## 3 Results and Discussion
### Metabolic By-Products  
Soluble metabolites identified in the effluent of the DF reactor across all operational stages included lactate, formate, acetate, propionate, and butyrate (Figure 3). Regardless of the nanoparticle concentrations tested (0, 50, 100, and 200 ppm), the predominant metabolites produced were acetate, butyrate, and propionate, as shown in Figure 3. Notably, a consistent lactate concentration, averaging around 1.0 g/L, was observed throughout all processes, independent of the nanoparticle condition. This consistency suggests that while nanoparticles may influence the enzymatic pathways associated with H\({}_{2}\) production, they do not disrupt or impact the metabolic pathway responsible for lactate production, nor do they affect the enzymes involved in its synthesis. \begin{table}
\begin{tabular}{c c c} \hline
**Condition** & **Average H\({}_{2}\) Productivity (NL H\({}_{2}\)/L-d)** & **Average H\({}_{2}\) Content (\%)** \\ \hline HRT 6 (RS) & 1.7 \(\pm\) 0.2 \({}^{\text{a}}\) & 50.7 \(\pm\) 1.4 \\ HRT 6 (RS) NPs 50 ppm & 2.4 \(\pm\) 0.2 \({}^{\text{b}}\) & 51.6 \(\pm\) 0.2 \\ HRT 6 (RS) NPs 100 ppm & 2.6 \(\pm\) 0.3 \({}^{\text{c}}\) & 50.5 \(\pm\) 0.3 \\ HRT 6 (RS) NPs 200 ppm & 1.6 \(\pm\) 0.2 \({}^{\text{a}}\) & 50.0 \(\pm\) 0.5 \\ \hline \end{tabular} Note: Within a specific condition, different letters in a column indicate values that are significantly different at the 95% confidence level.  
\end{table} 
Table 3: Summary of the process performance indicators during the continuous fermentative process for hydrogen production from RS hydrolyses with FeNPs addition. Conversely, acetate and butyrate have historically been closely associated with H\({}_{2}\) production. Notably, the introduction of 50 ppm FeNPs resulted in a 21% increase in butyrate concentration. When the nanoparticle concentration was further increased to 100 ppm, butyrate concentration significantly rose to 2.0 \(\pm\) 0.1 g/L, which is nearly 20% higher than the previous level and 43% higher than the concentration without nanoparticles. However, at the highest nanoparticle concentration of 200 ppm, there was a decrease in butyrate concentration to 1.8 \(\pm\) 0.1 g/L. In contrast, the addition of 50 ppm FeNPs did not result in an increase in acetate concentration, with levels remaining steady at 6.6 \(\pm\) 0.3 g/L. Moreover, when the nanoparticle concentration was increased to 100 ppm, acetate concentration decreased to 5.5 \(\pm\) 0.2 g/L, and this downward trend continued with the introduction of 200 ppm nanoparticles. The use of nanoparticles enhances H\({}_{2}\) production primarily through butyrate-type fermentation, with acetate-type fermentation playing a secondary role. Both routes are simplified and described in Equations (1) and (2), respectively. These reactions are exergonic, with standard free energy changes (\(\Delta\)G0) of \(-\)206 kJ/mol and \(-\)255 kJ/mol, respectively, indicating that both are thermodynamically favorable. \[\mathrm{C_{6}H_{12}O_{6}+2H_{2}O\to 2 CH_{3}COOH+2 CO_{2}+4H_{2}} \tag{1}\]  
\[\mathrm{C_{6}H_{12}O_{6}\to CH_{3}CH_{2}CH_{2}COOH+2 CO_{2}+2H_{2}} \tag{2}\]  
In these pathways, glucose is initially broken down into pyruvate, which is then oxidized to acetyl-CoA via the reduction of ferredoxin by the enzyme pyruvate-ferredoxin oxidoreductase. The reduced ferredoxin is subsequently oxidized by the hydrogenase enzyme to produce oxidized ferredoxin and H\({}_{2}\). Additionally, excess NADH produced during glycolysis can be oxidized by NADH-[Fe-Fe] hydrogenase to generate more H\({}_{2}\). Acetyl-CoA is then converted into acetate, butyrate, or ethanol, depending on environmental conditions and the specific microorganisms present [14]. However, the pH conditions in this study favored the butyrate pathway, which may explain why acetate formation does not directly correlate with increased H\({}_{2}\) productivity. Moreover, studies like those by [PERSON] et al.