The Separation Of Nicotine Degradation Bacteria Identification, Degradation Characteristics And The Preliminary Research Of The Degradation Pathway

Nicotine, is a major alkaloid in tobacco Plants. Excessively taking into nicotine will restrain central nervous system, paralyse the heart, and even cause death; In manuafeutring tobacco products, wastes with high content of nicotine as the main toxic compound are produced, which seriouly contaminate the environment; and the nicotine content in the upper tobacco leaf from our country are generally high so that unable to be used as tobacco blend, which caused waste of resource and economic losses. Thus exploring an effective way to reduce the nicotine content in cigarette and environment is of great significance to human health. Interestingly, microbe and its metabolized enzyme can degrade nicotine, which is not only helpful for human health, but also improve the quality of smoke and utility of tobacco leaves and bring a lot of economic benefits.This study utilized nicotine as the sole source of carbon, 29 bacterial strains was isolated from soil and tobacco waste. The degradation efficiency test showed that all of the stains’degradation efficiency were more than 70%, and 19 strains among the isolates whose degradation efficiency were more than 90%. Further study was basing on 12 strains which have been isolated, by the experimental results of the morphological,physiological and biochemical analysis and the comparison of their 16S rDNA analysis, these 12 strains were identified as Arthrobacter sp., Pseudomonas sp., Ochrobactrum intermedium and Agrobacterium tumefaciens.Studies on the Agrobacterium tumefaciens BKCER11 strain’s degradation capability and optimization of fermentation conditions were carried out. Finaly the results showed that the BKCER11 strain could tolerate high concentration of nicotine up to 5g/L, when the concentration of nicotine in the culture medium was greater than 5g/L, the BKCER11 strain’s growth was highly inhibited, and the nicotine in the medium could not be degraded. The BKCER11 strain not only has a wide tolerance to pH, but also possesses the ability to tolerate temperature. Lots of experiments showed that the BKCER11 strain was stable at pH5.5-8 and growed well, and the optimum pH for the strain’s growing and degradating nicotine was 5.5; moreover the BKCER11 strain was stable at 20℃-37℃and growed well, and the optimum temperature was 30℃. In order to define the strain’s optimum degradation condition orthogonal test was used in the next study. The results indicated that the BKCER11 strain degradation efficiency could reach to 96.63%, when the peptone and beef extract simultaneously used as the complex nitrogen sources of the medium and their content were 0.5% respectively; besides some microelement was at the content of 5mL/L and the medium’s pH was at the range of 5.5-6.0, moreover the most important is the original content of nicotine was 2g/L and kept a temperature at 30℃.The intermediate metabolite was detected by using the method of GC-MS. Two novel intermediate metabolites, 3-(2,3,4-dihydro-3H-pyrrol-5-y1)-pyridine and cotinine were detected in the culture medium. They were both different from the substances involed in the three everkown pathways of microbial metabolism of nicotine.