# Dark Fermentation of Fruit Juice Effluents: Effects of Substrate Concentration
## 3 Results and Discussion
### Hydrogen Production  
During the acidogenic fermentation of the substrates contained in the synthetic fruit juice wastewaters, H\({}_{2}\) and CO\({}_{2}\) were produced and accumulated in the gas phase. The evolution of cumulative H\({}_{2}\) and CO\({}_{2}\) during experiments with different initial sugar concentrations is presented in Figure 5. Cumulative gas production increased during acidogenic fermentation, but the trend and total amount of gas produced varied depending on the initial substrate concentration. When sugar concentrations were 0.30 and 0.91 C mol\(\cdot\)L\({}^{-1}\), gas production followed exponential trends, indicating optimal microbial growth. However, with initial substrate concentrations of 1.52 and 2.12 C mol\(\cdot\)L\({}^{-1}\), gas production exhibited two different trends in a similar way to the other fermentation products previously described. Figure 5: **(A)** Hydrogen production during acidogenic fermentation of synthetic wastewater with different substrate concentrations. **(B)** Hydrogen to carbon dioxide ratio during the fermentation tests. In the first stage, up to 50 h for the 1.52 C mol\(\cdot\)L\({}^{-1}\) initial concentration and up to 60 h for the 2.12 C mol\(\cdot\)L\({}^{-1}\) initial concentration, gas production rates were approximately linear, with H\({}_{2}\) and CO\({}_{2}\) production being very similar. In the second stage, gas production rates decreased over time, in a similar way to the acid production behavior previously. Additionally, CO\({}_{2}\) production exceeded the H\({}_{2}\) and production during the second phase. The behavior experienced can be explained by a slight initial substrate inhibition of bacterial biomass, taking place in the first stage, and product inhibition due to the accumulation of fermentation products in the second stage. The highest hydrogen production (6.1 L H\({}_{2}\)-substrate C mol\({}^{-1}\)) and the highest hydrogen percentage in the gas phase (57%) were achieved with a sugar concentration of 0.30 C mol\(\cdot\)L\({}^{-1}\). These results indicate that increasing the substrate concentration led to lower H\({}_{2}\) production and lower H\({}_{2}\) content in the gas phase. These reductions can be attributed to substrate and product inhibitions experienced in the system. Similar results have been reported in the literature [22; 41]. Considering that hydrogen is the most important product of acidogenic fermentation, its production was modeled using the modified Gompertz equation [42; 43]. This equation allows for the determination of key kinetic and stoichiometric parameters, as well as H\({}_{2}\) production rates and the potential for H\({}_{2}\) generation, in experiments conducted with different initial substrate concentrations. \[H(t)=P.exp\left\{-exp\left[\frac{R_{m}\cdot e}{P}.(\lambda-t)+1\right]\right\} \tag{3}\]  
where, \(H(t)\) is the cumulative H\({}_{2}\) production (mL); \(P\) is the maximum potential of H\({}_{2}\) production (mL); \(R_{m}\) is the maximum hydrogen production rate (mL\(\cdot\)h\({}^{-1}\)); \(\lambda\) is the duration of the lag phase (h); and \(t\) is time (h). Table 3 summarizes the values of the modified Gompertz equation coefficients for wastewaters with different initial substrate concentrations. Considering the correlation coefficients, the model accurately describes the experimental data in all cases, being the lowest correlation coefficient of 0.9701. This largest error was observed with an initial concentration of 2.12 C mol\(\cdot\)L\({}^{-1}\), which can be attributed to the significant inhibition caused by substrate and fermentation products experienced by the microorganisms when dealing with this very high initial concentration. Analyzing the results presented in Table 3, it must be highlighted that, on the one hand, the maximum H\({}_{2}\) potential production increased with initial substrate concentrations up to 0.91 C mol\(\cdot\)L\({}^{-1}\). However, at higher concentrations, the maximum H\({}_{2}\) potential decreased as substrate concentrations increased.