Studies On Soil Microbial Ecotoxicology And Microbial Degradation Of Nicotine

The dissertation is composed of two parts, the studies on the soil microbial ecotoxicology and the studies on microbial degradation of nicotine. Part 1. Studies on the soil microbial ecotoxicology of nicotineThe effects of nicotine on soil microbial community, soil respiration and soil enzymes were studied using the soil planted with vegetable. The culturable bacteria (plate counts), soil enzyme activities and changes in community structure (denaturing gradient gel electrophoresis (DGGE) analysis) were included for biological community assessments. The results will be valuable reference to building up alert index systems in nicotine-polluted soil, environmental quality evaluation and bioremediation of nicotine-polluted environments. The main results are reported as follows.1. The effects of nicotine at different concentrations on microbial population diversity, edaphon activity and enzyme activity of the soil planted with vegetable from Huajiachi compus of Zhejiang University, were studied systematically by the indoor culture method. The results suggested that every kind of microbial population, respiratory intensity, enzyme activity in the soil sample planted vegetable polluted by nicotine at different concentration, was varied with different degree, respectively, during incubation.The results showed that, the total number of bacteria in the soil samples polluted by nicotine, when the concentration of nicotine was higher than 0.008 μg.g~-1 dry soil (d. w.), was significant lower than that in the control group. The resumed rates of bacterial number were slower than that the control group during incubation, noteworthly. However, the nicotine at concentration of 0.004 μg.g~-1 d. w. might stimulate the growth of bacteria in the soil samples within initial short-term ofincubation. Actinomycete and aerobic nitrogen-fixing bacteria in the soil samples polluted by nicotine were inhibited evidently during incubation, while the fungi growth in the treatment soils was also inhibited by the nicotine within the initial two weeks, but then was stimulated, and its number was larger markedly than in the control group. The respiration intensity was different in the soil samples treated using nicotine from the control group within the initial four weeks of incubation. It was stimulated at first, and then restrained, such-and-such alternated twice, then resumed gradually. The results of enzymological studies indicated that the nicotine had a greateffect on proteinase activity in the soil samples planted with vegetable. The proteinase activity was inhibited by nicotine during the short-term of incubation, and there was a distinct dose-effect relationship between the nicotine concentration and the poteinase activity(R2=0.9485). But it was activated during the next term of incubation, and was significantly different from that of control soil. There was also obvious difference in the urease activity in the soil samples between the treatment soils by nicotine and the control soil. In the first two weeks, the urease activity was stimulated by nicotine with low concentration, but inhibited by nicotine of high concentration. However, all of the urease activities in each treatment soils were obviously higher than that in control soil after two weeks. In addition, the catalase activity in the soil samples polluted by nicotine was inhibited at first, and there was an obvious dose-effect relationship (R was 0.9222), and then it was activated. In contrast, the phosphatase activity in the soil was activated by nicotine at first, and there was a dose-effect relationship on day 7 (R2 was 0.8707). But all of the phosphatase activities in each treatment soils were inhibited on day 21, and then approached similarity after 28 days incubation.2. DGGE was applied to analyze the microbial gene diversity in the unpolluted and nicotine-polluted soils planted with vegetable from the profiles of nicotine concentration and acting time of nicotine. The results demonstrated that the similarity index of Jaccard between the treatment soils and the control soil decreased with the increase of nicotine concentration except for that from few treatment soils. There was an obvious dose dependent relationship between the nicotine concentration and the similarity index on day 7 and 35, but there did not appear a good linear.The results of time-effect relationship test suggested that the structure of microbial communities in the unpolluted soil had a similarity of more than 72 % to that of the original soilsamples during 42 days of incubation. The structure of microbial communities, however, varied in a major range in the nicotine-polluted soil, especially in the soils treated by the higher nicotine concentration, it was only 35.1 % where the similarity index of Jaccard between the origination and the soil sample incubated for 42days. In addition, the results showed that the variation of the structure of microbial communities in the soil samples polluted by nicotine were mostly occurred during the first stage of incubation. Therefore, the time-effect relationship of nicotine was not obvious.It was also found that the growth of some kinds of bacteria in the soil samples polluted by nicotine was inhibited by the analysis of their bands in DGGE gel during the incubation. With the increase of nicotine concentration or the prolong of polluted time, the color of their bands in DGGE gel changed from fuscous to undertone, even disappeared But other kinds of bacteria were enriched under the action of nicotine, and their bands changed from none to ens, or from undertone to deep color. Part 2. Studies on degradation of nicotine by microbe1. Study on microbiological degradation of tobacco tar. A bacterium, named strain MR4, was isolated from tobacco tar-contaminated soil and identified as Klebsiella oxytoca based on morphology, physiological and biochemical characteristics and phylogenetic analysis of 16S rDNA sequence. The organism grew optimally at 34 "C and pH 7.0 to 7.5. During growth on tobacco tar the isolate produced acidic materials, which caused the drop of pH in the cultures. GC/MS analysis indicated that the isolate had the ability to degrade phenolic compounds, heterocyclic compounds, especially nicotine in tobacco tar. The degradation rate of strain MR4 was 75.56% for nicotine, 35.84% to 58.16% for hydroxybenzenes and the other aromatic compounds, 29.15 % to 65.56 % for heterocyclics, and 35.17 % to 82.59 % for hydrocarbons.2. Study on microbiological degradation of nicotine. Two nicotine-degrading bacteria, strain HF-1 and strain HF-2, were isolated from tobacco waste-contaminated soil and identified as a member of Pseudomonas sp. and Arthrobacter sp., respectively, based on morphology, physiological tests, Vitek system (GN+), Biolog GN, 16S rDNA sequence and phylogenetic characteristics. Thermal denaturation test indicated that the G+C mol% of HF-1 and HF-2 was 62.7 and 63.5, respectively. The relationship between growth and nicotine degradation of the isolate suggested that both of strain HF-1 and strain HF-2 could utilize nicotine as sole source ofcarbon, nitrogen and energy. Viridescent pigment was observed during nicotine degradation by strain HF-1, and the color of strain HF-2's culture medium was turned to navy blue. Strain HF-1 grew optimally at 30 °C, initial pH of medium 6.5 to 7.5 and 1.3g I"1 of nicotine concentration in the nicotine inorganic salt medium. Strain HF-2 grew well at 20 to 33 °C, initial pH 6.5 to 8.0 and 0.5 to 2.0 g I'1 of nicotine concentration in the same media, and the maximum growth and nicotine degradation occurred at 30"C, initial pH 7.0 and 0.7 g I"1 of nicotine concentration in media under natural incubation condition, resulting in the determination of growth rates or the optical density (OD) of strain HF-1 and HF-2. Strain HF-1 could degrade 99.6% of nicotine under the optimized incubation conditions for 25 h, and strain HF-2 could degrade 100% of nicotine under the optimized incubation conditions for 43h. The concentrations of nicotine were monitored by high performance liquid chromatography. This work demonstrates both of Pseudomonas sp. strain HF-1 and Arthrobacter sp. strain HF-2 had a great ability to degrade nicotine, and they may be available for the application to the bioremediation of environments contaminated by tobacco waste.The metabolic intermediate of nicotine by Arthrobacter sp. strain HF-2 was determined by GC-MS, and the analysis result demonstrated that the nicotine-degradation pathway of strain HF-2 maybe is different from other bacterium of Arthrobacter sp. reported previously.