利用细胞计数手段和DGGE技术分析松花江干流部分地区的细菌种群多样性

松花江是我国东北地区的重要河流之一,为加强对其水环境微生物状况的了解,对松花江干流部分地区的微生物数量和多样性进行了分析。应用传统平板培养法和流式细胞技术测定水样中的细菌数;直接提取样品中的总DNA,以巢式PCR(Polymerase Chain Reaction)扩增细菌16SrDNA片段,应用聚丙烯酰胺凝胶电泳(Denaturing Gradient Gel Electrophoresis,DGGE)技术对扩增片段进行分离,研究水样和底泥样品细菌的种群多样性。实验结果显示,pH值为影响水环境中微生物总细胞数量的主要因素。水样中细菌群落多样性主要根据上下游分区,分区点在哈尔滨段附近,而底泥中细菌群落多样性的影响因素呈多样化,没有显示出较为明确的分区特征。

Analysis of bacterial diversity in the Songhua River based on nested PCR and DGGE

As the largest tributary of the Heilong River, the Songhua River flows about 1,434 kilometers from Changbai Mountains through Jilin and Heilongjiang provinces. It occupies a significant position in the ecological and commercial fields in North-east China. Suffering from complex contaminant conditions, the Songhua River aims for comprehensive environmental improvement and management. Thus, relatively accurate monitoring and investment are essential to provide basic data for decision and policy making. In this study, we aim to analyze the bacterial concentration and diversity in the river, along with their relationship to various environmental factors, in order to briefly evaluate the microbial condition in the Songhua River. Representative sampling points were chosen from upstream to downstream in the Songhua River. A total of fifteen water samples and ten sludge samples were collected. The bacterial concentration was assessed by both traditional heterotrophic plate counts (HPC) and a novel flow cytometric (FCM) technique. HPC used to be widely utilized as an approach to detect the cultivable bacterial communities; however, it has been the subject of some debate due to its non-negligible drawbacks and the development of new alternative tools. Results show that the order of magnitude of cultivable cells counted by HPC is about 106 while the order of magnitude of total cells counted by flow cytometry is about 104. It shows that traditional heterotrophic plant counts might easily neglect those cells that are uncultivable but possess physiological properties. Also, flow cytometry exhibits several advantages over HPC, for instance, more rapid rates and greater sensitivity. A regression analysis of environmental factors and microorganism concentrations was made based on four main factors: temperature, pH, dissolved oxygen and conductivity. Results show that pH has the most significant effect towards the total cells in each sample. The molecular technique using 16S rRNA profiles generated by PCR-DGGE was used in order to deter the variation in bacterial community structure of water and sludge samples from the Songhua River. Nested PCR and touch down PCR were utilized for the purpose of generating more DNA from samples, especially aquatic samples. A similarity analysis, cluster analysis and statistical analysis were performed according to the DGGE gels and the data output by Quantity One. The results showed that the bacterial diversity in water samples has an obvious discrimination between upper and lower zones, and the demarcation point is near the area of Harbin city. However, bacterial diversity in the sludge samples showed no significant differences along the river basin. The Shanno-Wiener Index, Uniformity Index and richness of water and sludge samples were calculated according to the DGGE profiles. The results showed that the bacterial diversity and richness decreases from downstream to upstream. The present study provides fundamental data for bacterial concentration and diversity, together with the influencing environmental factors, which could be used to support the future discrimination between functional zones in the entire Songhua River Basin.