Supplementation with iron oxide nanoparticles has been suggested as a potential method for improving energy generation through anaerobic digestion, specifically by enhancing the rate of methane production. This investigation examined the effects of iron oxide (Fe
3O
4) nanoparticles (NPs) on anaerobic co-digestion of cow manure (CM) and sewage sludge (SS) through batch testing conducted under mesophilic conditions (35 °C) using a RESPIROMETRIC Sensor System 6 Maxi—BMP (RSS-BMP). The use of Fe
3O
4 nanoparticles at doses of 40, 80, 120, and 160 mg/L (batches M1, M2, M3, and M5) was studied. The use of 160 mg/L Fe
3O
4 nanoparticles in combination with mixtures of different ratios (M4, M5, and M6) was further investigated. The findings indicate that the addition of Fe
3O
4 nanoparticles at a concentration of 40 mg/L to anaerobic batches did not significantly impact the hydrolysis process and subsequent methane production. Exposing the samples to Fe
3O
4 NPs at concentrations of 80, 120, and 160 mg/L resulted in a similar positive effect, as evidenced by hydrolysis percentages of approximately 94%, compared to 60% for the control (C2). Furthermore, methane production also increased. The use of Fe
3O
4 nanoparticles at a concentration of 160 mg/L resulted in biodegradability of 97.3%, compared to 51.4% for the control incubation (C2). Moreover, the findings demonstrate that supplementing anaerobic batches with 160 mg/L Fe
3O
4 NPs at varying mixture ratios (M4, M5, and M6) had a significant impact on both hydrolysis and methane production. Specifically, hydrolysis percentages of 94.24, 98.74, and 96.78% were achieved for M4, M5, and M6, respectively, whereas the percentages for the control incubation (C1, C2, and C3) were only 56.78, 60.21, and 58.74%. Additionally, the use of 160 mg/L Fe
3O
4 NPs in mixtures M4, M5, and M6 resulted in biodegradability percentages of 78.4, 97.3, and 88.3%, respectively. In contrast, for the control incubation (C1, C2, and C3) biodegradability was only 44.24, 51.4, and 49.1%.
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