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

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 bacte