## 2 Materials and Methods
### Experimental Design  
In order to determine hydrogen recovery using OFMSW as a substrate under conditions favoring the dark fermentation process, experiments were designed to evaluate organic loads of 5, 10, and 15 g of volatile solids per liter (gVS/L). The influence of the substrate-inoculum ratio (s/x) was also assessed, expressed in units of grams of volatile solids of substrate per gram of volatile solids of inoculum (gVSs/gVSi), with values of 1, 0.75, and 0.5 gVSs/gVSi. A total of 9 experiments were conducted, each in triplicate. Additionally, for each experiment, a blank was set up in which only the inoculum was applied. This was performed to identify the hydrogen recovery resulting from its biological activity, which was subtracted from the total hydrogen recovery. Thus, the hydrogen production contributed by OFMSW was obtained.  
Table 1 schematically illustrates the experimental design.  
\begin{table}
\begin{tabular}{c c c} \hline \hline
**Combination** & **Organic Load (gVS/L)** & **s/x Ratio (gVSs/gVSi)** \\ \hline C1 & 5 & 0.5 \\ C2 & 5 & 0.75 \\ C3 & 5 & 1 \\ C4 & 10 & 0.5 \\ C5 & 10 & 0.75 \\ C6 & 10 & 1 \\ C7 & 15 & 0.5 \\ C8 & 15 & 0.75 \\ C9 & 15 & 1 \\ \hline \hline \end{tabular}
\end{table}
Table 1: Experimental design.  
Experiment C7 was discarded due to the negative outcome in the water volume obtained after calculating the quantities of substrate, inoculum, and water necessary to meet the conditions. This is attributed to substrate characteristics such as the percentage of total solids and its density.  
The variables under control were pH and temperature. Considering the optimal pH range for maximum hydrogen yield falls between 5.2 and 6.0, a pH of 5.5 was utilized [35]. The assumed temperature for all experiments was 45 \({}^{\circ}\)C.  
The experimental setups employed amber glass bottles of 250 mL, containing a 200 mL volume composed of substrate, inoculum, distilled water, and 10 mL of a buffer solution constituted by 37% HCl for pH adjustment. The reactors were sealed and placed in a thermostat bath to initiate the test [28].  
For the measurement of hydrogen recovered during the FO phase, the volumetric displacement method was applied, where a liquid's volume is displaced by the generated volume of biogas [36]. Therefore, during the experiments, the biogas stream produced in the reactors was bubbled through a NaOH solution to capture the CO\({}_{2}\) present in the stream [28]. The bottle was connected to a third bottle through a micro-drip hose to collect and measure the volume of the displaced NaOH solution, which served as an indicator of the biogas volume produced during each trial [33].  
Given that the present study aimed to determine hydrogen production from OFMSW using the FO process, each trial concluded upon detecting traces of CH\({}_{4}\) in the chemical composition of the biogas. This signified the end of the FO phase, transitioning into the methanogenesis phase. To verify the traces of substances in the biogas stream, the biogas composition was characterized every 24 h using the biogas 5000 Landtec equipment, capable of detecting the presence of CH\({}_{4}\)[28].  
The experimental results were statistically processed (average, standard deviation, and Pareto analysis), and the conditions yielding the highest performance were employed in developing the conceptual design of the dark fermentation plant.