Isolation and Evaluation of Lignin-Degrading Bacteria from Decayed Wood in a Landfill

Lignin is a major biomass resource whose utilization is expected to contribute to renewable energy production and the reduction of greenhouse gas emissions. Biological treatment using lignin-degrading microorganisms has attracted attention as an environmentally friendly alternative to conventional chemical pretreatment methods. While previous studies have mainly focused on white-rot fungi, their slow growth rates and limited genetic tractability have prompted the search for bacterial alternatives. Therefore, this study aimed to isolate lignin-degrading bacteria from decayed wood collected from a landfill site. Wood samples were collected from a landfill site in Muroran, Hokkaido, Japan. Microorganisms were enriched in Nutrient Broth (NB) medium and screened on glucose–yeast extract–peptone (GYP) agar plates containing Remazol Brilliant Blue R (RBBR). Decolorizing colonies were selected and cultured in alkaline lignin medium (1.0–5.0 g/L) to evaluate their lignin decolorization ability. Strains showing decolorization were further subjected to adsorption assays using cells harvested after 5 days of cultivation. In addition, lignin degradation products were analyzed by high-performance liquid chromatography (HPLC). Approximately 200 isolates showed decolorization on RBBR plates, whereas only two strains showed decolorization in alkaline lignin medium. These strains were identified as Bacillus subtilis CTU-1 and Proteus sp. CTU-3. Bacillus subtilis CTU-1 exhibited higher decolorization activity compared to CTU-3 and was selected for further analysis. Adsorption assays indicated that lignin decolorization was not caused by adsorption. The lignin decolorization rate of Bacillus subtilis CTU-1 reached 79.9% after 5 days of cultivation in medium containing 1.0 g/L lignin. HPLC analysis of lignin degradation products revealed peaks corresponding to 4-hydroxybenzoic acid, vanillin, and vanillic acid. These results demonstrate that Bacillus subtilis CTU-1 possesses significant lignin-degrading potential, providing a foundation for the development of environmentally friendly bacterial pretreatment technologies for lignocellulosic biomass.