Elsevier

Water Research

Volume 74, 1 May 2015, Pages 203-212
Water Research

Nitrogen transforming community in a horizontal subsurface-flow constructed wetland

https://doi.org/10.1016/j.watres.2015.02.018Get rights and content

Highlights

  • The activity and abundance of nitrogen transforming bacteria were quantified.

  • Significant nitrate consumption under aerobic conditions was observed.

  • This nitrate consumption was due to aerobic denitrification.

  • Only short-term incubations should be used to measure denitrification turnover rates.

  • Anammox appears to be of low importance in nitrogen transformations in these ecosystems.

Abstract

Constructed wetlands are important ecosystems with respect to nitrogen cycling. Here we studied the activity and abundance of nitrogen transforming bacteria as well as the spatial distribution of nitrification, anaerobic ammonium oxidation (anammox), and denitrification processes in a horizontal subsurface-flow constructed wetland. The functional genes of the nitrogen cycle were evenly distributed in a linear way along the flow path with prevalence at the superficial points. The same trend was observed for the nitrification and denitrification turnover rates using isotope labeling techniques. It was also shown that only short-term incubations should be used to measure denitrification turnover rates. Significant nitrate consumption under aerobic conditions diminishes nitrification rates and should therefore be taken into account when estimating nitrification turnover rates. This nitrate consumption was due to aerobic denitrification, the rate of which was comparable to that for anaerobic denitrification. Consequently, denitrification should not be considered as an exclusively anaerobic process. Phylogenetic analysis of hydrazine synthase (hzsA) gene clones indicated the presence of Brocadia and Kuenenia anammox species in the constructed wetland. Although anammox bacteria were detected by molecular methods, anammox activity could not be measured and hence this process appears to be of low importance in nitrogen transformations in these freshwater ecosystems.

Introduction

Nitrogen (N) compounds, such as ammonium (NH4+) and nitrate (NO3), are some of the most widespread contaminants in ground- and wastewater (Harrington and McInnes, 2009, Singleton et al., 2007). For decades it was believed that the only N transformations responsible for returning fixed nitrogen to the atmosphere were nitrification, the aerobic stepwise oxidation of NH4+ via nitrite (NO2) to NO3, and denitrification, anaerobic NO3 or NO2 reduction with nitrous oxide (N2O) or dinitrogen gas (N2) as a final product. In the meantime, however, the process of anaerobic ammonium oxidation (anammox) was discovered, extending the N cycle by showing that NH4+ can also be oxidized under anaerobic conditions using NO2 as the electron acceptor to produce N2 (Mulder et al., 1995).

Constructed wetlands (CWs) are man-made systems designed to include specific modified characteristics of wetland ecosystems for improved treatment capacity (Kadlec and Wallace, 2008). CWs can provide an effective contaminant degradation zone due to enhancement of microbial activity in the plant's rhizosphere (Stottmeister et al., 2003). CWs can be used to treat a wide range of contaminants including N-containing compounds. To date, only few studies have focused on the microorganisms performing the nitrification and denitrification processes in CWs, despite their crucial role in N-removal (Chon et al., 2011, Correa-Galeote et al., 2013, Song et al., 2012). While the overall N turnover in ecosystems can be investigated by a wide range of methods, measurements of microbial activity and its correlation with the copy numbers of specific functional genes will add to our understanding of the systems (Ruiz-Rueda et al., 2009, Song et al., 2012, Wang et al., 2013). Furthermore, there are only a few reported measurements on nitrification and denitrification turnover rates in CWs using isotope labeling techniques (Erler et al., 2008, Scott et al., 2008, Stepanauskas et al., 1996).

Denitrification is commonly considered to be an anaerobic process and even small amounts of oxygen are reported to inhibit the activity of the denitrifying enzymes and suppress their synthesis in a stepwise manner (Bryan, 1981). However, a number of laboratory studies with batch cultures revealed that denitrification can also take place in the presence of oxygen, a process referred to as aerobic denitrification (Robertson et al., 1995, Robertson and Kuenen, 1984). In CWs, nitrification occurs in the plant's rhizosphere, as the plant's roots deliver oxygen, and/or in the top layer of the water body. This would also be the zone where aerobic denitrification could potentially occur. However, studies in natural and near-natural environments such as CWs to verify and quantify rates of aerobic denitrification are lacking (Gao et al., 2010).

Anammox accounts for over 50% of N loss in marine ecosystems (Kuypers et al., 2003, Thamdrup and Dalsgaard, 2002). However, to date, the role of anammox in freshwater ecosystems has not yet been explored in depth (Humbert et al., 2010, Jasper et al., 2014, Zhu et al., 2010). So far, anammox research has focused mainly on quantifying turnover rates (Burgin et al., 2011, Erler et al., 2008) and/or detecting anammox bacteria using molecular biological tools (qPCR, FISH) in various ecosystems in order to reveal if and where this process occurs naturally (Burgin et al., 2011). While anammox bacteria have been detected in a range of aquatic ecosystems (Jetten et al., 2009), almost no investigations were done in terrestrial ecosystems, despite molecular evidence of the presence of anammox bacteria in diverse soils (Hu et al., 2011, Humbert et al., 2010). Due to the mosaic of aerobic and anaerobic zones as well as the usually low dissolved oxygen concentration, CWs are assumed to offer favorable conditions for anammox (Zhu et al., 2010). Although anammox organisms have been detected in natural wetland soils (Humbert et al., 2012), information about their activity and contribution to N turnover, particularly in CWs, is rather limited. Some research has been done on enrichment and enhancement of anammox processes in CWs (Paredes et al., 2007, Tao et al., 2011, Wang and Li, 2011, Zhu et al., 2011). Anammox's contribution to total N2 production has also been investigated, but only in the sediments of full-scale surface flow CWs treating domestic sewage (Erler et al., 2008). Such systems are broadly representative of CWs together with horizontal subsurface-flow (HSSF) CWs, and the HSSF CWs are characterized by lower dissolved oxygen concentrations (Vymazal and Kröpfelová, 2008), so this could potentially increase anammox contributions. Thus the role of anammox in N removal in systems such as HSSF CWs as well as its relationship with other N transformations needs to be investigated (Zhu et al., 2010).

The objectives of the present study were: i) to explore the spatial distribution of both activity and bacterial abundance of nitrification and denitrification, ii) to quantify rates of aerobic denitrification, and iii) to investigate the role of anammox in N removal in HSSF CW.

Section snippets

Constructed wetland design

The CW in the study site was built as a part of CoTra (Compartment Transfer) project in 2007 at Leuna near Leipzig, Germany. Leuna has served as a major chemical manufacturing site since the beginning of the 20th century, and accidental spills, improper handling, and damages due to heavy bombing during the World War II have left the underlying groundwater highly contaminated with benzene, methyl-tert-butyl ether (MTBE), and NH4+ (Martienssen et al., 2006).

The CW consisted of a basin (length

Ammonia oxidizing bacterial abundance and activity

Abundance of ammonia oxidizing genes was quantified by qPCR targeting α subunit of ammonium monooxygenase (amoA), which is the key enzyme in the aerobic ammonia oxidation process. The genomic material of ammonia oxidizing bacteria was detected at all points in the CW with the highest abundance at the depth of 0.2 m, 2.2 × 106 copy number g−1 sample, and the lowest at 0.4 m depth, 6.8 × 104 copy number g−1 sample (F(2,33) = 12.968, p = 0.000) (Fig. 1). The abundance of the amoA gene varied with

Conclusions

In this study, the contribution and diversity of various nitrogen cycle processes in a horizontal subsurface-flow constructed wetland were determined with molecular markers and stable isotope tests. The functional genes of the nitrogen cycle were abundant along the flow path with prevalence at the superficial points. The same trend was observed for the nitrification and denitrification turnover rates. Furthermore, significant nitrate consumption under aerobic conditions was detected and this

Acknowledgments

This research was completed within the framework of the Marie Curie Initial Training Network ADVOCATE – Advancing sustainable in situ remediation for contaminated land and groundwater, funded by the European Commission, Marie Curie Actions Project No. 265063, SAFIRA project and the Helmholtz Interdisciplinary Graduate School for Environmental Research (HIGRADE). MSMJ was supported by ERC AG 232937 and 339880. We are also grateful to Ines Mäusezahl of the molecular biology laboratory for her

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