The aim of this study was to characterize the nitrogen transforming bacterial communities within a partially saturated vertical subsurface flow constructed wetland (VF) treating urban wastewater in southern Brazil. The VF had a surface area of 3144m, and was divided into four wetland cells, out of which two were operated while the other two rested, alternating cycles of 30days. The nitrifying and denitrifying bacterial communities were characterized in wetland cell 3 (764m surface area) over a period of 12months by using the FISH technique. Samples were collected monthly (from Feb 2014 to Feb 2015) from different layers within the vertical profile, during operation and rest periods, comprising a total of 6 sampling campaigns while the cell was in operation and another 6 when the cell was at rest. This wetland cell operated with an average organic loading rate (OLR) of 4gCODmd and a hydraulic loading rate of 24.5mmd. The rest periods of the wetland cell presented influences on the abundance of ammonia-oxidizing bacteria (AOB) (8% and 3% for feed and rest periods, respectively), and nitrite-oxidizing bacteria (NOB) (5% and 2% for feed and rest periods, respectively). However, there was no influence of the rest periods on the denitrifying bacteria. AOB were only identified in the top layer (AOB β-proteobacteria) in both operational and rest periods. On the other hand, the NOB (Nistrospirae and Nitrospina gracilis) were identified in feed periods just in the top layer and during rest periods just in the intermediate layer. The denitrifying bacteria (Pseudomonas spp. and Thiobacillus denitrificans) were identified from the intermediate layer downwards, and remained stable in both periods. Based on the identified bacterial dynamics, the partially saturated VF wetland operated under low OLR enabled favorable conditions for simultaneous nitrification and denitrification.

The elimination of organic carbon, nitrogen, five emerging organic contaminants (EOCs) and fluorescence signature was evaluated in two treatment lines comprising different constructed wetland (CW) configurations: (i) partially saturated vertical subsurface flow (SVF) wetland (treatment line 1) and (ii) unsaturated vertical subsurface flow (UVF), horizontal subsurface flow (HF) and free water surface (FWS) wetlands in series (treatment line 2). Results showed important differences between the different CW configurations. The highest removal of BOD5 (81%), COD (67%), TOC (72%) and fluorescing organic matter were observed in the UVF wetland, whereas the HF and FWS wetlands were the most efficient units for total nitrogen removal (60 and 69%, respectively).The SVF wetland showed a greater performance in the reduction of total nitrogen than the UVF bed (52 vs 35%). In addition, the SVF wetland exhibited a higher removal of the EOCs caffeine (95 vs 90%), trimethoprim (99 vs 87%) and sulfamethoxazole (64 vs 4%), as opposed to DEET (34 vs 63%), whose removal was superior in the UVF unit. Sucralose was negligibly removed in all the CWs. PARAFAC analysis of fluorescence measurements revealed that the proteinaceous tryptophan-like fluorescent component was the most highly removed one in all the investigated CWs (>28%) and, particularly, in the UVF wetland (66%), whereas humic and fulvic-like components resulted recalcitrant to decomposition. Increases of fluorescence intensities were often observed for fulvic-like substances in CWs operating with saturation of the bed, and these were particularly relevant in the SVF unit. Finally, important correlations (r>0.7) between the tryptophanlike fluorescent component and the water quality parameters COD and BOD5 suggest fluorescence spectroscopy as an useful monitoring tool for water treatment efficiency in CW systems.

The effect of effluent recirculation on the removal of total nitrogen (TN) and eight pharmaceuticals and personal care products (PPCPs) was evaluated during 9months in an experimental hybrid constructed wetland (CW) system applied in the treatment of urban wastewater. An Imhoff tank was followed by three stages of CWs (two 1.5-m vertical subsurface flow (VF) beds alternating feed-rest cycles, a 2-m horizontal (HF) and a 2-m free water surface (FWS) wetland in series). A fraction of the final effluent was recycled back to the Imhoff tank with a recirculation rate of 50% (hydraulic loading rate=0.37md). The system's performance varied throughout the study. In Period I (summer) consistently high load removal efficiencies of TN (89±5%) and a removal rate of 6.6±1.4gTNmd were exhibited. In Period II (fall), the poor performance of the FWS during the senescence of macrophytes caused a large increase in organic matter, solids and nutrient concentrations, drastically deteriorating water quality. The determination of PPCPs was conducted during this period. Recalcitrant compounds, namely sulfamethoxazole, carbamazapine, TCEP and sucralose were negligibly removed in all CWs. However, noteworthy was the ≈30% removal of sucralose in the VF wetland. Caffeine (80%) and fluoxetine (27%) showed similar elimination rates in both VF and HF units, whereas trimethoprim and DEET were significantly better removed in the VF than in the HF. The concentration of the four latter compounds showed a severe increase in the FWS, indicating possible desorption from the sediment/biomass during adverse conditions. Harvesting of the aboveground biomass in this unit returned the system's performance back to normality (Period III), achieving 77±7% TN removal despite the winter season, proving effluent recirculation as an effective strategy for TN removal in hybrid CW systems when stringent restrictions are in place.

Nitrogen dynamics and its association to metabolically active microbial populations were assessed in two vertical subsurface vertical flow (VF) wetlands treating urban wastewater. These VF wetlands were operated in parallel with unsaturated (UVF) and partially saturated (SVF) configurations. The SVF wetland exhibited almost 2-fold higher total nitrogen removal rate (5 g TN m d) in relation to the UVF wetland (3 g TN m d), as well as a low NO-N accumulation (1 mg L vs. 26 mg L in SVF and UVF wetland effluents, respectively). After 6 months of operation, ammonia oxidizing prokaryotes (AOP) and nitrite oxidizing bacteria (NOB) displayed an important role in both wetlands. Oxygen availability and ammonia limiting conditions promoted shifts on the metabolically active nitrifying community within 'nitrification aggregates' of wetland biofilms. Ammonia oxidizing archaea (AOA) and Nitrospira spp. overcame ammonia oxidizing bacteria (AOB) in the oxic layers of both wetlands. Microbial quantitative and diversity assessments revealed a positive correlation between Nitrobacter and AOA, whereas Nitrospira resulted negatively correlated with Nitrobacter and AOB populations. The denitrifying gene expression was enhanced mainly in the bottom layer of the SVF wetland, in concomitance with the depletion of NO-N from wastewater. Functional gene expression of nitrifying and denitrifying populations combined with the active microbiome diversity brought new insights on the microbial nitrogen-cycling occurring within VF wetland biofilms under different operational conditions.

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 (1.5m) operated under average loads of 130gCODmd and 17gTNmd in Period I, and 80gCODmd and 19gTNmd in Period II. The hydraulic loading rate (HLR) was 375mmd 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 2gTNmd for both periods) concomitant with NO-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 NO-N forms, to improve their capacity for total nitrogen removal.