The anaerobic co-digestion of microalga-bacteria biomass and papaya waste (MAB/PW) was evaluated under semi-continuous conditions. Microalgae-bacteria biomass was obtained from a high rate algal pond fed with municipal wastewater and artificially illuminated. The co-digestion of MAB/PW was evaluated using a 1:1 (w/w) ratio and an organic loading rate of 1.1 ± 0.1 g COD/L/d. Enzymatic activity assays of papain were performed in the feeding to determine the activity of this enzyme in the substrate mixture. A methane yield of 0.55 L CH4/gVS and 68% of total volatile solid removal were observed. The volumetric productivity was 0.30 ± 0.03 L CH4/L/d with a methane content of 71%. It was observed that papaya waste was a suitable co-substrate because it maintained a low ammonium concentration, decreasing the risk of inhibition due to ammonia and then increasing the methane yield of the microalgae-bacteria biomass compared to the biomass alone. The pretreatment effect by the addition of papaya waste on the microalgae-bacteria biomass was supported by the papain activity remaining in the substrate.

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