In this study, bacterial community structure in a horizontal subsurface flow constructed wetland (HSF-CW) planted with Phragmites australis was investigated using the 16S rRNA cloning-sequencing technique. Two layer depths were considered: the rhizosphere zone (RH) and the deep-layer zone (DL) in different sampling periods. Bacteria-specific primers 008F and 1492R were used to amplify the 16S rRNA genes and construct six clone libraries. A total of 1,284 individual sequences were used to assess the HSF-CW diversity. Phylogenetic analysis of RH and DL clone libraries shows that 41.57 and 42.17 % of the 16S rRNA sequences are affiliated with the Proteobacteria in the RH and the DL, respectively. The remaining major phylogenetic groups are Bacteroidetes, Planctomycetes, and Chloroflexi with 11.78, 9.36, and 7.6 %, respectively, in the RH and 11.38, 6.48, and 7.65 % in the DL, respectively. Minor divisions such as Verrucomicrobia, TM7, Nitrospira, and Gemmatimonadetes represented <6 % of the total sequences, while 14.2 % were unidentified Bacteria. Among the Proteobacteria, the Alphaproteobacteria subclass is represented in both locations, while the Deltaproteobacteria and Gammaproteobacteria subclasses were predominant in the RH and the DL, respectively. Results suggest that Archaea and Bacteria in the HSF-CW are the essential actors in the nitrogen cycle and that the established microbial community is efficient in nitrogen removal from wastewater.

Wastewater microbiota represents important actors of organic depollution. Nowadays, some species used as bioindicators of the effluent quality are still identified by microscopy. In the present study, we investigated eukaryotic diversity at the different steps of the treatment process of a wastewater treatment plant (aerobic, anaerobic, clarifier basins and anaerobic digester) using the 18S rRNA gene sequencing approach. Of the 1519 analysed sequences, we identified 160 operational taxonomic units. Interestingly, 56.9% of the phylotypes were assigned to novel phylogenetic molecular species since they show <97% sequence identity with their nearest affiliated representative within public databases. Peritrichia ciliates were the most predominant group, with Epistylis as the most common genus. Although anaerobic, the digester appears to harbor many unclassified phylotypes of protozoa species. Novel lineages such as LKM11 and LKM118 were widely represented in the digester. Diversity values given by Shannon indexes show that the clarifier is the most diversified. This work will help designing molecular tools that are fast, reliable, and reproducible for monitoring wastewater depollution and studying phylogenetic relationships among the wonderful world of protists within this anthropogenic ecosystem.