## 3 Results and Discussion
### Dark Fermentation Reactor Performance Evaluation
#### 3.2.2 FeNPs Nanoparticles Evaluation  
After applying the overlining pretreatment, continuous operation was resumed to assess FeNPs performance. Initially, the reactor was operated in batch mode for two days. Following this, the system transitioned to continuous operation with an HRT of 6 h. The reactor responded favorably, achieving peak H\({}_{2}\) productivity of approximately 2.6 NL H\({}_{2}\)/L-d and stabilizing at an average productivity of 1.7 \(\pm\) 0.2 NL H\({}_{2}\)/L-d, with the H\({}_{2}\) content in the biogas at 50.7\(\%\)\(\pm\) 1.4 (Figure 2 and Table 3). After a two-day period without nanoparticle addition (approximately six steady-state cycles), the evaluation of FeNPs (magnetic) commenced with the introduction of 50 ppm directly into the feed tank. Following the nanoparticle addition, a significant increase in H\({}_{2}\) productivity was observed, with the system reaching a steady state of 2.4 \(\pm\) 0.2 NL H\({}_{2}\)/L-d with an associated H\({}_{2}\) content of 51.6\(\%\)\(\pm\) 0.2 (Figure 2). This represented a productivity increase of approximately 41.2\(\%\). After six cycles of stable operation, the FeNP concentration was increased to 100 ppm. Initially, a rapid increase in productivity was observed, followed by a brief decrease during the first HRT (6 h). However, productivity soon increased again, stabilizing at 2.6 \(\pm\) 0.3 NL H\({}_{2}\)/L-d, which corresponds to an approximate 53\(\%\) increase compared to the period without nanoparticle addition. During this period, a slight decrease in H\({}_{2}\) content was noted, with the value reaching 50.5\(\%\)\(\pm\) 0.3 (Table 3). Following six stable cycles, the FeNP concentration was further increased to 200 ppm. This increase led to an immediate drop in  
Figure 2: Overall continuous operation for hydrogen production. Hydrogen productivity (\(\bullet\)), hydrogen content in the evolved off-gas (\(\blacksquare\)). H\({}_{2}\) productivity, returning to nearly the initial performance observed without nanoparticles at 1.6 \(\pm\) 0.2 NL H\({}_{2}\)/L-d. The H\({}_{2}\) content remained steady at 50.0% \(\pm\) 0.5. Although previous studies have often identified 200 ppm as the optimal Fe nanoparticle concentration, with some systems showing tolerance up to 700 ppm before experiencing inhibition [18; 35], the results of this study suggest that inhibition began to occur at magnetic nanoparticle concentrations above 100 ppm. The surface of FeNPs can enhance the activity of ferredoxin oxidoreductase by accelerating electron transfer from NADPH to [Fe-Fe] hydrogenases, thereby boosting H\({}_{2}\) production [36; 37]. Moreover, nanoparticles possess an electron affinity that facilitates the reduction of protons (H+) to H\({}_{2}\), effectively acting as an electron sink [38]. However, at high concentrations, nanoparticles can induce toxicity and generate reactive oxygen species, which negatively impact microbial growth [39]. As a result, while a FeNP concentration of 200 ppm initially appeared promising for enhancing H\({}_{2}\) productivity, it ultimately led to a significant reduction in productivity compared to the 50 and 100 ppm concentrations. This behavior can be directly attributed to the specific system and operational conditions. In continuous operation, the constant supply of nanoparticles can lead to a chronic effect that exceeds the microbial utilization rates. In addition, the magnetic properties of FeNPs can cause them to accumulate in the magnetically stirred reactor, thereby increasing their concentration in the system. The results obtained in this study align with those reported in the literature. For example, [PERSON] et al. [35] investigated the use of acid hydrolyses of sugarcane bagasse for fermentative H\({}_{2}\) production under mesophilic conditions at various pH levels. They found that the addition of Fe\({}^{2+}\) nanoparticles at a concentration of 200 ppm increased H\({}_{2}\) productivity by nearly 32%. On the other hand, [PERSON] et al.