Studies On The Isolation And Identification Of The Nicotine-degrading Strain ND1and Its Fermentation System

Nicotine, the main alkaloid produced in tobacco plant, can be as high as more than95%of the total alkaloid in tobacco. It is synthesized in the roots and transported into vacuole of leaves by vessel, which can resist biotic and abiotic stresses. Biosynthesis and accumulation of Nicotine could be induced by bad climate and environment conditions, infection of diseases and insect pests, mechanical damage, inadequacy nutrient and water management, and so on. As a heterocyclic compound, nicotine is hard to degrade by microbes. The nicotine produced by tobacco products and its wastes could accumulated in environment and cause harm to human health and environmental safety through "soil-plant-human body" or "soil-water-human body" or other pathways.Hunan is a major province cultivating and processing tobacco, and the tobacco acreage is over145mu for years. The extreme heat of Hunan in June and August is negative factor to normal maturity of tobacco, ripens tobacco quickly, causes the high content of nicotine in upper leaves of tobacco, downgrades the level of tobacco and affect the economic benefit of farmers. In order to solve this problem, we isolated endophytes from the upper leaves of tobacco collected from Changsha and Chenzhou cities, then screened endophytes which can degrade nicotine through using the nicotine as the only carbon source and nitrogen source. Then, we detected and compared the ability of these endophytes in degrading nicotine, the results are as following.1. The upper leaves (i.e., the4th and5th leaves under the heart leaves) from tobacco cultivar K326planted in Changsha and Chenzhou were collected to isolate the endophytes. Then the nicotine was used as selection pressure to screen the cultivable endophyte strains which can degrade the nicotine. There are419and458cultivable endophyte strains were isolated from the tobacco planted in Hunan Agricultural University base and Chenzhou, respectively. What is more, there are109of the419strains and120of the458strains could be cultivable in selection medium. After classification based on morphological characteristics,40of the109strains and42of the120strains were selected to proceed phylogenetic analysis based on16S rDNA. The results showed that48.6%of the strains are the dominant strains belonging to Pseudomonas. And the others are Stenotrophomonas (26.4%), Bacillus (15%), Erwinia (2.5%), Cupriavidus (2.5%), Enterbacter (2.5%) and Mangrovibacter sp.(2.5%). There is no obvious difference for the population diversities between the cultivable endophyte strains from Changsha and Chenzhou.2. Detected80endophyte strains’degradation ability of nicotine through analyzing the degradation rate of nicotine and growth increment of bacteria. It displayed that there were7strains isolated from Changsha, which could degrade nicotine more than76%, and11strains isolated from Chenzhou could degrade nicotine more than76%. Among of these strains, the stain NDl possesses strongest degradation ability that could degrade82.6%of nicotine (0.5g/L) in the medium in24h. Furthermore, this strain was identified as Pseudomonas fluorescens based on16S rDNA and BIOLOG methods.3. Preliminarily optimized the fermentation conditions of the ND1strain through single-factor experiment. The culture conditions for fastest speed and highest degradation rate of ND1degrading nicotine (0.5g/L) are6%inoculum amount,30℃,130rpm/min, and pH6.0-7.0. The orthogonal experiment showed that:after adding3.0g/L of corn starch, the NDl strain could degrade98.85%of0.3g/L nicotine in72h.