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The present work proposes the optimization of the co-digestion of vinasse, filter cake, and deacetylation liquor in a continuous reactor by adding iron(III) oxide (Fe₃O₄) nanoparticles (NPs), comparing the results with a previous reactor operation without NPs. Initially, tests were carried out in batches with different NP concentrations, resulting in 5 mg L⁻¹ as the best concentration to be added in the continuous reactor along the increments of the applied organic load rate (OLR). Methane (CH₄) production reached a maximum value of 2.8 ± 0.1 NLCH₄ gVS⁻¹ (normal liter methane per gram of volatile solids), and the organic matter removal reached 71 ± 0.9% in phase VI (OLR of 5.5 gVS L⁻¹ day⁻¹). This production was 90% higher than the reactor co-digestion operation without NPs. The anaerobic digestion (AD) development was stable with stable organic acid (OA) concentrations, indicating the predominance of the propionic acid route to produce CH₄. The main methanogenic Archaea identified was <i>Methanoculleus</i>, indicating that the predominant metabolic route was that of acetate (SAO) coupled with hydrogenotrophic methanogenesis. The use of Fe₃O₄ NPs managed to improve the AD from the first-generation and second-generation (1G2G) ethanol production residues and stimulated microbial community growth, without modifying the preferable metabolic pathways.