# Dark Fermentation of Fruit Juice Effluents: Effects of Substrate Concentration
## 3 Results and Discussion
### Volatile Fatty and Other Acids Production  
During acidogenic fermentation, microorganisms metabolize substrates to produce volatile fatty and other acids, hydrogen, and carbon dioxide. Figure 4 presents the VFA and other acids production as well as the substrate concentrations during the tests conducted with different initial substrate concentrations. As shown in Figure 4, the main acids produced were acetic, butyric, and lactic acids, which is consistent with findings reported in the literature [25]. Formic acid was also produced, but only during the initial stages of the fermentation process, maintaining its concentration thereafter. Figure 4: Volatile fatty and other acids and substrate concentrations during acidogenic fermentation of wastewater with different initial substrate concentrations. (**A**) Substrate concentration: 0.30 C mol\(\cdot\)L\({}^{-1}\); (**B**) substrate concentration: 0.91 C mol\(\cdot\)L\({}^{-1}\); (**C**) substrate concentration: 1.52 C mol\(\cdot\)L\({}^{-1}\); and (**D**) substrate concentration: 2.12 C mol\(\cdot\)L\({}^{-1}\). In all tests conducted, the organic substrates contained in the synthetic wastewater (fructose, glucose, and sucrose) were fully fermented. When comparing the substrate fermentation rates, it was observed that the fructose fermentation rate was consistently higher than that of glucose and sucrose. This could be attributed to the higher initial concentration of fructose, which facilitates mass transfer, as well as the higher affinity of the microbial culture used in this study for fructose. It is also important to note that the time required for complete fermentation of the sugars in the different effluents increased exponentially, from 12.67 h to 191.58 h, as the sugar concentration in the wastewater increased linearly from 0.30 to 2.12 C mol\(\cdot\)L\({}^{-1}\). Thus, higher sugar concentrations in wastewater make sugar fermentation more difficult for the microorganisms. Taking into account that this effect was mainly experienced after 40 h of operation, the inhibition could be mainly explained by a product inhibition event [23]. In the first case, as shown in Figure 4A, when the sugar concentration was 0.30 C mol\(\cdot\)L\({}^{-1}\), glucose and sucrose were completely fermented simultaneously after approximately 10 h, while fructose required more time, about 12 h, due to its higher concentration. The main acids produced were lactic, acetic, and butyric acids, with final concentrations of 80, 35, and 32 C mmol\(\cdot\)L\({}^{-1}\), respectively. Thus, lactic acid was the primary product obtained during the acidogenic fermentation when dealing with low initial substrate concentration. As illustrated in Figure 4A, both sugar consumption and acid production followed an exponential trend. The rates of sugar fermentation and acid production were lower during the initial phase due to the low biomass concentration at the early stages, but these rates increased over time. This exponential trend can be explained by the exponential growth of the biomass, which is associated with optimal growth conditions. Therefore, no limitations or inhibitions were detected when dealing with the substrate concentration of 0.30 C mol\(\cdot\)L\({}^{-1}\). In the case of feeding wastewater with 0.91 C mol\(\cdot\)L\({}^{-1}\) mM of sugar, as shown in Figure 4B, fructose was completely fermented before glucose and sucrose, taking approximately 34 h. Complete fermentation of glucose and sucrose required 37 h. The production of lactic acid was about 250 C mmol\(\cdot\)L\({}^{-1}\) butyric and acetic concentrations were similar, with final concentrations of about 175 C mmol\(\cdot\)L\({}^{-1}\). It is important to note that acid production exhibited a slightly linear trend, rather than the expected exponential one, indicating the presence of some sort of inhibition. Finally, when dealing with wastewaters containing substrate concentrations of 1.52 and 2.12 C mol\(\cdot\)L\({}^{-1}\), two phases in acid production were observed, as shown in Figure 4C,D.