The dynamics of the active microbial populations involved in nitrogen transformation in a vertical subsurface flow constructed wetland (VF) treating urban wastewater was assessed. The wetland (1.5 m2) operated under average loads of 130 g COD m- 2 d- 1 and 17 g TN m- 2 d- 1 in Period I, and 80 g COD m- 2 d- 1 and 19 g TN m- 2 d- 1 in Period II. The hydraulic loading rate (HLR) was 375 mm d- 1 and C/N ratio was 2 in both periods. Samples for microbial characterization were collected from the filter medium (top and bottom layers) of the wetland, water influent and effluent at the end of Periods I (Jun–Oct) and II (Nov–Jan). The combination of qPCR and high-throughput sequencing (NGS, MiSeq) assessment at DNA and RNA level of 16S rRNA genes and nitrogen-based functional genes (amoA and nosZ-clade I) revealed that nitrification was associated both with ammonia-oxidizing bacteria (AOB) (Nitrosospira) and ammonia-oxidizing archaea (AOA) (Nitrososphaeraceae), and nitrite-oxidizing bacteria (NOB) such as Nitrobacter. Considering the active abundance (based in amoA transcripts), the AOA population revealed to be more stable than AOB in both periods and depths of the wetland, being less affected by the organic loading rate (OLR). Although denitrifying bacteria (nosZ copies and transcripts) were actively detected in all depths, the denitrification process was low (removal of 2 g TN m- 2 d- 1 for both periods) concomitant with NOx-N accumulation in the effluent. Overall, AOA, AOB and denitrifying bacteria (nosZ) were observed to be more active in bottom than in top layer at lower OLR (Period II). A proper design of OLR and HLR seems to be crucial to control the activity of microbial biofilms in VF wetlands on the basis of oxygen, organic-carbon and NOx-N forms, to improve their capacity for total nitrogen removal.

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