Biomethane Production from Stillage: The Role of Temperature, Initial Substrate Load, and Inoculum Variability

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.