## 3 Results and Discussion
### Microbiological Data
As expected, HB counts were higher than those found for total coliforms and _E. coli_ in inlet and outlet zones (Table 3), both for substrate and roots. The enumeration of HB followed the same trend as the indicator organisms, with regard to the reduction in counts for the inlet to the outlet and also to the lower prevalence both in substrate and roots during the colder seasons, in particular in the last two years. Nonetheless, the range assessed was within references in literature [39]. Substrate-associated bacterial communities from the inlet and outlet zones of the CW were evaluated by DGEg fingerprinting during five seasons (Figure 3). DGEg patterns showed that species richness varied among samples, without a trend, with the higher number of bands (37) found in the substrate collected from the inlet zone during the hot season of the second year (H2_in), and the lower (27) recorded in the C3_out. Figure 3: Cluster analysis generated by the 16S rRNA DGE patterns of bacterial communities associated to the substrate in the inlet (in) and outlet (out) zone of the CW, in cold (C) and hot (H) seasons across three consecutive years (1, 2, 3). The dendrogram was created using the UPGMA clustering method based on Jaccard coefficient. The species richness (S) and the Shannon-Wiener index (H\({}^{\prime}\)) is presented. Besides the band number, some differences were also observed in band intensity and position, indicating the existence of different substrate-associated bacterial communities, and corroborated by the Shannon-Wiener index. The dendrogram and PCO constructed based on DGGE data showed differences in the structure and composition of substrate bacterial communities (Figures 3 and 4). PCO explained 60.3% of the total variance between samples on the two first components. Bacterial communities were split into two main groups between samples collected in the cold and hot seasons of first/second year and third year (with a similarity of about 50%), and the samples collected from the inlet zone in the C season of the first year (C1_in). The first group includes all samples from the third year, while the second group aggregates samples collected in the second year and the sample C1_out. These results indicate that the structure and composition of bacterial communities were mainly influenced by the year, rather than the season or the CW zone. The statistical analysis (PERMANOVA) also showed that the composition of substrate-associated bacterial communities was significantly (\(p<0.05\)) influenced by year. [PERSON] et al. [16] analyzed the effect of different types of bed materials used in a horizontal subsurface flow CW for tannery wastewater treatment had on the dynamics of the substrate-associated bacterial communities. Results showed that the temporal factor had less influence on bacterial communities than the place of collection (substrate or root) and the type of substrate. [PERSON] et al. [39] also studied the microbial community structure in a horizontal subsurface flow CW planted with sand filter, treating domestic wastewater. [PERSON] et al. [40] found diverse and distinct bacterial communities associated with different CW systems. In this study, the most relevant spatial pattern found in microbial community structure was related to the depth gradient, followed by differences between the CW inlet and outlet. Figure 4: Principal coordinate analysis (PCO) of the substrate associated bacterial communities based on Bray–Curtis similarity matrix showing samples colored by year.