Polyurethane (PU), a widely used and durable plastic, persists in the environment, resulting in significant waste management challenges. Therefore, developing eco-friendly degradation technologies, such as screening for efficient biodegrading microorganism strains, is urgently needed to address this issue. Bacillus velezensis MB01B, an efficient polyester PU-degrading bacterium, was isolated from landfill soil and demonstrated the ability to degrade 91.4% of 0.75% Impranil DLN within 24 h under the optimal conditions (30.5 °C and initial pH 6.5). To assess the degradation capability of MB01B, three PU substrates of increasing complexity—Impranil DLN film, polyester thermoplastic polyurethane (TPU) film, and commercial PU desk mat—were tested; after 30 days, weight losses of 24.8%, 18.3%, and 5.4% were observed, respectively. In addition, SEM images showed significant morphological changes on the surface of these PU materials after treatment with MB01B. FTIR analysis of Impranil DLN films following degradation showed reductions in key functional groups (ester and urethane); and the identification of neopentyl glycol and 1,6-hexanediol as degradation intermediates suggested MB01B possesses the capability to hydrolyze ester and urethane bonds. Concurrently, genome sequencing combined with RT-qPCR identified several enzymes, including urethanases and esterases/lipases, involved in PU degradation. Based on these results, the pathway for MB01B to degrade Impranil DLN was inferred. Finally, MB01B was successfully formulated into a solid microbial inoculum with favorable storage properties and used for in-situ degradation of the commercial PU materials (Impranil DLN films, TPU films and PU desk mat) in landfill soil, underscoring its potential for the in-situ biological treatment of PU plastic wastes.
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