Pathways regulating the removal of nitrogen in planted and unplanted subsurface flow constructed wetlands

• Nitrification-denitrification route regulates N removal in subsurface flow CWs.
• Ammonia oxidation archaea contribute to nitrification and that effect depends on plant species.
• Denitrifiers composition shifted toward communities with a lower genetic potential to emit N2O.
• Anammox process does not have a strong role in N cycling in subsurface flow CWs.
• Plant species controls treatment performance of CWs probably through its effect on O2 supply.

Single-stage constructed wetlands (CWs) are characterized by a low potential for N removal. Understanding the pathways regulating N cycling as well as their dependence on environmental variables might improve the potential of CWs for N removal and results in more accurate simulation tools. In this study we employed qPCR targeting marker functional genes (amoA, nirK, nirS, clade I and II nosZ) or microorganisms (anammox) regulating key pathways of N cycling to unravel their relative importance. Furthermore, the influence of plant species on treatment performance was studied. Our findings indicated nitrification-denitrification as the principal route of N removal in CWs, while anammox did not have a strong contribution. Evidence was also arisen that ammonia oxidizing archaea (AOA) contributed on NH3 oxidation. Overall, plant species had a weak effect on the abundance of N functional genes (amoA of AOA), but it strongly affected the performance of CWs in terms of N removal in the following order: unplanted < Phragmites communis < Typha latifolia. These findings suggest that plant species stimulate N removal by upregulating the rates that the responsible biochemical pathways operate, probably by increasing O2 supply. In addition, our study revealed differences in indicators linked to N2O emissions. The abundance of clade II nosZ genes remained low across the season scaling down a strong contribution in the reduction of the emitted N2O. The increasing ratios of nosZ/Σnir and nirS/nirK with the progress of season indicate a shift in the composition of denitrifiers towards strains with a lower genetic potential for N2O release. Similar trends were observed among the treatments but the mechanisms differed. The planted treatments stimulated an increase in the ΣnosZ/Σnir ratio, while the unplanted an increase in the nirS/nirK ratio.

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