Isolation And Characterizations Of A High-Efficiency Alkane-Degrading Bacterium XCZ And Functional Analysis Of Its Monooxygenase Genes AlkB

A high-efficiency alkane-degrading bacterium XCZ was isolated from crude oil. It was identified as Pseudomonas aeruginosa according to its morphological, physiological and biochemical features and analysis of 16S rDNA sequences. XCZ could grow in the mineral medium containing 500mg/L n-octadecane as sole carbon source and degrade 97.5% n-octadecane after 40h of incubation. The most suitable temperature and pH for the degradation of n-octadecane by XCZ was 37℃and 7.0 respectively. XCZ was able to degrade C₁₃-C₃₂ n-alkanes in high concentration crude oil (2g/L) and the degrading rates of C₁₃-C₂₄ n-alkanes were more than 80%.Alkane monooxygenase genes alkB1 and alkB2 were amplified by polymerase chain reaction from the genome of P. aeruginosa XCZ. Both genes were above 99% identical to the counter genes in the type strain P. aeruginosa PAO1. In order to evaluate the activity of the two alkB genes in P. aeruginosa XCZ, two suicide recombination vectors were constructed: pEXBl'K for the disruption of alkB1 and pJQB2'T for the disruption of alkB1. The suicide recombination vectors were introduced into P. aeruginosa XCZ by triparental mating and alkB1 single mutant B1M, alkB2 single mutant B2M were successfully obtained. alkB1/alkB2 double mutant B12M was finally obtained by introducing pJQB2'T into B1M. Southern blot analysis indicated that there was only one copy of alkBl and alkBl respectively in the genome of P. aeruginosa XCZ. alkB1 in the mutants B1M, B12M and alkB2 in the mutants B2M, B12M had already been successfully mutated.Comparison of the growth patterns on different n-alkanes between the mutants and the wild strain was conducted for identifying the degradation range and ability of n-alkanes by AlkB1 and AlkB2. The experiment showed that the mutant B12M had lost its ability utilizing the C₁₃-C₁₆ alkanes but could still degrade the alkanes longer than C₁₇, which indicated that there was other hydroxylase(s) in P. aeruginosa XCZ for the degradation of alkanes longer than C₁₇, alkB1 and alkB2 were essential for the degradation of C₁₃-C₁₆ alkanes. B1M and B2M could both grow on C₁₃-C₃₀ alkanes. B1M and B2M grew faster than B12M and slower than the wild strain on C₁₄-C₂₀ alkanes as sole carbon source and had similar growth patterns with the wild and B12M strains on C₂₂-C₃₂ alkanes, which implicated that the alkane monooxygenase AlkBl and AlkB2 could both degrade the alkanes ranging from C₁₃-C₂₀ and were not able to degrade the alkanes longer than C₂₂. alkB2 may play more important role in the degradation of C₁₄-C₂₀ alkanes than alkB1 because B2M grew faster than B1M on C₁₄-C₂₀ alkanes as sole carbon source. The low efficiency of the degradation of C₁₃ and C₁₄ alkanes by B2M demonstrated the central role of AlkB2 on their degradation.