Selective plating medium and PCR-DGGE techniques were applied to analyze the seasonal changes in the number and diversity of micro-organisms in the rhizosphere of three sequenced cells in a vertical subsurface-flow wetland system constructed for municipal sewage treatment. The relationship between the community structures of the microorganisms and their functions in removal of the organic pollutants was also analyzed. The results obtained from the two applied methods showed that with the seasonal changes in the sequence of autumn-winter-spring-summer, the number of the micro-organisms displayed a general "up-down-up" pattern. The largest number of microorganisms from the rhizoshpere in three treatment cells detected by the selective plating medium method was in the autumn while that by the PCR-DGGE techniques was in the summer. Correlation analysis indicated that the seasonal change in temperature was the main factor that determined the observed pattern by affecting the growth of the microorganisms. In a given season, the number of micro-organisms obtained from the two applied methods displayed a different pattern in the three sequenced treatment cells. Towards the flow direction in the sewage treatment process, the number of micro-organisms increased from the first cell to the second cell and then decreased in the third cell, accordingly, with most of the organic pollutants being removed in the first two treatment cells.
Based on the structure comparability of the microbial community among treatment cells, all soil samples collected in the same season, except for sample no. 11, were clustered in the same branch, with all DGGE comparability values being higher than 20%, which can thus be classified into the same group. The DGGE spectrograms of the soil microorganisms from different seasons are different. The highest comparability value, 40%, was found in the spring, while the lowest, about 25%, was in the summer, indicating that the level of the microbial diversity was higher in the summer season under the tested conditions.
Four different sequences(YP1-YP 4)were obtained, and their closest known-origin groups were identified by BLASTX analysis as Enterobacter sp., Vibrio sp., Erwinia sp. and Klebsiella oxytoca, respectively. The BLASTX comparison tests showed that YP1 played an important role in decomposing organic matters; YP2 was fund to be related to the life activities of the Cerea Spore Bacilli with functions in phosphate dissolution; YP3 had functions similar to the apolipoprotein e (APOE) that involved in catabolism and metabolism of micro-organisms; and YP4 had an effect on nitrogen removal subject to microbial activities.