## 3 Results  
### Chemical Composition of Substrates and Inoculum after Pretreatment Processes  
Although the organic load of the whole autotrophic alga (AA) is lower than the heterotrophic alga (H) (Table 1), AA has a more favorable pH for the AD process than H of 7.0 vs. 5.9, respectively. The neutral pH is preferable for the proper development of the AD process, whereas an acidic pH provides conditions for increasing the acid formation and creating an imbalance between the population that produces and consumes them. In an extreme situation, the excessive accumulation of acids causes a decrease in pH and a blockage of the AD process.  
The substrates HPA and HPU have the highest organic loads (23-24 g/L COD, 9-10 g/L TS, and 8-10 g/L VS, Table 1), followed by HPEnz and, finally, HExt with the lowest concentrations, corresponding to about half of the higher ones. In fact, as HExt is a residue that results from a process that removes a substantial part of its organic potential (the lipid fraction), it would be expected that this would be the comparatively most diluted substrate. A pH value of 6.2 was registered in the HPU, which was the most favorable when compared to the other substrates with slightly lower values (5.6-5.8).  
Under microscope observations, the structure of the materials to be digested revealed the extent the of cell wall disruption compared with the whole cells. It can be observed that H (Figure S1a) presents defined cell walls with slight changes due to the algae preservation method (freeze-dried after being stored in the freezer), as was expected. The structure of HExt (Figure S1b) shows extensive disruption with cellular burst and the agglutination of the cellular components. As for the heterotrophic alga pretreated with the enzyme (HEnz, Figure S1c), it is possible to observe some cellular disruption, although much less extensive than the previous case. The microscopic observations of the cellular structure of the algae after the pretreatment either with autoclave or with ultrasound show the identified zones with the cellular disruption and the introsimatic part of the cells (Figure S1d and Figure S1e, respectively). The autotrophic algae (Figure S1f), which were not subjected to any pretreatment, show whole cells with the characteristic green coloration, and few empty cells, which may also be the result of the preservation method.  
To assess the amount of dissolved organic compounds such as protein, carbohydrates, and DNA resulting from the applied pretreatment, the absorption spectra were measured at wavelengths from 190 to 290 nm, as shown in Figure S2. The greatest cellular disruption resulted from autoclave pretreatment, followed by ultrasound and untreated algae. The extracted algae were suspended in water, after lipid extraction; therefore, the release of components into the medium was moderate. In the case of algae pretreated with lysozyme, the resulting spectrum corresponds to the low cellular disruption observed at the microscope and presents the lowest intensity among all pretreatments. According to other research [25] involving cellulase, protease, and pectinase, in addition to lysozyme, the multiple-enzyme combination showed better results than the single-enzyme treatment. On the other hand, the costs of using multiple enzymes are higher.  
\begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Substrate** & **pH** & **COD (g/L)** & **TS (g/L)** & **VS (g/L)** \\ \hline H & 5.87 & 22.5 \(\pm\) 0.2 & 8.40 \(\pm\) 0.1 & 8.0 \(\pm\) 0.0 \\ HExt & 5.76 & 9.70 \(\pm\) 0.2 & 5.30 \(\pm\) 0.3 & 5.0 \(\pm\) 0.3 \\ HPEnz & 5.61 & 16.1 \(\pm\) 0.0 & 7.20 \(\pm\) 0.1 & 6.9 \(\pm\) 0.0 \\ HPA & 5.63 & 23.4 \(\pm\) 0.2 & 10.2 \(\pm\) 0.1 & 9.7 \(\pm\) 0.1 \\ HPU & 6.23 & 23.8 \(\pm\) 0.0 & 8.80 \(\pm\) 0.2 & 8.4 \(\pm\) 0.2 \\ AA & 7.04 & 16.4 \(\pm\) 0.0 & 9.50 \(\pm\) 0.2 & 7.6 \(\pm\) 0.0 \\ I & – & 17.6 \(\pm\) 0.4 & 12.5 \(\pm\) 0.1 & 9.1 \(\pm\) 0.0 \\ \hline \hline \end{tabular}
\end{table}
Table 1: Substrate and inoculum chemical composition: pH, COD, TS, and VS.