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0 g L?1 (Figure ?(Figure2B).2B). This accumulation of VFA, but especially of NH3, resulted in a sharp drop of the daily biogas production in Alk-HRT due to inhibition of the anaerobic microbial community. To recover the biogas production it was necessary to reduce the levels of inhibitory substances present in the reactor's sludge by ALPI stopping the feeding and gradually replacing sludge for fresh medium [see Section Determination of the Optimal Hydraulic Retention Time (HRT)]. This approach reduced the free NH3 content to 850 mg L?1, as well as the concentration of VFAs, allowing the anaerobic microbial community to recover and for the biogas production to resume (Figure ?(Figure2).2). By setting the HRT to 15 days the accumulation of NH3, and VFAs was prevented and the inhibitory effect reduced, which resulted in a stable biogas production. Figure 2 Parameters affecting the anaerobic digestion of Spirulina at alkaline conditions in the Alk-HRT reactor. Daily biogas production (gray line��left axis). (A) Nitrogen profile (right axis): Total Nitrogen (?), Total Ammonium Nitrogen (TAN) ... The biogas production in the Alk-OLR was influenced by the accumulation of OM (measured as CODT and CODS) which drastically affected its performance (Figure ?(Figure3).3). When the OLR was increased to 1.0 g Spirulina L?1 day?1, the CODT rapidly increased, from 10 to 17 g O2 L?1. A similar trend was observed for the soluble organic matter (CODS) which increased from 6 to 10 g O2 L?1. This accumulation of both OM, in the form of CODT and CODS, lead to the inhibition of biogas production due to substrate overload (Figure ?(Figure33). Figure 3 Parameters affecting the anaerobic digestion of Spirulina at alkaline conditions in the Alk-OLR reactor. Organic matter profile (right axis): Total (?) and Soluble (?) Chemical oxygen demand. Dashed vertical lines indicate a change in ... In contrast to what was observed in Alk-HRT, in Alk-OLR free NH3 and VFAs did not reach levels as high as in the Alk-HRT reactor. The NH3 concentration in Alk-OLR reached its maximum, 0.73 g L?1, in P-III when 1.0 g Spirulina L?1 day?1 was fed as substrate while the concentrations of VFAs remained low throughout the experiment with a slight increase in P-III when 1.0 g Spirulina L?1 day?1 was fed. When the reactor was operated under optimal conditions, Alk-Opt, no accumulation of NH3, VFAs, or OM was observed during the 67 days of operation. The three parameters remained under controlled levels throughout the experiment and did not affect the biogas production (Figure ?(Figure44). Figure 4 Main sludge components from the anaerobic digestion of Spirulina at alkaline conditions in the Alk-Opt reactor. Daily biogas production (gray line��left axis). Free Ammonia (NH3) (?); Total (?) and Soluble (?) Chemical Oxygen ...