Simplified Anaerobic Cultivation of Acetivibrio cellulolyticus and Methanosarcina barkeri: Implications for Lignocellulosic Biomethane Research.

Conventional anaerobic digestion relies on diverse inocula present in sludge-based systems. A defined consortium approach was investigated as an alternative. Acetivibrio cellulolyticus was chosen as the cellulose degrader, and two strains of Methanosarcina barkeri were selected as methane producers. Initial cultivation following manufacturer protocols highlighted significant challenges in maintaining strict anaerobic conditions, particularly in the absence of specialized infrastructure. A simplified anaerobic cultivation workflow was therefore evaluated for pure cultures of the selected anaerobes and subsequently used to evaluate a defined consortium using microcrystalline cellulose (MCC) and industrial lignocellulosic biomass (LB) residue as growth substrates. The workflow enabled successful cultivation of pure cultures in their recommended nutrient media without detectable contamination, as assessed by microscopy, aerobic contamination checks, and gas chromatography analysis. Growth-associated observations were obtained on MCC after prolonged incubation (~30 days); however, a metabolically active cellulolytic-methanogenic consortium was not established, as methane was not detected and no activity was detected on the LB substrate. This study demonstrates that anaerobic cultivation of fastidious microorganisms is feasible using a simplified method without fully controlled anaerobic environments and highlights inherent challenges associated with the defined consortium on substrates such as MCC and complex LB residue. The simplified workflow may provide an accessible approach to anaerobic cultivation for sustainable biomethane research in laboratories lacking specialized anaerobic infrastructure. Further work is required to determine conditions supporting methane production using the defined consortium.