Dionisi Hebe M, Layton Alice C, Harms Gerda, Gregory Igrid R, Robinson Kevin G, Sayler Gary S
Department of Microbiology, The University of Tennessee, Knoxville, Tennessee 37996, USA.
Appl Environ Microbiol. 2002 Jan;68(1):245-53. doi: 10.1128/AEM.68.1.245-253.2002.
Utilizing the principle of competitive PCR, we developed two assays to enumerate Nitrosomonas oligotropha-like ammonia-oxidizing bacteria and nitrite-oxidizing bacteria belonging to the genus NITROSPIRA: The specificities of two primer sets, which were designed for two target regions, the amoA gene and Nitrospira 16S ribosomal DNA (rDNA), were verified by DNA sequencing. Both assays were optimized and applied to full-scale, activated sludge wastewater treatment plant (WWTP) samples. If it was assumed that there was an average of 3.6 copies of 16S rDNA per cell in the total population and two copies of the amoA gene per ammonia-oxidizing bacterial cell, the ammonia oxidizers examined represented 0.0033% +/- 0.0022% of the total bacterial population in a municipal WWTP. N. oligotropha-like ammonia-oxidizing bacteria were not detected in an industrial WWTP. If it was assumed that there was one copy of the 16S rDNA gene per nitrite-oxidizing bacterial cell, Nitrospira spp. represented 0.39% +/- 0.28% of the biosludge population in the municipal WWTP and 0.37% +/- 0.23% of the population in the industrial WWTP. The number of Nitrospira sp. cells in the municipal WWTP was more than 62 times greater than the number of N. oligotropha-like cells, based on a competitive PCR analysis. The results of this study extended our knowledge of the comparative compositions of nitrifying bacterial populations in wastewater treatment systems. Importantly, they also demonstrated that we were able to quantify these populations, which ultimately will be required for accurate prediction of process performance and stability for cost-effective design and operation of WWTPs.
利用竞争性聚合酶链反应（PCR）原理，我们开发了两种检测方法来计数类似寡营养亚硝化单胞菌的氨氧化细菌和属于硝化螺菌属的亚硝酸盐氧化细菌：针对amoA基因和硝化螺菌16S核糖体DNA（rDNA）这两个目标区域设计的两组引物的特异性通过DNA测序得以验证。两种检测方法均经过优化，并应用于全尺寸活性污泥污水处理厂（WWTP）的样本。如果假设在总菌群中每个细胞平均有3.6个16S rDNA拷贝，并且每个氨氧化细菌细胞有两个amoA基因拷贝，那么在所检测的城市污水处理厂中，氨氧化菌占总细菌菌群的0.0033%±0.0022%。在一家工业污水处理厂中未检测到类似寡营养亚硝化单胞菌的氨氧化细菌。如果假设每个亚硝酸盐氧化细菌细胞有一个16S rDNA基因拷贝，那么硝化螺菌属在城市污水处理厂的生物污泥菌群中占0.39%±0.28%，在工业污水处理厂的菌群中占0.37%±0.23%。基于竞争性PCR分析，城市污水处理厂中硝化螺菌属细胞的数量比类似寡营养亚硝化单胞菌细胞的数量多62倍以上。本研究结果扩展了我们对污水处理系统中硝化细菌菌群相对组成的认识。重要的是，这些结果还表明我们能够对这些菌群进行定量，而这对于准确预测工艺性能和稳定性以实现污水处理厂经济高效的设计和运行最终是必不可少的。
