Selection of Soil- and Wastewater-Derived Indigenous Anaerobic Bacterial Isolates for Enhanced Lignocellulosic Substrate Degradation and Methane Production

Lignocellulosic biomass is an abundant renewable resource, yet its effective utilization remains limited due to its structural recalcitrance, primarily attributed to lignin. While aerobic lignin-degrading microorganisms, particularly fungi, have been extensively studied, much less is known about bacteria capable of lignin depolymerization under low-oxygen conditions. This study focused on the isolation and evaluation of native anaerobic bacterial cultures capable of degrading lignin-derived compounds to enhance biogas production. Soil samples from decaying vegetation and olive mill wastewater were used as microbial sources. Enriched cultures were developed anaerobically using kraft lignin and <i>p</i>-coumaric acid as sole carbon sources. Twelve pure bacterial strains were isolated and screened for their ligninolytic activity. All strains were able to degrade <i>p</i>-coumaric, with the highest biomass concentration reaching 387 mg L^-1 and maximum substrate consumption rate at 438 mg L^-1 d^-1. When kraft lignin was used as sole carbon source, 9 out of 12 strains showed growth, with a maximum of 55 mg L^-1 over 11 days. Enzyme activity assays confirmed the production of lignin peroxidase and laccase, with highest values at 2.10 and 0.15 U mL^-1, respectively, even under conditions of limited oxygen. The enriched cultures were applied in biomethane potential (BMP) batch tests, resulting in increased methane production. The best performing culture resulted in a bioaugmentation percentage of 174% compared with control. These findings suggest that native ligninolytic bacteria can serve as promising bioaugmentation agents in anaerobic digestion of lignocellulosic waste.