Isolation, Identification And Nicotine Metabolism Pathways Analysis Of Two Nicotine-degrading Bacteria

Base on the status of nicotine pollution and the requirement of treatment, with the aim of microbial nicotine degradation, the research was carried out through such aspects as isolation of high effective nicotine-degrading bacteria, their degrading charactractics and the nicotine degrading pathway analysis of isolated bacteria. The main results of this study were as follows:(1) Two novel nicotine-degrading bacterial strains were isolated from tobacco waste and identified as Acinetobacter sp. TW and Sphingomonas sp. TY, respectively, based on morphology, physiological and biochemical tests, Biolog analysis and 16S rRNA sequencing. The best culture conditions for nicotine degradation were 25-37℃and initial pH 7.0-8.0 for strain TW and 25-30℃and initial pH 6.0 for strain TY. Under the best conditions, the cell growth and nicotine-degrading kinetics of the two isolates were assessed, and 1.0 g/L nicotine was completely degraded within 12 h and 18 h for strain TW and TY, respectively. Moreover, the presence of four widely-used commercial neonicotinoid insecticides in the medium had no effects on nicotine degradation by strain TW; among the four tested neonicotinoids, only thiamethoxam significantly delayed nicotine degradation by strain TY. Strain TW and TY were also able to degrade selected neonicotinoids. This is the first report of bacterial members belonged to Acinetobacter sp. and Sphingomonas sp. having the ability to degrade nicotine. This study showed that these two newly isolated bacteria may be suitable for the disposal of tobacco waste and the reduction of nicotine in tobacco leaves.(2) Two newly isolated strains Acinetobacter sp. TW and Sphingomonas sp. TY capable of degrading nicotine were used to investigate nicotine metabolic pathway. Several intermediates from nicotine degradation by strain TW and TY were isolated via semi-preparative HPLC, and identified based on the results of NMR, MS/MS, FT-IR (KBr), HPLC, HPLC-DA, LC-MS and GC-MS analysis. The nicotine degrading pathway in strain TW was proposed to adopt pyrrolidine one which was reported in Pseudomanas sp., from nicotine to 6-hydroxy-3-succinoylpyridine through the intermediates N-methylmyosmine, pseudooxynicotine and 3-succinylpyridine. In addition, nicotyrine, myosmine and its precursor nornicotine were verified as metabolites of nicotine degradation in strain TW. The nicotine degrading pathway in strain TY was also proposed to be from nicotine to HSP, but through the unlike intermediates as 6-hydroxynicotine,6-hydroxy-N-methylmyosmine and 6-hydroxypseudooxynicotine. Therefore the nicotine degrading pathway in strain TY was satisfactorily verified different from either the pyridine one in Arthrobacter sp. or the pyrrolidine one in Pseudomonas sp.. Additionally, myosmine, cotinine and 2, 3-dipyridyl were verified as metabolites of nicotine degradation in TY. It was interesting that a newly found compound,3-(1-methyl-2,3-dihydro-lH-pyrrol-2-yl)-pyridine was produced during nicotine degradation by both strain TW and TY. These findings provide new insights into the microbial metabolism of nicotine and the environmentally friendly route of nicotine degradation.