One of the factors that has a direct impact on anaerobic digestion is the applied organic loading rate (OLR). Increasing OLR can boost methane production but can also cause process failure. As a result, establishing the appropriate OLR for the procedure is critical. This study evaluated the effect of increasing the OLR using soybean molasses in a thermophilic anaerobic reactor (R-Thermo), as well as the effect of feeding strategy and co-processing with okara. Furthermore, the performance versus stability trade-off between R-Thermo and mesophilic anaerobic digestion (R-Meso) was investigated. The increase of OLR from 10 to 15 and 20 kg-COD/m³/d led to a decrease in COD removal efficiency (90, 86, and 75%), methane yield (12.0, 11.6, and 9.9 mol-CH/kg-COD) and an increase in total volatile acids concentration (251, 456, and 1393 mg-HAc/L, respectively). At 15 kg-COD/m³/d, R-Meso performed similarly to R-Thermo, and at 20 kg-COD/m/d, R-Meso outperformed (81% COD removal efficiency, 9.3 mol-CH/kg-COD and 154.5 mol-CH/m/d). Temperature greatly influenced the distribution of metabolic pathways, as shown by thermodynamic and kinetic analyses, thus impacting bacterial diversity. At 55 °C, amongst the bacterial genera, Tepidiphilus stood out (>28.2%), followed by Acetomicrobium, Coprothermobacter and Candidatus_Caldatribacterium. The OLR clearly impacted the archaeal community; Methanothermobacter (77.4%) was favored over Methanosarcina (14.8%). Under thermophilic temperature, it seems that syntrophic acetate oxidation (SAO) bacteria might have competed for substrate with acetoclastic methanogens, while in R-Meso microorganisms responsible for the initial steps of organic matter breakdown, such as members of the Firmicutes and Proteobacteria phyla (at least 67%), were dominant. In summary, R-Meso, characterized by a more uniform distribution of metabolic pathways, as well as a diverse and well-adapted microbial consortium, have exhibited enhanced stability and outperformed R-Thermo at high-loads.

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