Isolation,Identification And Degradation Of A Nicotine-degrading Strain

Nicotine was classified as an important biological insecticide which used alone or as a pre-mixed agent with other biological and chemical insecticides.It is mainly controlled for Aphidoidea,Pieris rapae,Heliothis assulta,Aphis gossypii,Dendrolimus and America's moth.Nicotine is a major toxic alkaloid in wastes generated from cigarette manufacturing processes.Nicotine pollution in the environment is derived from widely used insecticides and a large amount of high-concentration wastes by tobacco manufacture and processing activities.Due to its high toxicity,solubility,and persistence in the environment,nicotine caused the pollution of groundwater and soil.There is therefore an increasing concern on detoxifying nicotine.Microbial remediation has been frequently applied to remove different environmental pollutants because of its low cost,no secondary pollution and eco-compatibility.In this study,a bacteria strain S-1 capable of degrading nicotine was isolated from tobacco powdery wastes.Morphological,physiological,biochemical characteristics and 16S rRNA sequence of strain S-1 were identified.Degradation characteristics and metabolic pathway of nicotine by S-1 were also investigated.1.A nicotine-degrading bacterial strain S-1 was isolated from Chinese Tobacco Research Institute(Qingdao,China),can utilize nicotine as a sole source of carbon and energy.The isolate was identified as Pseudomonas sp.S-1 based on morphology,physiology and biochemical and 16S rRNA gene sequence analysis.The optimal growth temperature and pH of Pseudomonas sp.S-1 in beef extract-peptone medium are 30? and 7.0.2.Degradation rates of nicotine by strain Pseudomonas sp.S-1 in mineral salt medium(MSM)is relevant to temperature,pH and initial concentration of nicotine.A 100 mL flask that contained 20 mL of MSM with the addition of 400 mg/L of nicotine was inoculated with 0.6 mL of fresh cell suspensions(5.3 × 108 cfu/mL)of Pseudomonas sp.S-1 and incubated at pH 7.0.After incubation for 10 h,degradation percentages of nicotine were 73.5%,97.5%,98.7%,12.6%and 8.3%at 20,25,30,37 and 42? respectively.The degradation percentages of nicotine at pH 5.0,6.0,7.0,8.0 and 9.0 were 97.1%?98.6%?100%?100%and 5.3%,respectively after 10 h of incubation at 30?.The extent of nicotine degradation accounted for 100%,97.8%and 60.2%at initial concentrations of 100,400 and 800 mg·L-1,respectively after 12 h of cultivation at pH=7.0,30? and 0.6 mL of inoculating biomass(5.3×108 cfu/mL).These results indicated that the optimal degradation temperature and pH of Pseudomonas sp.S-1 are 30? and 7.0 and that the favorable concentration for nicotine degradation is 400 mg·L-1.3.Metabolic intermediates of nicotine were isolated with preparative-HPLC and characterized with liquid chromatography high-resolution mass spectrometry(LC-HRMS)and nuclear magnetic resonance(NMR).The main metabolites included 1-methyl-2-(pyridin-3-yl)-3,4-dihydro-2H-pyrrolium,cotinine,N-methyl-4-(pyridin-3-yl)butanamide,N-methyl-4-oxo-4-(pyridin-3-yl)butanamide,3-succinyl pyridine,6-hydroxy-3-succinylpyridine and 2,5-dihydroxypyridine.Nicotine was first postulated to dehydrogenation of its pyrrolidine moiety to produce a positively charged intermediate by protonation,1-methyl-2-(pyridin-3-yl)-3,4-dihydro-2H-pyrrolium.And then this metabolite was oxidized at position 5 of the pyrrolidine to yield cotinine.Cotinine was degraded to a new intermediate N-methyl-4-(pyridin-3-yl)butanamide by carbon-nitrogen bond cleavage of pyrrole ring between positions 1 and 5.N-methyl-4-(pyridin-3-yl)butanamide was further oxidized to form another new intermediate N-methyl-4-oxo-4-(pyridin-3-yl)butanamide.Subsequently,N-methyl-4-oxo-4-(pyridin-3-yl)butanamide was hydrolyzed and released methylamine to form 3-succinyl pyridine which was hydroxylated at position 6' of the pyridine to form 6-hydroxy-3-succinylpyridine.6-Hydroxy-3-succinylpyridine was further degraded to produce 2,5-dihydroxypyridine that would be entered citric acid cycle and further mineralized.Nicotine catabolism in strain S-1 undergoes a new pyrrolidine pathway that differs from the other catabolic pathways in bacterial species.This work sheds light on catabolic diversity of nicotine and heteroaromatics.The genes and enzymes responsible for nicotine degradation are needed to further studied in the future.