## 3 Results and Discussion
### Initial and Final Characterization of the Mixtures Tested in the Biochemical Methane Potential  
The mixtures fed to the BMP tests, as well as their digestates, were characterized and compared with the characteristics of the substrates used (see Table 1).  
At the beginning of the BMP, the mixture of the inoculum (with a pH of 8.33) and the effluents (with a pH around 5.5 due to their origin from an initial TPAD stage in the acidogenic reactor) resulted in a pH close to 7.5, which is optimal for BMP tests. The mixing of the inoculum with the different substrates resulted in mixtures with the characteristics presented in Table 2.  
\begin{table}
\begin{tabular}{c c c c} \hline \multirow{2}{*}{**Parameter**} & **MC** & **MN** & **MNO** \\  & **Mean (SD 1 \%)** & **Mean (SD 1 \%)** & **Mean (SD 1 \%)** \\ \hline pH & 7.25 (0.80) & 7.30 (0.90) & 6.87 (1.25) \\ TS (g/kg) & 23.30 (11.98) & 24.72 (12.72) & 23.88 (5.62) \\ VS (g/kg) & 16.11 (0.86) & 17.32 (0.33) & 17.03 (0.45) \\ TCOD (mgO\({}_{2}\)/L) & 39,074.87 (5.90) & 45,288.61 (2.79) & 45,129.28 (4.51) \\ SCOD (mgO\({}_{2}\)/L) & 26,169.41 (8.99) & 31,666.18(8.78) & 23,779.51 (2.80) \\ DOC (mg/L) & 4191.50 (1.84) & 4144.50 (2.48) & 4354.17 (1.04) \\ TN (mg/L) & 1513.33 (1.84) & 1486.67 (2.48) & 1499.50 (1.04) \\ \hline \end{tabular} \({}^{1}\)SD: standard deviation (%).  
\end{table}
Table 2: Characterization of the initial mixtures of Biochemical Methane Potential. Control (MC), nanoparticles (MN), and nanoparticles together with ozone pre-treatment (MNO).  
In this table, it can be observed that the addition of iron nanoparticles in the acidogenic reactor, mainly in MN, favored an increase in TS and VS, together with a high SCOD. As previously mentioned, Fe\({}^{0}\) facilitates the solubilization of organic matter [15]. In addition, their combination with ozone pre-treatment (MNO) resulted in a significant increase in DOC. This effect has been observed previously by [PERSON] and [PERSON] [28], where biological sludge from the municipal wastewater treatment plant was ozonated. However, the mixture (MC, MN and MNO) shows a high TCOD due to the significant addition of particulate matter with the inoculum (Table 1).  
Table 3 shows the results of the physico-chemical characterization parameters of the substrates once the BMP was finished. This characterization allows us to observe the changes experienced during the BMP test, as well as the influence of iron nanoparticles and iron nanoparticles combined with ozone pre-treatment on the anaerobic co-digestion process.  
A slight increase in pH has been observed at the end of the BMP tests. The pH of all the mixtures increased and reached values close to 8, a trend that has also been documented by [PERSON] et al. [29]. It can be seen that all the values of the parameters analyzed (Table 3) decreased with respect to the initial values (Table 2). This indicates that organic matter degradation took place during the BMP test. However, TN showed an increase in concentration during the process in all the experiments, possibly due to the fixation of inorganic nitrogen into organic forms by microorganisms.  
At the end of the BMP, MN continues to show high concentrations in the digestate of TS and VS, TCOD, SCOD and DOC. This finding highlights the potential for the digestate to be used in agriculture due to its nutrient-rich composition enhanced by the presence of iron nanoparticles.