Molasses stillage, with its high organic content and nutrient-rich composition, represents a promising feedstock for biogas production. This study systematically evaluated its biochemical methane potential (BMP) using two inocula (wastewater treatment plant vs agricultural biogas plant) across three temperatures (40, 50, 60 °C) and initial substrate load (ISL: 2, 5, 10 g·Lincolum -1). The wastewater inoculum achieved superior methane yields (0.262-0.477 N m3·kgvs -1), peaking at 50 °C with a 22% increase over agricultural systems (0.192-0.378 N m3·kgvs -1). 16S rRNA sequencing revealed the wastewater treatment plant inoculum's superior functional diversity, dominated by syntrophic Chloroflexota and Acidobacteriota alongside methanogenic Methanobacterium (52-61% relative abundance) and acetoclastic Methanothrix (18-23%). In contrast, agricultural biogas plant inocula showed specialized thermophilic communities dominated by Bacillota (68-72%) and hydrogenotrophic Methanoculleus (29-34%). Both systems exhibited inhibition at 60 °C/ISL10 (yields reduced by 34-42%), correlating with declining diversity and Methanofastidiosum proliferation. These findings provide two key operational insights: wastewater inocula offer greater process stability due to microbial diversity, and 50 °C represents the thermal optimum for stillage codigestion, balancing yield and community resilience.

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