## 2 Materials and Methods
### Experimental Setup  
In a previous stage of this study, the effect of nanoparticles and ozone pre-treatment on the thermophilic acidogenic stage (first stage of TPAD configuration) of the TPAD process was evaluated using a Continuous Stirred Tank Reactor (CSTR) (Scharlab, Barcelona) operating in an acidogenic thermophilic range (55 \({}^{\circ}\)C). This reactor, with a total volume of 4 L, maintained a hydraulic retention time (HRT) of 6 days. The reactor was fed with a mixture of biosolids and vinasses (BV) in a ratio of 50:50 (\(v/v\)) and subjected to pre-treatment with nanoparticles and nanoparticles combined with ozone. The effluents obtained from  
\begin{table}
\begin{tabular}{c c c c c} \hline \hline \multirow{2}{*}{**Parameter**} & \multicolumn{1}{c}{**1**} & \multicolumn{1}{c}{**C**} & \multicolumn{1}{c}{**N**} & \multicolumn{1}{c}{**NO**} \\  & **Mean (SD 1 \%)** & **Mean (SD 1 \%)** & **Mean (SD 1 \%)** & **Mean (SD 1 \%)** \\ \hline pH & 8.33 (0.22) & 5.12 (0.78) & 5.12 (0.97) & 5.07 (1.18) \\ TS (g/kg) & 24.05 (0.27) & 19.50 (11.98) & 26.50 (12.72) & 22.16 (5.62) \\ VS (g/kg) & 15.41 (0.39) & 14.52 (14.49) & 18.51 (0.74) & 17.30 (6.77) \\ TCOD (mgO\({}_{2}\)/L) & 32,263.65 (4.12) & 51,980.32 (2.99) & 51,183.69 (5.70) & 46,045.10 (2.10) \\ SCOD (mgO\({}_{2}\)/L) & 19,119.20 (12.55) & 326,617.97 (15.13) & 35,696.68 (5.36) & 36,047.66 (3.93) \\ DOC (mg/L) & 1062.00 (0.90) & 7575.03 (0.16) & 9140.98 (2.66) & 7547.73 (1.06) \\ TN (mg/L) & 2332.67 (2.99) & 869.00 (0.57) & 1054.75 (1.71) & 899.75 (0.59) \\ VFA (g equiv acetic/L) & 0.34 (10.92) & 5.54 (0.56) & 5.84 (0.14) & 5.62 (3.42) \\ Acetic acid (mg/L) & 181.87 (40.88) & 1333.47 (2.06) & 2540.29 (1.04) & 2317.66 (15.42) \\ Propionic acid (mg/L) & 101.94 (53.56) & 796.61 (8.98) & 1069.07 (1.86) & 844.48 (12.75) \\ Butyric acid (mg/L) & 103.80 (31.68) & 4763.22 (1.98) & 3159.08 (2.46) & 3407.94 (12.33) \\ \hline \hline \end{tabular}  
* SD: standard deviation (%).  
\end{table}
Table 1: Characteristics of inoculum (I) and substrates used: effluent from reactor without pre-treatment (C), effluent from reactor with nanoparticles (N), and effluent from reactor with nanoparticles and pre-treated with ozone (NO).  
these experiments were used as substrates in the BMP tests carried out to mimic mesophilic methanogenic conditions (35 \({}^{\circ}\)C) corresponding to the second stage of TPAD configuration. The BMP tests were conducted in small, amber-colored reactors with a total volume of 250 mL and an active volume of 120 mL, operated in batch mode. Each reactor received a 50/50 (\(v\)/\(v\)) addition of inoculum and the substrate to be tested (60 mL each). In addition, a control test was performed using 60 mL of inoculum mixed with 60 mL of distilled water. The tests were carried out at pH 7.5 and in the mesophilic range (35 \({}^{\circ}\)C) by placing the reactors in a thermostatic stirred bath stirred at 40 rpm.  
In Figure 1, a diagram of the experimental setup used in this study is presented.  
In order to ensure the reproducibility of the experiments, each test was conducted in triplicate. For monitoring purposes, analyses included total chemical oxygen demand (TCOD), soluble chemical oxygen demand (SCOD), dissolved organic carbon (DOC), total nitrogen (TN), total solids (TS), volatile solids (VS), pH, total volatile fatty acids (VFAs) and individual acid concentrations. Analyses were performed both at the beginning and the end of the tests. Daily, the volume of biogas production (based on headspace pressure in the vial) and its composition were measured. The test length was defined by the evolution of methane production. The experiments were supposed to have finished once the increase in production was lower than 1% in two consecutive measurements [19].