## 3 Results
### Anaerobic Digestion of Microcalga Chlorella Protothecoides  
Biogas production was observed from the beginning in all the substrates tested, with no lag phase evidenced, as shown in Figure 1a. After the first week of the trial, the difference in the biogas production capacity of the tested substrates began to be noticed, and after 25 days it was possible to observe three distinct levels of accumulated biogas volume, namely: 125, 118, and 110 mL (HPA, AA, and HPEnz, respectively); 94 mL (HExt); and 59 mL (H and HPU). However, from this point onwards, the biogas volume collected in HExt increased the most, evolving positively over time until it equaled the volume recorded in HPA at 35 days of the trial (144 mL). The successive increases in the biogas volume in this unit suggest that the material being digested--the extracted heterotrophic alga (HExt)--was degraded in a more phased and gradual way throughout the experiment, compared to the others, but achieved the highest biogas volume (168 mL). Figure 1: Volume of gas produced during anaerobic digestion of algae: **(a)** accumulated biogas; **(b)** accumulated methane. STP—standard temperature and pressure. It is interesting to note the occurrence of a \"restart\" process observed in several units. For AA, the sharp increase in the biogas volume during the first 17 days of the test (6.3 mL/d, Figure 1a) was followed by prolonged small increments and, finally, about 25 days after, a further increase was observed in the biogas volume (4.0 mL/d). The other cases concern H and HPU units, which presented similar behavior to each other during the first 28 days of operation. However, on the following days, the HPU unit showed successive increases in the biogas volumes that end up being equal to those obtained in AA and close to the HPA production by the end of the experiment. These observations can be understood as a process of microbial consortium adaptation to the material being digested. In the case of HPU and AA, the microbial development in HPU occurred at a later stage than in AA. Nevertheless, the AA showed a higher biogas production than H.  
The behavior comparison of whole autotrophic and heterotrophic algae (AA vs. H) showed that the former reached a gas production of 113 mL in the first 18 days and that amount was never reached by H until the end of the run. It should be noted, however, that both AA and H showed marked increases in the volume of biogas in the test's final phase, which indicates that the gas potential production of these substrates may be higher than those recorded. This study showed that the lipid-extracted _Chlorella protothecoides_ provided the best accumulated methane production (HExt, 117 mL, Figure 1b) due to the important action of prior grinding before extracting the oil, and that a good evaporation of the solvent guaranteed the absence of solvent residues in the algal material. In addition, the pretreatment effect on methane production showed that either the autoclave (HPU, 115 mL) or ultrasound (HPA, 107 mL) processes are great methods to disrupt the walls of microalgal cells, allowing aqueous solubilization of organic matter, as referred to by other authors [26]. [PERSON] et al. [27] reported the highest accumulated methane production of 121 mL obtained with the microalgae biomass pretreated using the autoclave method. [PERSON] et al. [28] demonstrated the impact of ultrasound pretreatment on methane production, increasing the sonication time and improving the anaerobic degradability of algal biomass. All the above-mentioned methods originated substrates that provided the highest methane volumes compared with the enzyme pretreatment (HPEnz, 78 mL). Effectively, the units digesting the heterotrophic algae pretreated by an enzymatic process were the only ones that showed a decrease in biogas production in the assay's final stage, indicating the exhaustion of the process (Figure 1a). Microalgae like _Chlorella_ sp. contain complex cell walls that are composed of an outer layer, which may be resistant to the AD process [29].