| In this paper, basic characters of nicotine degradation by a novel nicotine-degrading bacterium, Ochrobactrum intermedium DN2, was studied. Medium and cultural conditions for strain DN2 growth and kinetics of co-metabolism of nicotine and glucose was investigated; Further, metabolites of nicotine degradation were determined by GC-MS and pathway of nicotine degradation was proposed. Strain DN2 also was used to degrade nicotine in tobacco extract, tobacco waste extracts for making reconstituted tobacco and upper cured tobacco. Main results of this paper were followed as:1. A novel bacterial strain which was able to utilize nicotine as its sole carbon source was isolated from soil in which tobacco had grown at Sanming region in FUjian Province and named as DN2. on the basis of phenotypic characterization, chemotaxonomic characterization and 16SrRNA gene analysis, strain DN2 was identified asα-proteobacteria, Ochrobactrum intermedium. Using 500mg·L-1 nicotine as its sole carbon, the strain was able to degrade 15mg of nicotine per liter per hour and 97.65% of nicotine was degraded within 36 h.2. In order to reduce biodegradation time, optimal factors are determined. The optimal conditions for nicotine degradation by strain DN2 were found to be 30°C, pH 6.5, 0.1% glucose, 0.1% yeast extract, 2000 mg·L-1 nicotine and 10% inoculum. 98.3% of nicotine was degraded by no-induced strain DN2 within 60 h under optimal conditions and process of nicotine degradation followed zero-order reaction kinetics of Monod model, while 99.2% of nicotine was degraded by induced cells of strain DN2 within 24 h and process of nicotine degradation followed also zero-order reaction kinetics of Monod model. The results also showed that tolerance of strain DN2 to nicotine was up to 5000 mg·L-1 when 0.1 % of glucose was added.3. The kinetics of co-metabolism of nicotine and glucose by strain DN2 was studied. The experimental results demonstrated that both nicotine and glucose could be degraded simultaneously. The results also showed that the biodegradation rate of nicotine was accelerated or inhibited by the presence of glucose, while glucose degradation was inhibited by the presence of nicotine. At relatively low concentration of nicotine (50 mg·L-1) and glucose (30, 60,120 mg·L-1), biodegradation kinetics of both nicotine and glucose could be described by first-order reaction kinetics model. It was also found that at higher substrates concentrations (nicotine 500 mg·L-1; glucose 120, 1000, 2000 mg·L-1), nicotine biodegradation followed zero-order reaction kinetics model. When glucose concentration was 120 mg·L-1, its degradation followed the first-order reaction kinetics model, however with the increase of glucose concentrations to 1000 and 2000 mg·L-1, the biodegradation of glucose followed the zero-order reaction kinetics equation.4. Plackett-Burman design was used to evaluate importance of the selected fifteen internal and external factors on the growth of Ochrobactrum intermedium DN2. Results showed that tryptone, MgSO4·7H2O, temperature, the initial pH value, volume of medium and incubation time were the main factors affecting the growth of strain DN2. Then, the central composite design and response surface analysis were used to determine the optimal levels of the main factors. The optimized medium was determined as follows: tryptone 11.34 g·L-1, beef extract 3.00 g·L-1, NaCl 5.00, MgSO4·7H2O 3.71 g·L-1, pH value 7.23; and the optimal cultural conditions were: cultural temperature, 32℃; rotatory speed, 120 r·min-1; volume of medium, 88 mL/250 mL Erlenmeyer and incubation time, 34 h. Under these conditions above, it allowed cell density of strain DN2 to be increased from 7.35×108 cfu·mL-1 to 6.49×109 cfu·mL-1 and time obtaining maximal cell production decreased from 48 h to 36 h. the experimental data have validated the theoretical values.5. The metabolites of nicotine degradation by strain DN2 were determined and analyzed. The first metabolite of nicotine degradation by strain DN2 was isolated from enzymatic reaction mixtures using sorption of Dowex 50 resin in the acid form. Based on the analysis of GC-MS, paper chromatography, ultraviolet spectrophotometry, color reaction and melting point determination, melting point of the product was 120-121℃, molecular weight was 178, Rf=0.12-0.13, the maximum absorbance of UV spectrum was 232nm and 295nm, A232/195=2.167, orange-red was showed when it reacted with FeCl3 and red with CeNO3 which is accordance with that of 6-hydroxynicotine. So it was identified as 6-hydroxynicotine. Using GC-MS analysis, seven kinds of metabolic intermediates of nicotine by strain DN2 were determined as 6-hydroxy-nicotine, 6-hydroxy-N-Methylmyosmine, 6-hydroxy-oxynicotine, 6-hydroxy-pseudooxynicotine, 2,6-dihydroxy-pseudooxynicotine, 2,6-dihydroxypyridine and 2,3,6-trihydroxypyridine, which may be consistent with A. oxydans P-34. The pathway of nicotine degradation by strain DN2 was also proposed.6. The effects of the initial pH value, levels of inoculumand culture temperature on nicotine degradation in tobacco waste extract were investigated in flasks using strain DN2. A Box-Behnken experimental design was applied in the design of experiments and in the analysis of the experimental data. Whenα=0.05, the results that three linear coefficients and all quadratic coefficients were significant. Among the interaction effects, interaction coefficients of the initial pH value vs. levels of inoculum, levels of inoculum vs. culture temperature were significant, while interaction coefficient of the initial pH value vs. culture temperature was not significant. The experimental data also allowed the development of an empirical model describing the interrelationship between independent and dependent variables. By solving the regression equation, the optimal values of the variables were determined as: the initial pH value of 7.27, levels of inoculum of 14.93%, temperature 31.85℃, with the corresponding Y=66.13%, while actual value was 64.82%, indicating that the model is satisfactory and practicable. Under these conditions, process of nicotine degradation in tobacco waste extract was also carried out. The results showed that 86.58 % of nicotine was degraded in 16 h, the degradation process of nicotine followed zero-order reaction of Monod model. These results indicated that strain DN2 is of application value in nicotine pollution treatment.7. In order to enhance further rate of nicotine degradation in tobacco waste extract and reduce the time of nicotine degradation, a medium for nicotine degradation by strain DN2 was optimized in presence of yeast extract, glucose and Tween 80 using response surface methodology. A full factorial central composite design was applied in the design of experiments and in the analysis of the experimental data. The results showed that the most significant variable influencing nicotine degradation was yeast extract, followed by glucose, and then Tween 80. Moreover, these three factors may interact with each other and combine to produce positive effects on nicotine degradation. The experimental data also allowed the development of an empirical model (P<0.0001) describing the interrelationship between independent and dependent variables. By solving the regression equation, the optimal values of the variables were determined as: yeast extract 0.94 g·L-1 glucose 1.01 g·L-1, and Tween 80 0.80 g·L-1. The rate of nicotine degradation with strain DN2 in the medium above was up to 95.55 % within 9.8 h. Response surface methodology proved reliable in developing the model, optimizing factors and analyzing interaction effects. The results provide better understanding on the interactions between yeast extract, glucose and Tween 80 for the nicotine degradation.8. Tobacco waste extracts for making reconstituted tobacco (TWE) contain high content of nicotine as the toxic compound. Strain DN2 was used to degrade nicotine in the extract and examined factors effecting nicotine removal. The results showed that it was advantageous to degrade nicotine by strain DN2 using ammonia solution. The results also showed that the optimal conditions of nicotine degradation by train DN2 was 30°C, 7.0, 15% inoculum, addition 0.1% yeast extract, respectively. Further, under conditions obtained above, the average degradation rate of nicotine was up to 140.55 mg·L-1·h-1 by three fed-batches of nicotine degradation in 30-L reactor. These results of this study indicated that strain DN2 was useful for reducing nicotine content in reconstituted tobacco. This is the first report of microbially mediated nicotine biodegradation in TWE used for manufacturing reconstituted tobacco.9. Preliminary studies on biodegradation of nicotine in the upper flue-cured tobacco by strain DN2 was carried out. The results showed that the tobacco treated by steam was propitious to nicotine degradation by strain DN2 and different between two methods, which spraying directly culture and cell suspension used for degrading nicotine was indistinctive. According to results of studies on nicotine degradation in the upper flue-cured tobacco (BF3) from AnHui Province by strain DN2, optimal conditions for nicotine degradation was obtained: moisture level of 75%, 0.1% yeast extract and glucose added, pH7.15, inoclum level of 15%, 30-37℃, which nicotine content reduced from 3.92% to 1.83% within 3d, and 56.18% of nicotine was degraded. These results indicated that strain DN2 was potential application in reducing nicotine content in upper flue-cured tobacco.
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