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Tobacco wastes that contain nicotine alkaloids are harmful to human health and the environment. In the investigation, a novel nicotine-biodegrading bacterium TND35 was isolated and identified as Pseudomonas plecoglossicida on the basis of phenotypic, biochemical characteristics and 16S rRNA sequence homology. We have studied the nicotine biodegradation potential of strain TND35 by detecting the intermediate metabolites using an array of approaches such as HPLC, GC–MS, NMR and FT-IR. Biotransformation metabolites, N-methylmyosmine, 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) and other three new intermediate metabolites namely, 3,5-bis (1-methylpyrrolidin-2-yl) pyridine, 2,3-dihydro-1-methyl-5-(pyridin-3-yl)-1H-pyrrol-2-ol and 5-(pyridin-3-yl)-1H-pyrrol-2(3H)-one have been identified. Interestingly, these intermediate metabolites suggest that the strain TND35 employs a novel nicotine …