## 3 Results
### Quantification of VFAs in the Effluent  
Regarding the effluent resulting from the biogas production in the experimental phase, the overall quantification of VFAs production was conducted to define the potential metabolic pathway followed by the reactors under the evaluated conditions of temperature, initial organic load, and s/x ratio. Figure 3 contrasts the levels of biogas production with the associated production of VFAs without detailing the specific substance.  
Reviewing the behavior of the combinations in Figure 3, for combinations with an s/x of 1, the increase in initial organic load meant an increase in the production of VFAs, which is in line with the results obtained by other studies [52]. This situation repeats for combinations with an s/x of 0.5, where the variation in produced VFAs is more evident with an increase in organic load. Therefore, it is evident that low organic loads are considered a limiting factor in VFAs production due to the small amount of substrate [20]. On the other hand, combinations with an s/x of 0.75 showed a different behavior. Specifically, C2, with a load of 5 gVS/L, had a higher production than C5, with a load of 10 gSV/L. Regarding the biogas produced, the conditions in C2 were more favorable for  
Figure 3: Biogas and volatile fatty acids production.  
Figure 2: Pareto chart on biohydrogen production.  
as both biogas and VFAs production were higher, indicating better substrate digestion and therefore a better process performance.  
The cases of C4 and C8 are noteworthy, as both show the lowest biogas production and, at the same time, the highest levels of VFAs. This indicates that fermentation under the values of the analyzed combinations leaned almost entirely towards acidogenic pathways. Considering that undissociated VFAs can penetrate the cell membrane of the microorganisms composing the inoculum, causing a pH reduction and leading to unfavorable physiological conditions, this can result in process inhibition [53]. While this could explain the behavior in both combinations, it is necessary to consider that in both cases and despite the low yields, total inhibition did not occur. This may be because, during the laboratory tests, the digestive process was completed to the methanization point for all combinations tested. In the case of mixed-culture inoculum (WWTP sludge), there could have been the presence of microorganisms that competed with H\({}_{2}\)-producing bacteria, such as those associated with propionic acid production [54]. This is reflected in the high concentrations of VFAs recorded by the combinations in general and particularly by C4, C8, and C9, which coincidentally were the combinations with the lowest biogas production.  
While all experimental phase tests generated high concentrations of VFAs, the biogas yield associated with the combinations C1 to C3 is mainly determined by the limited availability of substrate, considering significantly low initial organic loads and s/x. In other words, the substrate is the limiting factor of the process, a situation that extends to the combinations C5 and C6. On the other hand, fermentation in C4, C8, and C9 took acidogenesis as the main route, favoring the production of VFAs at the expense of consuming and/or inhibiting H\({}_{2}\) production. It was observed that this situation occurred when increasing the initial organic load while maintaining a low s/x, aligning with the results obtained in other studies [52].