## 3 Results
### Identification of Rhodobacter and Pigments in Anaerobic Digestion of Heterotrophic Algae  
A sample was collected at the end of AD to identify the reddish-pigmented cells developed in H and HPU units. The pigmented cells and typical reddish-like clusters were seen under an optical microscope (Figure 5). Figure 4: Relative abundance of archaeal communities and taxonomic classification at genus level. The analysis of the photosynthetic pigments was performed by spectrometry, and the absorption spectrum is presented in Figure 6. As expected, the absorbance maxima of whole cells were found at 862, 806, 592, 528, and 490 nm, corresponding to the presence of characteristic pigments of PNSB, as bacteriochlorophyll \(a\) and carotenoids of the spirilloxanthin series [46,47]. Figure 5: A liquid sample of culture medium observed under an optical microscope. Bar, 5 \(\upmu\)m. The black arrow shows the reddish-like clusters that contain pigmentation. Figure 6: Absorption spectrum of the whole-cells sample. The wavelength (nm) of the absorption maxima is shown at the top of the peaks. According to the literature, the color change resulted from an increase in phototrophic purple non-sulfur bacteria (PNSB) populations in anaerobic conditions [47; 48]. PNSB are found among the _Rhodospirillates_ order, including the genus _Rhodospirillum_, the _Rhizobiales_ order, which includes _Rhodpseudomonas_ and _Rhodomicrobium_, and the _Rhodobacteraceae_ family including _Rhodobacter_[49]. Concerning the source of PNSB in our study, 16S rRNA gene sequences were found to be affiliated with _Rhodobacter_ and _Rhodpseudomonas_, which were detected with a relative abundance of 0.48% and 0.26%, respectively, of the total bacterial sequences in the inoculum (Figure 7). At the end of AD, _Rhodobacter_ populations were not detected with extremely low relative abundance values in H (OUT) with 0.02% and in HPU (OUT) with 0.04% (Figure 7a) of total bacterial sequences. Thus, as previously mentioned, the changes observed in the samples collected after AD, from both H (OUT) and HPU (OUT), which revealed a dominance in \(\alpha\)-Proteobacteria (Figure 3d), may be related to the high relative abundance values of _Rhodpseudomonas_ populations (38.5% and 52%, respectively, Figure 7b), which are responsible for the production of the reddish pigmentation in the digestates. In addition, the prevalence of these microorganisms, which produce nitrogenase, the key enzyme for hydrogen generation, under nitrogen-limited conditions, and in a phototherapeutic mode using organic carbon as a source of electrons [50], is consistent with the simultaneous development of the hydrogenotrophic methanogens _Methanospirillum_ and _Methanobacterium_ in both H and HPU units during the AD process. The fact that the process is carried out in transparent glass vials, in daylight, and the presence of PNSB in the inoculum may explain the favorable conditions for the growth of _Rhodobacter_ and _Rhodpseudomonas_ populations and the production of reddish pigments in AD. According to [PERSON] et al. [49], the primary driving force for PNSB to obtain energy is the light, since they have light-dependent metabolism units as the pigments. In addition, the formation of a pigment system needs a reductive environment to prevent damage, so an anaerobic digester level provides good operating conditions [51]. In our study, these conditions were conducive to the development of PNSB, and it was observed that the biogas/methane yields were not much affected by this phenomenon. The highest value of 290 L CH\({}_{4}\)/kg VSin obtained with HExt showed only a small decrease to 263 L CH\({}_{4}\)/kg VSin in HPU units (Table 3), which means that it is possible to produce valuable pigments and simultaneously carry out the AD to produce high methane yields. Figure 7: Relative abundance (%) in total bacteria sequences, in each sample collected from inoculum, H, and HPU units, for the taxonomic classification of bacterial reads at (**a**) _Rhodobacter_ and (**b**) _Rhodpseudomonas_ genus level.