1. Bacterial communities composition in a full-scale pilot plant constructed wetlands for municipal wastewaters treatment Paula Arroyo HernándezGemma Ansola González Luis Saénz de MieraEstanislao de Luis Calabuig Jorge Falagán Instituto de Medio Ambiente Universidad de León Análisis de las comunidades bacterianas presentes en un humedal artificial piloto a escala real para el tratamiento de agua residual urbana

2. CONSTRUCTED WETLANDS Natural wetlands are a complex ecosystem of plants, microorganisms and substrata that together act as a biogeochemical filter. The capacity of theses ecosystems to improve water quality has been recognized for many years. For that, since the 1950s, it has been developed an alternative technology named Constructed Wetlands (CWs), which are man-made ecosystems that imitate their natural counterparts. These CWs have been utilized to treat polluted water in a great variety of places and water characteristics. Jorge Falagán

3. Microorganisms are the primary agent for removal of organic matter from wastewaters in constructed wetlands. As the wastewater flows along the spaces between the rhizomes and the surrounding planting medium it is biochemically oxidized by microorganisms. These communities are also implicated in the removal of metals through bacterially-mediated reactions, especially oxidation. In spite of the interest of these communities, few studies have been carried out on a full scale pilot plant and most of them are focused on a determined group of bacteria. CONSTRUCTED WETLANDS

4. A FULL SCALE PILOT PLANT CONSTRUCTED WETLANDS Bustillo de Cea

5. The Hierarchical Mosaic of Aquatic Ecosystems (HMAE®) The system is made up of four tanks of artificial aquatic ecosystems. The first basin is formed by a stabilization pond of up to 2m in depth where inflow enters and 1.5m where the out flow goes out; the second and the third basins are semi-aquatic ecosystems with a surface flow, where Typha latifolia and Iris pseudacorus were planted in them respectively. Basin four, which acts as a gravel filter, was planted with Salix atrocinerea (terrestrial ecosystem). Further details on the process are related elsewhere, Ansola et al (2003). 1.Laguna 2.Ecosistema semiacuático con Typha latifolia 3.Ecosistema semiacuático con Iris pseudacorus 4.Filtro de gravas con Salix atrocinera

6. BUSTILLO DE CEA (HMAE®)

10. SAMPLE COLLECTION Wastewater samples were collected aseptically from the different ecosystems of the HMAE ® in Bustillo de Cea, once in a month. Autumn bacteria communities collected from the rhizosphere of Typha latifolia and Salix atrocinerea and the surrounding planting medium were analyzed. Communities of the sludge of the stabilization pond were also studied. One sample was collected in the entry part of the lagoon (P sludge) and the other one in the last part of the lagoon (S Sludge).

11. Physicochemical analysis of the wastewater

12. Bacterial communities 1. ENVIRONMENTAL SAMPLES 2. EXTRACTED NUCLEID ACIDS 3. AMPLIFIED rDNA 4. rDNA CLONES 5. NUCLEOTIDE SEQUENCE 6. DNA DATASET DNA extraction PCR Transform to E. coli vector Sequencing E.M.B.L. database search 7. ALIGNEMENT Clustal-W Phylogenetical analysis 8. PHYLOGENETICAL TREES: LogDet Distance, Minimum evolution trees

13. RESULTS: Physicochemical characteristics of the wastewater Physicochemical characteristics of the influent show that wastewater presents a low pollution and a domestic origin. The results obtained in the HMAE ® in Bustillo de Cea are quite similar to other studies. SST (mg/l) BOD5 (mg O2/l) QOD (mg O2/l) NTK (mgN/l) NH4+ (mg NH4/l) TP (mgP/l) 62.7962.1148.6247.4763.2261.22 REMOVAL %

14. RESULTS: ANALYSIS OF BACTERIAL COMMUNITIES Phylogenetic tree showing genetic relationship of the Deltaproteobacteria rhizosphere bacteria based on 16S ribosomal gene sequences. Bacteria were named with letters and numbers. Letters explained the origin of the clone: “T” clones from the rhizosphere of Typha and “S” clones form the rhizosphere of Salix. Phylogenetic tree showing genetic relationship of the Deltaproteobacteria rhizosphere bacteria based on 16S ribosomal gene sequences. Bacteria were named with letters and numbers. Letters explained the origin of the clone: “T” clones from the rhizosphere of Typha and “S” clones form the rhizosphere of Salix.

15. The results of the study show the quantitative and qualitative composition of the bacteria communities present in the different environments of the HMAE ®. On a Phyllum level, there were found bacteria belonging to fourteen different Phyllum. The most frequent in the four samples was the Phyllum Proteobacteria RESULTS: ANALYSIS OF BACTERIAL COMMUNITIES

17. - Rhizosphere of Typha. The most abundant group were aerobic chemoheterotrophic bacteria of the Order Pseudomonadales (Gammaproteobacteria), which play an important role in decomposition, biodegradation, and the C and N cycles. RESULTS: ANALYSIS OF BACTERIAL COMMUNITIES

18. - Rhizosphere of Salix. The Order Rhizobiales (Alphaproteobacteria) constituted by nitrogen-fixing bacteria, was the dominant group of this community. Bacteria included in Burkholderiales order (Betaproteobacteria) were also abundant. These bacteria are involved in aerobic degradation of organic matter.

19. RESULTS: ANALYSIS OF BACTERIAL COMMUNITIES - Primary and secondary sludge. Sulphate-reduction bacteria, typically found in anaerobic sludge, were the most frequent in the sludge, bacteria such us Desulfovibrio sp., Desulfurobacter sp. or Desulfuromonas sp. These bacteria play an important role in reducing methane generation.

20. The methodology of characterizing bacteria communities using the direct sequencing of the 16S ribosomal RNA codifying genes makes possible to study the complete bacterial communities of the HMAE® The methodology of characterizing bacteria communities using the direct sequencing of the 16S ribosomal RNA codifying genes makes possible to study the complete bacterial communities of the HMAE®. The results suggest a relationship among wastewater characteristics and bacterial communities. The study is focus on bacterial communities of the rhizosphere of the plants and of the sludge, not in free water such us other studies. Bacterial communities in the sludge are dominated by anaerobic microorganisms (Deltaproteobacteria), while those communities associated with the rhizosphere of plants are dominated by aerobic bacteria (Alpha y Gammaproteobacteria. CONCLUSIONS

21. GRACIAS POR SU ATENCIÓN Thanks for your attention Paula Arroyo Hernández Gemma Ansola González Luis Saénz de Miera Instituto de Medio Ambiente Universidad de León Fotos: Jorge Falagán