Analysis of Genetic Diversity and Functional Identification of Micro-organism Species in the Rhizosphere in Constructed Wetlands
|School||Central South University of Forestry Science and Technology|
|Keywords||constructed wetland denaturing gradient gel electrophoresis(DGGE) 16S rDNA Sequencing analysis microbial diversity|
Over the last two decades, constructed wetland systems, attributed to their special purification mechanisms and functions, have been widely used in municipal sewage treatment. As rapidly developed technologies with characters of low energy consumption, low construction and operation cost and simple procedure in maintenance and management, constructed wetlands have received great attention in the field of environmental protection. The samples used in the present study were collected from the constructed wetland system established in 2002 in Zhuzhou campus of Central South University of Forestry and Technology. The system was used since for treatment of both the sewage from the student dormitory and the wastewater from laboratory.It has been proved that constructed wetland is a complecated system with integrated functions combining various physical, chemical and biological reactions, of which the plant-microorganism interaction plays a very important role. It has been recognized that in the constructed wetland, as in all ecosystems, microorganisms are the main decomposer responsible for decomposition of the organic pollutants. The diversity and stability of the microbial structure and the restoration of the microbial community are key factors in maintaining the treatment efficiency and stability of the constructed wetland system. Thus the analysis of the microbial community structure in the filler environment of the wetland system and the identification of the species and functions of dominant bacteria in the rhizosphere areas are of high values in particular for better understandings of the pollutant removal functions and mechanisms of the constructed wetland system.The selective culture method and PCR-DGGE technology were applied in the present study to analyze the diversity of the microorganisms with main objectives to determine the evolution pattern and tendency of microbial community in the filler layers of the established wetland system using soil samples collected from different treatment cells. The results are summerized as follows:1. Optimization of DGGE technology1.1 Optimization of DNA extraction methods:The total DNA of soil bacteria was directly extracted without culture. The improved SDS method was shown to be better then other applied methods as it can generate the largest and highest purity DNA fragments which can be directly used for PCR amplification. As it is rapid and simple in operation with no need for special treatment the improved SDS is an ideal method for extraction of microbial DNA.1.2 Optimization of PCR-DGGE:Use of PCR-DGGE for determination of microbial genetic diversity, with F357GC and R518 primers,55℃of pathogenic fire temperature and 35 cycles, yielded the ideal products. The optimal conditions for Parallel DGGE electrophoresis were determined by time processing. The best separation of PCR fragments was obtained at 5h and 150V.1.3 Application of the PAGE silver staining saved high amounts of reagents and materials. The method was short time consuming and simple processing with low background staining from the glue and high resolution rate. Use of the improved boiling method to recovere the differential bands from PAGE simplified the procedure and reduced the loss of information in the second PCR amplification process.2. Selective culture method and PCR-DGGE experiments2.1 Selective culture method:The number of microorganisms in the cells both with and without plants changed with the seasons. Following the "Autumn-Winter-Spring-Summer" change sequence, two variation patterns, "high-low-high" and "high-low-high-low", were observed. In the same season, the number of microorganisms in two treatment units exhibited generally the "low-high-low" changing trend.2.2 PCR-DGGE:The number of species of soil microbial community was found to be apparently greater in summer than in other seasons, the lowest number was found in winter and no signifiant difference observed between spring and autumn. The number of microbial community was much greater in plant zones than in the control. Results from the UPGMA cluster analysis of sample basis on DGGE fingerprints showed that the structure of bacteria in soil in rural communities in different seasons was obviously different. Shannon index analysis showed that the diversity of microorganisms in the two treatment units (with plants and without plants) in the same season first increased and then decreased along the flow direction.2.3 Cloning and sequencing:From selected part of the predominant bands by cloning and sequencing,5 different sequences were obtained. Through NCBI homology analysis and retrieval, the nearest known origin groups were located as Enterobacter, Vibrio, Erwinia and Klebsiella oxytoca, respectively,. The BLASTX comparison test showed that the five samples were most similar to 3,4-dihydroxy-2-butanone-4- phosphate, hypothetical protein, low-quality protein and conserved hypothetical protein.