## 1 Introduction
At least three groups of microorganisms are involved in the AD process: primary fermenting bacteria, anaerobic oxidizing bacteria, and methanogenic archaea. Bacteria degrade biomass primarily to acetate and hydrogen. The acetoclastic and hydroperotrophic methanogens are responsible for the conversion of the acetate or the hydrogen into carbon dioxide and methane, respectively [10]. Recently, most studies have used molecular analysis to investigate the composition and structure of microbiota in anaerobic digesters and to correlate them with the use of different substrates [11; 12; 13]. Although the results still depend on factors such as DNA extraction, primers, and amplification region, the application of next generation sequencing (NGS), which includes metagenome analysis, has been shown to be of great value in investigating the specific genomes, cultured and uncultured, that are present in an environmental community. In addition, these studies have contributed to increasing the genome repository in the collections of public databases [14]. In an earlier study, a reddish pigmentation was observed inside anaerobic digester units that were kept under light and AD anoxygenic conditions. That population of bacteria was identified as \"purple non-sulfur bacteria\" (PNSB) after analyzing the nucleotide sequences of the 16S rRNA genes [15]. Other authors have also spotted PNSB in the strictly anaerobic sewage sludge digester and in waste logoons. They observed a pigmented bloom of PNSB with an intense red color in logoons, and the bloom was associated with a significant reduction in odor [16]. These bacteria are photoheterotrophic organisms, i.e., they can use organic carbon compounds under light. At low levels of sulfide (H\({}_{2}\)S), below 0.5 mM, purple non-sulfur bacteria are able to oxidize sulfide to sulfur (S\({}^{0}\)), thiosulfates (S\({}_{4}\)O\({}_{6}\)\({}^{2-}\)), or sulfate (SO\({}_{4}\)\({}^{2-}\)). They were named PNSB because when sulfur is formed it is deposited extracellularly, and so when observed under the microscope they do not show sulfur globules, as compared with PSB [16]. Another study used PNSB as a bioaugmentation strategy in AD, which resulted in the enhancement of the overall performance of their indigenous methanogenic culture through the attained upgrade to both the organic load removal efficiency and the yielded biogas quantity and quality [17]. PNSB has bacteriochlorophyll \(a\) and \(b\) and carotenoid pigments, the latter including spirilloxanthin, spheroidene, lycopene, and rhodopsin [18]. These pigments can give them colors ranging between purple, red, brown, and orange, depending on the relative content. Lycopene is an important carotenoid with applications as food additives, drugs, and cosmetics due to its anti-oxidative, anti-cancer, and anti-inflammatory activities, but its extraction from plants suffers from seasonality. The proposed alternative for the sustainable production of lycopene has been through the growth of PNSB. The accumulation of carotenoids during the growth of PNSB can be exploited for biotechnology applications [19; 20]. Moreover, there is evidence that the application of PNSB in plant growth has benefits due to the accumulation of polyphosphate, pigments, and vitamins and the production of plant-growth-promoting substances (PGPSs) [21]. The main purpose of this study is the application of microalgae for energy purposes with the simultaneous production of energy carrier gas, digestate, and pigments, using microalgae materials of _Chlorella protothecoides_ as AD process substrates, within the biorefinery concept framework. As far as the authors are aware, the production of these products simultaneously and during the same digestive anaerobic process/anaerobic reactor has never been the subject of any investigation by other researchers.