Development of Population Dynamics Analysis Methods of Soil Microbes for Optimizing Bioremediation of Contaminated Soil

本研究引入微生物醌类、基因和微生物细胞介电特性作为分析土壤微生物种群动态的指标，以优化污染土壤的生物修复。建立了土壤微生物呼吸类醌的分析方法，并成功地将醌谱定义为各醌类的分数含量，作为分析土壤微生物相变化的工具。采用醌谱法研究了五氯酚等杀菌剂对土壤微生物相的影响。采用PCR技术分析了混合培养体系中特定细菌的数量。16S rRNA扩增量的对数与PCR周期呈线性相关，表明PCR技术可用于特定细菌的定量。在细菌混合培养系统中，还提出了16S rRNA限制性内切酶切割和不切割模式来对微生物进行分类。结果表明，YAG激光可以捕获大肠杆菌和pombe裂糖菌的细胞，并在高频交变电场下进行旋转。活细胞在1MHz处有一个临界旋转的峰值，而死细胞则有不同的临界旋转频率，这取决于杀死的过程。这些特征表明，峰值临界旋转频率的测量反映了电池的状况。研究了基于梯度力的净化方法。构建了0.2mm间隙的梳状电极。对含有大肠杆菌的样品溶液进行处理，去除率几乎达到100%。研究了非热放电等离子体在土壤灭菌中的应用。以O_2或Ar为载气，雾化H_2O_2，在常压下产生脉冲放电等离子体，在方波电压急剧上升的同心圆柱形电极上对气体进行处理。暴露在等离子体激发气体下，枯草芽孢杆菌孢子在小于10^<-6>的薄膜上存活60秒后的存活率降低。少

In the present study, microbial quinones, genes and dielectric characteristics of microbial cells were introduced as indices for analyzing population dynamics of soil microbes for optimizing bioremediation of contaminated soil.Analysis method of respiratory quinones of soil microbes was developed, and the quinone profile defined as fractional content of each quinone specie was successfully used as a tool to analyze the change of soil microbial phase. The effect of bactericide such as pentachlorophenol on microbial phase of soil was investigated by using the method of quinone profile developed in this study.PCR technique was introduced to analyze the population of specific bacterium in a mixed culture system. A linear correlation between the logarithm of amplification of 16S rRNA and PCR cycle was found, which showing that PCR technique could be used to quantify specific bacterium. Cutting and no-cutting patterns by restriction enzyme of 16S rRNA was also suggested to classify microbes … More in a mixed culture system of bacteria.It was demonstrated that Escherichia coli and Schizosaccharomyces pombe cells were trapped with YAG laser and rotated by a high frequency alternating electric fields. The living cells had a peak critical rotation at 1MHz, however dead cells showed different peak critical rotation frequencies, which depended on the process of killing. These characteristics suggest that the measurement of the peak critical rotation frequency reflects cell condition. Purification method based on gradient force was also studied. Comb like electrodes with 0.2mm gap were constructed. Sample solution containing Escherichia coli was treated, and removal efficiency achieved almost 100%.Application of non-thermal discharge plasma for sterilization of soil was also studied. Using O_2 or Ar as a carrier gas, H_2O_2 was nebulized and the gas was treated by pulsed discharge plasma generated under atmospheric pressure in a concentric cylindrical electrode energized with a sharp-rising square wave voltage. Exposure of the plasma excited gas reduced the survivability of Bacillus subtilis spores on a film of less than 10^<-6> after 60sec. Less