# Dark Fermentation of Fruit Juice Effluents: Effects of Substrate Concentration
## 3 Results and Discussion  
### Biomass Growth and Substrate Fermentation  
Substrate fermentation, product generation, and biomass growth were evaluated during the acidogenic fermentation tests conducted with the different initial substrate concentrations. The results obtained in the four experimental series are presented in Figure 1.  
As shown in Figure 1, the initial evolution of biomass concentration was similar across all cases. However, a slight decrease in biomass growth rate was observed when increasing the substrate concentration during the first 15 h of fermentation. To accurately determine whether substrate inhibition of bacterial biomass occurred, the experimental results were analyzed using mathematical modeling by using the Haldane-Andrews model [21]. This model described the substrate consumption, and the biomass growth experienced with all the substrate concentrations tested, offering accurate predictions. The results of the fitting are presented in Figure 2, where the Haldane-Andrews modelling results, as well as the experimental data points, are presented. The maximum initial biomass growth rate was observed with a substrate concentration of 0.30 C mol\(\cdot\)L\({}^{-1}\). This rate slightly decreased when increasing the substrate concentration. From this fitting, the value of the models' parameters were determined, their values being those presented in Table 2.  
As shown in Figure 2, and according to the Haldane-Andrews kinetics presented in Figure 3, there is an optimal substrate concentration range of 0.25-0.65 C mol\(\cdot\)L\({}^{-1}\). When the substrate concentrations in the reactor fall within this range, the maximum substrate conversion rate is achieved, and biomass growth occurs at the maximum growth rate. Lower initial substrate concentrations lead to mass transfer limitations, while higher initial substrate concentrations result in substrate inhibition of bacterial biomass [21; 32]. Based on the presented results and modeling data, it can be concluded that, when operating with high substrate concentrations, a slight substrate inhibition of bacterial biomass was experienced within the first 10 h of operation. Similar results have been described in the literature when dealing with high initial substrate concentrations. However, a markedly different behavior was observed after 40 h of operation. As shown in Figure 1, biomass growth was negligible in all cases after this period. This can be attributed to the complete consumption of the substrate at concentrations of 0.30 and 0.91 C mol\(\cdot\)L\({}^{-1}\). In contrast, when operating with concentrations of 1.52 and 2.12 C mol\(\cdot\)L\({}^{-1}\), the substrates were fermented without net biomass growth. The different trend observed after 40 h of operation cannot be solely attributed to substrate inhibition of bacterial biomass. This is because, after 40 h, the substrate concentration was lower than at the beginning. For example, for the initial concentration of 1.52 C mol\(\cdot\)L\({}^{-1}\), the substrate concentration was about 0.6 C mol\(\cdot\)L\({}^{-1}\), which falls within the non-substrate inhibition zone according to the Haldane-Andrews model previously fitted. In the literature, this phenomenon hasbeen explained by product inhibition of biomass [23]. This type of inhibition is caused by the accumulation of fermentation products, which can only occur after several hours of operation. In this case, the undissociated fermentation products can permeate across the membrane increasing the maintenance requirements of microorganisms and reducing the other metabolic routes [12; 23; 26]. When operating at high initial substrate concentrations, a significant amount of fermentation product is generated. The high concentration of these products can reduce and even stop the fermentative process. The consumption of substrates without biomass growth can be explained by increased maintenance requirements, which can reduce and even make biomass yield negligible.