The Metabolomic Analysis Of The Response Of Pseudomonas Sp.Hf-1 To Nicotine Stress And Its Degradation Mechanism And Bioremediation Of The Tobacco Waste Contaminated Soil

The study investigated the metabolomic response of Pseudomonas sp. HF-1 to nicotine stress, its biodegradation of nicotine, the ecological and the molecular mechanisms of bioremediation of tobacco waste contaminated soil. The main results were as follows:(1) One of the predictive gene coding amine oxidase in the nicotine catabolic gene cluster of pMHl was amplified by PCR and subcloned into prokaryotic expression vector pET-41b (+) to construct the recombinant pET-41b-amo. Transforming pET-41b-amo into E. coli BL21 (DE3) constructed the expression strain BL21-pET-41b-awo. The hypothetical protein in the form of inclusion body was highly expressed inducing by IPTG. According to SDS-PAGE, the molecular weight of recombinant protein was about 55 kDa. Also, the amine oxidase activities were determined using nicotine as the sole substrate, and the results showed that the amine oxidase induced by nicotine had higher enzymatic activities than that induced by glucose. It can be inferred that the oxidase encoded by the amo played an important role in he ncotine metabolic pdthway.(2)A standard one-dimensional NMR spectra was performed for each sample using a 90°radio-frequency pulse, with 90°pulse length of approximately 10μs, an additional relaxation delay of 2 s, a mixing time of 100 ms and t1 of 6.5μs. With the NMR spectroscopic analysis, NMR data processing and multivariate data analysis, the results showed that the NMR-based metabolomic technique provided an efficient method to identify nicotine-induced stress effects at the level of metabolites. The metabolome of Pseudomonas sp. HF-1 is largely dominated by 42 metabolites, including 8 aliphatic organic acids,6 amino acids,3 sugars and 11 nucleotides. The higher nicotine concentration resulted in the delayed exponential phase of bacterial cells accompanied with retarded nucleotide biosynthesis. Nicotine caused the decreased glucose catabolism, increased succinate accumulation and severe disturbance in osmoregulation and complex antioxidant strategy. A further increase of nicotine level was a critical threshold value that triggered the change of metabolic flow. Further investigation of the information at these levels will provide the universal insights into the molecular mechanism responsible for nicotine stress to Pseudomonas sp.HF-1.(3) The highly effective nicotine-degrading bacterium Pseudomonas sp. HF-1 was augmented in the tobacco waste contaminated soil to evaluate the effects of bioremediation. In comparison with non-adding (NA) systems, the ones with addition of stain HF-1 (AT) exhibited considerably stronger pollution disposal abilities, higher stability of pH value and moisture content, especially in groups with large quantity of pollutants. The microbial community structures during bioaugmentation were systematically investigated using denaturing gradient gel electrophoresis (DGGE). The fingerprint profiles showed that the Shannon-Wiener index decreased with increasing tobacco wastes in the NA treatments. However, a stepwise increase of the Shannon-Wiener index was found in the AT groups. A comparison of sequences excised from DGGE gels demonstrated that there were differences in the dominant microbial species between the two treatments, and suggested that AT could enhance the whole disposing efficiency. The results of real-time qPCR indicated that stain HF-1 existed in the AT systems and grew with relative high quantities. The oxidase stress of the microbial community during the process of the nicotine stress was detected by the SOD and CAT enzymatic activity and GSH and MDA content. At the beginning of the stage, the SOD and CAT enzymatic activity was suppressed by high concentration of pollutants, while the content of MDA increased. With the degradation of nicotine, the enzymatic activity and GSH content increased. So, it can be concluded that microbes could adjust their mechanism to protect themselves when poisoned by pollutants.