【作者】 张汉波；

【导师】 段昌群；

【作者基本信息】 云南大学 ， 生态学， 2003， 博士

【摘要】 土壤微生物群落的生物量和物种多样性变化一直是评价重金属急性毒害效应的重要指标,然而由于重金属长期留存于环境中,因此分析重金属的长期生态效应,尤其是对微生物种群生理特征和遗传结构的深远影响将具有重要的理论意义和实践价值。不仅可以揭示微生物对重金属污染环境的适应进化策略,为生物多样性保护提供理论支持,还可以指导污染环境的生物修复。本研究以堆积时间为10 年、20 年和近100 年的铅锌矿渣堆中的微生物类群为对象,在群落水平和种群水平上分析了矿渣堆中的微生物进化生态特征,得出如下结果和结论: 理化研究表明矿渣是一种极端重金属污染环境。它们具有很高的铅、锌、镉重金属含量,总量分别达到4481.08±716.23、3590.03±112.63 和63.31±7.82mg kg-1,有效态含量也分别达到172.95±49.73、27.62±0.23 和14.49±1.97mg kg-1。随堆积年代延长矿渣发生明显酸化,导致可溶性Pb 含量增加。同一个矿渣堆不同深度层次之间的差异主要也是可溶性Pb 含量。在空间上,年轻矿渣堆三个深度层次的重金属总量没有差异,但年代稍长的矿渣堆表层重金属总量较低,中层和下层相对较高,这种差异随年代增加而增大。此外,矿渣堆组成颗粒大,植被少,持水能力低。在群落水平上微生物总生物量、抗性细菌比例以及物种多样性指标在矿渣堆和同一矿渣堆不同深度层次之间有较大差异。同校园土壤相比,矿渣中可培养真菌数量差异不大,但细菌和放线菌均减少近一个数量级;矿渣堆具有较高Zn、Pb 耐受细菌比例,且大部分样品抗0.5mmol l^-1 Zn 或Pb 以及同时抗Zn 和Pb 的细菌比例大于100%;堆积时间长的矿渣堆C 表层具有相对低的抗性细菌比例。在同一矿渣堆中,25-30cm 层比5-10cm 层有较高的微生物DNA含量,但平板分离技术则显示样品5-10cm 层的可培养微生物多。此外下层比上层有较多抗性细菌。根据优势菌落形态判断,三个矿渣之间以及同对照样品之间,细菌、放线菌和真菌的Shannon-Wiener 多样性指数没有显著差异,但矿渣同对照样品之间的微生物种类并不相同,且三个矿渣堆的25-30cm 层的物种多样性都比5-10cm 层高。经分析讨论认为,抗重金属细菌比例大于100%的现象暗示有部分细菌的生长对一定浓度的重金属产生了依赖,矿渣DNA 含量与平板计数的不一致性表明25-30cm 层有更多不能培养的微生物,而下层具有较高的抗性细菌比例有可能是矿渣上层较强的干旱胁迫导致。老矿渣更多还原

【Abstract】 Microbial biomass and biodiversity in soil have been widely used in the evaluation of acute toxicity of heavy metals. However, due to evolution of the resistance to toxic metals in microbial population, a long-termed ecological response to heavy metals should be considered, and especially the physiological and genetic characteristic should be described in order to reveal the changes in microorganisms under chronic pollution. This not only can address some adaptive strategies of microorganisms to heavy-metal-contaminated environments and offer important theoretical background for biological conservation, but also can provide the guidelines for the bioremediation of polluted environments in future. In this study, microorganisms were sampled from three lead-zinc mine tailings with about 10, 20 and 100 years of deposited history respectively, and evolutionary ecological characteristics of the microorganisms were studied at the levels of community and population. Some results and conclusions could be summarized as follows. Physiochemical analysis revealed lead-zinc tailing soil could be viewed as the extreme heavy-metal-contaminated environments. These environments contained extremely high total Pb, Zn and Cd, with concentrations of 4481.08±716.23, 3590.03±112.63 and 63.31±7.82mg kg-1, respectively. The soluble concentration of Pb, Zn and Cd in tailings were 172.95±49.73、27.62±0.23 and 14.49±1.97mg kg-1, respectively. The gradually acidification and long-term rainwater erosion increased soluble Pb, which mainly explained the differences between three tailing piles and between the layers of 5-10cm and 25-30cm. In the tailing soil with short time duration, total metals were with no much difference between three layers of 5-10cm, 25-30cm and 55-60cm, but with long time duration it was lower in the layer of 5-10cm than in both layers of 25-30cm and 55-60cm, and this difference enhanced with longer duration. Additionally, these environments in tailing soil had sparse vegetation and mainly consisted of large waste ore, thus showed a low capacity of water. At the level of community, among three tailing soils and between different layers there were significant differences in microbial biomass, proportions of tolerant bacteria and species diversity. Compared with campus soils, there was no much difference in the amount of fungi, but ten times less in the amount of actinomyces and bacteria in tailing soil. Moreover, soils sampled from tailings had much more proportion of heavy-metal-tolerant bacteria and most of them had more than 100% of tolerant bacteria with the MIC (minimum inhibitory concentration) of 0.5mmol l-1 of Zn or Pb, and Zn plus Pb. Relatively, the tailings C showed lower proportion of tolerant bacteria. In each tailing pile, the soil from the layer of 25-30cm had higher microbial DNA content and proportion of tolerant bacteria but lower number of cultivable microorganisms, in comparison of the soil at the layer of 5-10cm. Species number and Shannon diversity index of fungi, actinomyces and bacteria determined by the morphology of colony showed no significant difference between three tailings, even between tailings and control sample. However, the species of microbe were significantly different between tailings and control sample. It was explained that, the variation of microbial biomass revealed by DNA extracted directly from tailings and the enumeration of solid plates suggested that the layer of 25-30cm had more uncultivable microbes than the layer of 5-10cm. In this case, these uncultivable species maybe need supplement of some heavy metals. Spatially, that most tolerant bacteria tend to live in the deeper layer indicated that more intensified desiccation limits their accumulation in the upper layer. On the other hand, lower proportion of tolerant bacteria in old tailings, especially in the layer of 5-10cm of tailings C, is presumably related to the living status of microbes, and that means the microbes maybe live as microcolony or biofilm, and to a great degree, maybe mainly live in rhizosphere, where are generally enriched nutrients but low bioavailability of heavy metals which can support the growth of more sensitive bacteria. Correlation analysis showed the proportion of tolerant bacteria increased with more amount of soluble Cd, and the reduction of cultivable actinomyces and bacteria may be related to the increase of soluble Zn and species diversity was affected positively by water content of tailings. Phylogenetic analysis based on the partial 16S rRNA gene sequences indicated that two morphologically distinct types of colonies would be the undescribed species. The sixty numerically dominant bacteria belonged to three phylogenetically distinct groups G1, G2 and G3, and displayed detectable sequence distances to other species of genus Arthrobacter. Interestingly, their cell wall lacks lysine, an amino acid commonly found in all previously described species of this genus.Another 10 tailing strains belonged to genus Agromyces but displayed only 95.8% sequence similarity to the closest relative Agromyces mediolanus. Together with the facts of no formation of filaments and no fermentation of glucose, these bacteria were supposed as one novel species of genus Agromyces, and the name Agromyces tailingsis, sp. nov. was proposed for the typical strain CM01. Physiological and genetic analysis showed Arthrobacter G1 groups differentiated in integrated adaptation. Among 36 interspecies distinct characteristics (IDC), 6 were different between geographically distinct populations of G1, but among 27 interspecies nondistinct characteristics (INC), 14 were different. Moreover, the population from environment with high soluble Pb showed relatively low genetic diversity that was revealed by RAPD (Randomly Amplified polymorphic DNA) analysis. Additionally, different relationships were observed between resistant characteristics of adaptive population. For example, tolerance to desiccative stress was negatively correlated with resistant level of Cd and Kan, but there were positive correlations between tolerant levels of Cd and Zn, and between tolerances to NaCl stress and to H2O2 stress. The facts stated above suggested three points. Firstly, the effect of multiple ecological factors on biological characteristics of Arthrobacter population was nonsymmetrical in heavy metal tailing soil, and bacteria tend to regulate the INC relatively to IDC when facing the natural selective pressure. Secondly, adaptive costs occurred in these already-evolved Arthrobacter strains tolerant to heavy metal. Decreased genetic diversity of population living in the tailings with increased soluble Pb implied their evolutionary costs. Moreover, that heavy-metal-tolerant bacteria increased its sensitivity to desiccative stress also demonstrated the existence of adaptive costs. Thirdly, the occurrence of cross adaptation was also demonstrated by the positive correlation between tolerances to NaCl stress and to H2O2 stress. Therefore multiple stress ecological factors in tailings could play different roles in affecting the amount of actinomyces and bacteria, the population genetic diversity of Arthrobacter, species diversity and tolerant bacterial proportions. A model was proposed to explain the nonsymmetrical responses between different bacterial groups and different ecological factors. Totally, these results indicated that tailing pile could be the new source for finding newheavy-metal-tolerant microorganisms. Two morphologically distinct types of colonies investigated in this study may include more than 4 undescribed bacterial species. Meanwhile, several evolutionary ecological phenomena requires further investigation: (1) strong selective pressure and isolation imposed by tailings could facilitate formation of microbial species during a relatively short time; (2) the natural selection can work on the level above species; (3) different environmental factors could play different roles in microbial biomass, tolerant bacterial proportions, species diversity, and physiological and genetic diversity in these studied bacteria; (4) different biological characteristics also may have different responses to environmental modification.更多还原