Effect of plants and filter materials on bacteria removal in pilot-scale constructed wetlands

Due to the lack of testing units or appropriate experimental approaches, only little is known about the removal of bacteria in constructed wetlands. However, improved performance in terms of water sanitation requires a detailed understanding of the ongoing processes. Therefore, we analyzed the microbial diversity and the survival of Enterobacteriaceae in six pilot-scale constructed wetland systems treating domestic wastewater: two vertical sand filters, two vertical expanded clay filters and two horizontal sand filters (each planted and unplanted). Samples were taken from the in- and outflow, from the rhizosphere, and from the bulk soil at various depths. Colony-forming units of heterotrophic bacteria and coliforms were analyzed and the removal of bacteria between the in- and outflow was determined to within 1.5-2.5 orders of magnitude. To access the taxon-specific biodiversity of potential pathogens in the filters and to reduce the complexity of the analysis, specific primers for Enterobacteriaceae were developed. While performing PCR-SSCP analyses, a pronounced decrease in diversity from the inflow to the outflow of treated wastewater was observed. No differences were observed between the bulk soil of planted and unplanted vertical filters. Some bands appeared in the rhizosphere that were not present in the bulk soil, indicating the development of specific communities stimulated by the plants. The fingerprinting of the rhizosphere of plants grown on sand or expanded clay exhibited many differences, which show that different microbial communities exist depending on the soil type of the filters. The use of the taxon-specific primers enabled us to evaluate the fate of the Enterobacteriaceae entering the wetlands and to localize harboring in the rhizosphere. The most abundant bands of the profiles were sequenced: Pantoea agglomerans was found in nearly all samples from the soil but not in the effluent, whereas Citrobacter sp. could not be removed by the horizontal unplanted sand and vertical planted expanded clay filters. These results show that the community in wetland system is strongly influenced by the filtration process, the filter material and the plants.