline
**Electrode-Oxygen Level** **\% (r/v)** & **Discharge Power/Input** **Voltage** & **Peak-to-Peak Output** **Voltage (kV)** & **Dissipated Power (W)** \\ \hline DBD-0\% & 13.88 & 4.5 \(\pm\) 0.2 & 2.6 \(\pm\) 0.1 \\  & 21.88 & 6.4 \(\pm\) 0.0 & 7.0 \(\pm\) 0.3 \\ \hline DBD-1\% & 13.88 & 6.3 \(\pm\) 0.1 & 1.9 \(\pm\) 0.2 \\  & 21.88 & 7.8 \(\pm\) 0.1 & 5.9 \(\pm\) 0.2 \\ \hline SBD-0\% & 13.88 & 2.4 \(\pm\) 0.1 & 1.6 \(\pm\) 0.0 \\  & 21.88 & 3.5 \(\pm\) 0.1 & 3.2 \(\pm\) 0.1 \\ \hline SBD-1\% & 13.88 & 2.8 \(\pm\) 0.1 & 1.9 \(\pm\) 0.0 \\  & 21.88 & 3.9 \(\pm\) 0.1 & 3.8 \(\pm\) 0.1 \\ \hline \end{tabular}
\end{table}
Table 1: Monitored output voltages (kV) and dissipated power (W) values as function of the applied CAP treatment conditions (i.e., electrode configuration, oxygen level of the gas flow, and discharge power/input voltage).  
each possible combination of plasma characteristics. To determine the nitrite and nitrate concentration, another colorimetric method was applied using the nitrite/nitrate assay kit (Sigma-Aldrich, St. Louis, MO, USA). As for the H\({}_{2}\)O\({}_{2}\) assay, a calibration curve was again developed to convert absorbance values into nitrite and nitrate concentrations. Three independent replicates (\(n=3\)) were again used to determine (i) the equations of the absorbance values as function of the nitrite and nitrate concentrations and (ii) the nitrite and nitrate concentrations of the CAP treated water using each possible combination of plasma characteristics. The authors refer to the manufacturer's protocol for the exact procedures.  
For the determination of the pH of the CAP treated water, again three independent replicates were used (\(n=3\)). These values were measured using a pH-electrode for small volumes (pH electrode InLab Micro, Mettler Toledo, Zaventem, Belgium).