## 2 Materials and Methods
### Setup and Operating Parameters  
Figure 1 illustrates the reactor setup used for the continuous H\({}_{2}\) production system, which consisted of a polyvinyl chloride fermenter with a total capacity of 1.2 L with a 0.4 L headspace. The setup was equipped with a pH controller (BSV, Spain) and a pH probe (HO35-BSV01, Spain), along with gas and liquid phase sampling ports. The system was also equipped with an integrated continuous gas flow meter (see Figure 1). Prior to introducing the inoculum into the reactor, a 0.1 L portion of the acidogenic inoculum was activated in a 2.1 L gas-tight fermenter (1 L working volume) for 24 h at 37 \(\pm\) 1 \({}^{\circ}\)C, maintained at approximately 150 rpm. During this activation period, pH control was not applied, and the inoculum was cultured in mineral growth medium supplemented with 10 g/L lactose as described in Section 2.1. The reactor was initially inoculated with 10% w/o of fresh, actively cultured inoculum and operated in batch mode for approximately 8 h before transitioning to continuous operation. Lactose and mineral media were added solely during this initial batch phase to support microbial activity. The continuous operation included a start-up and stabilization period, followed by four operating conditions. The first condition assessed the system without the addition of nanoparticles (NPs) at an HRT of 6 h, corresponding to an OLR of 100.4 g COD/L-day, after which increasing concentrations of FeNPs (iron (II, III) oxide nanopowder, Sigma, USA) were introduced at 50, 100, and 200 mg/L. According to the specifications provided by the supplier, the nanopowder used had a particle size distribution ranging from 50 to 100 nm. During the start-up phase, the hydrolysate was prepared for operation. Briefly, the reactor was inoculated as described above and operated on a 24 h HRT. Once the reactor reached a stable production state, the HRT was reduced to 12 h, resulting in system \begin{table}
\begin{tabular}{l l} \hline \hline
**Parameter** & **RS Hydrolysate** \\ \hline TCOD (g/L) & 33.7 \(\pm\) 1.3 \\ SCOD (g/L) & 28.5 \(\pm\) 0.9 \\ TOC (g/L) & 8.9 \(\pm\) 0.3 \\ TN (g/L) & 0.4 \(\pm\) 0.0 \\ Total solids (g/L) & 53.7 \(\pm\) 2.3 \\ Volatile solids (g/L) & 47.5 \(\pm\) 3.1 \\ Reducing sugars (g/L) & 22.1 \(\pm\) 0.6 \\ \multicolumn{2}{l}{* COD yield (\%)} \\ \hline \hline \end{tabular}  
* TCOD: Total chemical oxygen demand. SCOD: Soluble chemical oxygen demand. TOC: Total organic carbon. TN: Total nitrogen.
* Ratio of TCOD in hydrolysates to the total solid concentration used.  
\end{table} 
Table 2: Physicochemical characterization of hydrolysates obtained from RS pretreated by dilute acid-assisted thermal pretreatment. Figure 1: General scheme of the dark fermentation system. inhibition after two days of operation. In response, the reactor was run in batch mode for two days with lactose added for recovery. Due to the poor performance of the reactor, bioaugmentation was required. A significant improvement in reactor productivity within 8 h allowed a switch to continuous mode (HRT 12 h), which was maintained for two days while still feeding lactose. Once production stability was established, there was a shift to using RS hydrolyses as the substrate, with an increased HRT of 18 h. However, after 8 days of operation, the reactor activity decreased, eventually leading to total inhibition. Consequently, a new start-up strategy was implemented. To reduce the presence and accumulation of inhibitors, the hydrolyses were subjected to the following over-limiting treatment: Calcium hydroxide (Ca(OH)\({}_{2}\)) was added to the hydrolyses heated at 50 \({}^{\circ}\)C and stirred continuously until a pH of 10 was reached, and the mixture was allowed to stand for 30 min. The supernatant was then filtered and cooled to 30 \({}^{\circ}\)C for subsequent neutralization to pH 6 with 1N sulfuric acid (H\({}_{2}\)SO\({}_{4}\)). Activated carbon was added at a concentration of 1.5% _w/o_ and mixed for 1 h, followed by further filtration.