尾矿区不同植被恢复模式下高效固氮菌的筛选及Biolog鉴定

为了从铁尾矿中获得高效土著固氮菌,采用选择性无氮培养基从不同恢复模式下铁尾矿土样中筛选高效固氮菌,并且通过乙炔还原法对分离出的48株固氮菌进行固氮酶活性测定。结果表明:筛选出两株具有较高固氮活性菌株Db1与Ec1,Biolog鉴定为固氮菌属(Azotobacter),固氮比活力分别为203.20 nmol·mg-1·h-1和307.23 nmol·mg-1·h-1。不同植被恢复模式、物种多样性、尾矿坡向、工程措施、恢复年限均对尾矿固氮菌株数量有影响。样地处于尾矿阳坡,或属于人造混交林,或植被恢复年限长,又或者实施六孔砖、坡岩造地等工程措施的尾矿植被恢复模式下筛得的固氮菌菌株数较多或固氮活力较高。

Isolation and Biolog identification of the high-efficiency azotobacter from iron tailing under different vegetation restoration modes

Iron tailings are the wastes discharged after selecting the useful components from the grinded ore in dressing plants. The chemical composition mainly comprised iron, silicon, magnesium, calcium, aluminum, as well as a small amount of phosphorus, sulfur etc. Iron tailings are the special matrices with barren soil nutrients. A metal mine waste tailing dump is a typical degenerative ecosystem. Owing to their bad characteristics on the physical chemistry, the waste tailing dumps are going to become infertile lands generally. The wastes, such as tailings and acid mine wastewater produced during the ore dressing, not only destroy and occupy a lot of land resources but also result in serious pollution of the soil and water resources. Therefore, the research related to mining ecological restoration has become a focus. Phytoremediation is the key method for mining ecological restoration. Soil microorganism is the absolutely necessary biological factor on improving the soil environment and on further vegetation restoration in iron tailing dumps.There is a correlation between the microorganism quantity, its enzyme activity and the soil nutrient content. Some efficient native azotobacteria are able to accommodate and improve the particular iron tailings conditions, promote the soil microbe enzyme activity and activate the mineral nutrients in the soil. Azotobacteria can promote the absorption of nutrients in plant root system, enhance the host plant disease resistance and other stress resistances, and improve the soil structure. Of the microorganisms, azotobacteria are the dominant population in soils. It is possible to screen and isolate some efficient azotobacteria in the iron tailings. However, there have not been any publications of azobobacteria in the iron tailings. Moreover, the kinds of azobobacteria and their ability of nitrogen fixation are affected by plants, revegetation modes and site conditions. In the paper, the iron tailings soils were used for screening, isolating, analyzing of enzyme activity and Biolog identifying of azotobacteria. Soil samples were taken from the direct vegetation restoration forest (Sabina vulgaris forest, Amorpha fruticosa forest, Populus tomentosa forest and multi species mingled forest), the vegetation restoration forest via water and soil conservation engineering measures, the vegetation restoration forest via artificial afforestation construction, and the forest soils from different slope direction site conditions. It is the foundation for understanding the interaction mechanism between azotobacteria and host plants, and the tailings remediation mechanism. It is the base for trees selection of reforestation in iron tailings, and helpful for the improvement and development of mining areas.Acetylene reduction method was used to determine nitrogen-fixation activity of azotobacteria. Molecular nitrogen is reduced to ammonia by nitrogenase. Some compounds with N-N,N O, C N and C C bond also can be reduced by nitrogenase. The nitrogenase activity is analyzed according to the diversity of nitrogenase substrates. The process of acetylene being reduced to methane by nitrogenase can be quickly checked by gas chromatography simply and sensitively. The indirect determination method of nitrogenase activity has been widely applied.In order to obtain efficient native azotobacteria for iron tailings recovery, high-efficiency azotobacteria were screened by a nitrogen-free selective medium from tailing samples of different recovery modes, and the nitrogenase activities of the 48 strains isolated were measured by acetylene reduction. Db1, Ec1 with high nitrogenase activity screened from 48 strains by acetylene reduction, were identified as Azotobacteria by Biolog analysis, and their nitrogenase specific activities were 203.20 nmol·mg^-1·h^-1 and 307.23 nmol·mg^-1·h^-1 respectively. The numbers of strain were affected by different recovery modes, species diversity, slope direction of tailings and engineering measures. More strains with higher nigrogenase activity can be obtained from soils of the sample plots which covered by plants for longer time, or located in the sunny slope of tailings, or adopted artificial mixed forests, or with the engineering measures of six holes brick and slope plantation.