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Figure 8 Active methanogenic pathways. Schematic representation of the specific enzymes of the three methanogenic pathways, hydrogenotrophic (CO2), acetoclastic (Acetate) and methylotrophic (MeOH) based on the KEGG pathway modules M00567, M00357, and M00356. For ... Discussion In the work presented here we show that it is possible to use a haloalkaline anaerobic microbial community for the anaerobic digestion of the microalgae Spirulina at alkaline conditions (pH 10 and 2.0 M Na+), and that the obtained biogas is rich in methane. The study of the haloalkaline anaerobic community indicates that Bacteroidetes and Methanocalculus play a key role in the anaerobic digestion at these extreme conditions. Biogas rich in methane As expected, by applying alkaline conditions in our anaerobic digester, methane rich biogas was obtained (Figure ?(Figure1).1). This was due to the fact that the solubility of carbon dioxide in a solution is determined mainly by the pH of the solution and its buffering capacity. Because of the high pH, the (bi)OTX015 nmr carbonate concentration in the medium can be high while a driving force for carbon dioxide absorption is maintained. Since all absorbed/hydrated carbon dioxide immediately reacts with OH? to form (bi)carbonate, CO2 absorption kinetics are faster than at neutral pH. With the high pH and alkalinity used in our experiments, the reactor's medium acted as a CO2 scrubber and the carbon dioxide remained in solution as carbonates (alkalinity) which resulted in a low percentage of CO2 in the headspace (Figure ?(Figure1).1). In all three experiments, this scrubber effect produced biogas with a high percentage of methane that ranged from 77 to 88% (Table ?(Table3)3) with peaks up to 96% in the Alk-HRT (Figure ?(Figure1A),1A), 92% in the Alk-OLR (Figure ?(Figure1B)1B) and 90% in the Alk-Opt (Figure ?(Figure1C).1C). These values for methane content are higher than the 78% obtained in the study of Van Leerdam et al. (2008), which is, to date, the only other study of anaerobic digestion at high pH. A further interesting and important observation was that, as in the case of van Leerdam et al., no H2S was detected in the biogas during all three experiments. This high methane content and the absence of H2S make this biogas suitable to be used as biomethane for vehicles and national gas supply grids with none or only a minor upgrade. For example, in Germany the minimum required content of methane in biomethane is 96%, in Norway 95% and in Netherlands 88% (Persson et al., 2006). Biogas production Daily biogas production ranged from 35 to 60 ml per day in all three reactors Alk-HRT, Alk-OLR, and Alk-Opt (Figure ?(Figure1),1), comparable to what was found previously with methanethiol as substrate which, to date, is the only other known substrate digested at alkaline conditions (Van Leerdam et al., 2008).