Biodegradation of benzo(a)pyrene by a genetically engineered Bacillus licheniformis: Degradation, metabolic pathway and toxicity analysis

Benzo(a)pyrene (BaP), the polycyclic aromatic hydrocarbons (PAHs), is known for its high carcinogenic, low bioavailability, and resistance to removal. To enhance the removal efficiency of BaP, a screened BaP-dominant degrading bacterium identified as Bacillus licheniformis, was utilized to construct a genetically engineered Bacillus licheniformis, named GEM-NY2. Through successive subculturing, it was observed that the genetic stability of the first 20 generations of GEM-NY2 remained good, and the removal rate of BaP was 78.21-80.16 %. Compared to the recipient strain NY2, GEM-NY2 achieved a BaP removal rate of 78.61 %, showing a 10.36 % improvement. Then the optimal degradation parameters of the GEM-NY2 for BaP were investigated by the response surface methodology (RSM): salinity of 30 g/L, temperature of 37.5celcius, pH of 6, and microbial inoculum of 3.5 %. Furthermore, UPLC-MS analysis confirmed that GEM-NY2 initiated the attack on BaP at sites C4 and C5, C7 and C8, and C9 and C10, leading to the formation of dihydroxy-BaP, then generating ring-cleavage products. Eight metabolites were obtained, and the ring-opening process of BaP by GEM-NY2 was revealed. The results of EcoSAR and acute toxicity experiments of Photobacterium phosphoreum showed as the benzene rings of BaP were cleaved, the toxicity of the metabolites gradually decreased. The luminescence inhibition rate decreased significantly from 82.89 % to 52.36 % within 14 days. In summary, the genetically engineered Bacillus licheniformis (GEM-NY2) constructed in this work exhibited promising bio-degradation potential towards BaP, highlighting its favorable prospects for the effective removal of PAHs.