# Dark Fermentation of Fruit Juice Effluents: Effects of Substrate Concentration
## 3 Results and Discussion
### Hydrogen Production
This effect can be attributed to a slight substrate inhibition of bacterial biomass, but primarily to product inhibition. As previously mentioned, product inhibition reduces H\({}_{2}\) yield, leading to lower H\({}_{2}\) generation despite consuming the same amount of substrate. Consequently, the highest maximum H\({}_{2}\) potential production, 12.46 L H\({}_{2}\), was achieved with a substrate concentration of to 0.91 C mol\(\cdot\)L\({}^{-1}\). Taking into account the volume of the reactor, 3 L, the specific hydrogen production was about 4.15 Nm\({}^{3}\) H\({}_{2}\cdot\)m\({}^{-3}\) reactor. A similar value has been reported in the literature by [PERSON] et al., who studied hydrogen production via acidogenic fermentation as a function of organic load (from 0 to 1.6 \(\times\) 10\({}^{3}\) mM glucose), finding the highest maximum H\({}_{2}\) potential \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Initial Sugar Concentration** & \(P\) **(mL H\({}_{2}\))** & \(R_{m}\) **(mL H\({}_{2}\cdot\)h\({}^{-1}\))** & \(\lambda\) **(h)** & \(r^{2}\) \\ \hline
0.30 & 5482.77 & 444.08 & 2.71 & 0.9996 \\
0.91 & 12,456.73 & 404.22 & 4.78 & 0.9970 \\
1.52 & 8908.81 & 163.33 & 2.60 & 0.9970 \\
2.12 & 5217.24 & 93.06 & 0 & 0.9701 \\ \hline \hline \end{tabular}
\end{table} 
Table 3: Modified Gompertz equation coefficients when dealing with different initial substrate concentrations. at a substrate concentration of approximately 0.85 C mol\(\cdot\)L\({}^{-1}\) glucose [31]. This initial substrate concentration closely aligns with the results obtained in this study. On the other hand, the highest maximum H\({}_{2}\) production rate, 444.08 mL\(\cdot\)h\({}^{-1}\), was obtained when dealing with a substrate concentration of about 0.30 C mol\(\cdot\)L\({}^{-1}\). This parameter decreases with increasing initial substrate concentration. However, the highest H\({}_{2}\) production was obtained when dealing with a substrate concentration of about 0.91 C mol\(\cdot\)L\({}^{-1}\). Because of the different results obtained with the maximum potential of H\({}_{2}\) production and the maximum hydrogen production rate, a new parameter was selected to establish the comparison. The parameter selected was the Maximum Specific Hydrogen Production Rate (MSHPR). The MSHPR can be defined as the maximum hydrogen production per unit of time and biomass in the reactor and measured as ml H\({}_{2}\)-biomass C mol\({}^{-1}\)\(\cdot\)L\({}^{-1}\). This parameter was calculated by dividing Rm by the biomass concentration. The results obtained for the MSHPR when feeding different initial substrates concentrations are presented in Figure 6.  
As can be seen in Figure 6, the highest MSHPR was obtained when operating with a substrate concentration of 0.30 C mol\(\cdot\)L\({}^{-1}\), 5.63 L H\({}_{2}\)-biomass C mol\({}^{-1}\).h\({}^{-1}\). This value was very similar to that obtained with 0.91 C mol\(\cdot\)L\({}^{-1}\), 5.50 L H\({}_{2}\)-biomass C mol\({}^{-1}\).h\({}^{-1}\). In these cases, almost any substrate or product inhibition was experienced, justifying the similar values obtained. However, the value of the MSHPR significantly decreases when increasing the substrate concentration to 1.52 and 2.12 C mol\(\cdot\)L\({}^{-1}\). The significantly lower MSHPR values obtained when operating with those concentrations could be explained by the inhibition effects caused by the very high initial substrate concentration as well as by the very high concentration of fermentation products accumulated during the fermentation, as previously described. Based on the results presented, it can be stated that the best initial substrate concentrations to maximize the hydrogen generation were within 0.30 and 0.91 C mol\(\cdot\)L\({}^{-1}\). With the aim to extrapolate these results to industrial facilities dealing with fruit juice wastewaters, the potential hydrogen generation was determined.