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Environmental Processes

Microbial nitrogen cycle hotspots in the plant-bed/ditch system of a constructed wetland with N2O mitigation Shanyun Wang, Weidong Wang, Lu Liu, Linjie Zhuang, Siyan Zhao, Yu Su, Yixiao Li, Mengzi Wang, Cheng Wang, Liya Xu, and Guibing Zhu Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b04925 • Publication Date (Web): 11 May 2018 Downloaded from http://pubs.acs.org on May 13, 2018

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Environmental Science & Technology Microbial N-cycle hotspots in CW with N2O mitigation

Microbial nitrogen cycle hotspots in the plant-bed/ditch system of a constructed wetland with N2O mitigation

Shanyun Wang1,3, Weidong Wang1,3, Lu Liu1, Linjie Zhuang1, Siyan Zhao1, 2, Yu Su1, 2, Yixiao Li1, Mengzi Wang1, Cheng Wang1, 2, Liya Xu1, Guibing Zhu1, 2 *

1. Key Laboratory of Drinking Water Science and Technology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, China 2. University of Chinese Academy of Sciences, Beijing 100049, China 3. These authors contributed equally to this work.

* Corresponding author Dr. Guibing Zhu Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, China. E-mail: [email protected]

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ACS Paragon Plus Environment


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Abstract

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Artificial microbial nitrogen (N) cycle hotspots in the plant-bed/ditch system were developed and

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investigated based on intact core and slurry assays measurement using isotopic tracing technology,

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quantitative PCR and high-throughput sequencing. By increasing hydraulic retention time and

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periodically fluctuating water level in heterogeneous riparian zones, hotspots of anammox,

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nitrification, denitrification, ammonium (NH4+) oxidation, nitrite (NO2-) oxidation, nitrate (NO3-)

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reduction and DNRA were all stimulated at the interface sediments, with the abundance and activity

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being about 1-3 orders of magnitude higher than those in non-hotspots. Isotopic pairing experiments

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revealed that in microbial hotspots, nitrite sources were higher than the sinks, and both NH4+

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oxidation (55.8%) and NO3- reduction (44.2%) provided nitrite for anammox, which accounted for

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43.0% of N-loss and 44.4% of NH4+ removal in riparian zones but didn't involve nitrous oxide (N2O)

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emission risks. High-throughput analysis identified that bacterial quorum sensing mediated this

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anammox hotspot with B.fulgida dominating the anammox community, but it was B.anammoxidans

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and Jettenia sp. that contributed more to anammox activity. In the non-hotspot zones, the NO2-

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source (NO3- reduction dominated) was lower than the sink, limiting the effects on anammox. The

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in-situ N2O flux measurement showed that the microbial hotspot had a 27.1% reduced N2O

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emission flux compared with the non-hotspot zones.

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Keywords: Microbial N-cycle; abundance; activity; hotspot; mechanism; N2O; riparian zones;

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N-tracing;

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TOC art

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Introduction

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Anthropogenic modification of the earth’s biogeochemistry has been increasingly manifesting

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in the changes occurring in the nitrogen (N) cycle. Human activities are now responsible for greater

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than 50% of the reactive-N input to the biosphere1,2. The consequential impacts have become an

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important global concern including atmospheric acidification, aquatic eutrophication, groundwater

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nitrate (NO3-) pollution, greenhouse gas nitrous oxide (N2O) emission, and climatic changes3-7. As a

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result, development of sustainable methods for N mitigation has become quite urgent.

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Microbe-mediated N cycling has been generally regarded as the most economic and effective

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approach to N remove from water systems. These microbe-driven N transformations are mainly

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carried out through a series of electron transfers between different valences of N forms. It could

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then be expected that in a highly heterogeneous zone where substrate levels vary and microbial

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biomass are generally high, significant N transformation processes would also be present, making it

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a “hotspot” of various N cycling processes8-10. Riparian zone, the interface between terrestrial and

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aquatic ecosystems, plays an important role in N removal despite the small land area that it

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covers11-13. The tidal fluctuation of water levels in riparian zones allow periodic flooding (anoxic)

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and emergence (re-oxygenation) of the shore, resulting in a highly heterogeneous condition with

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anoxic-oxic alternation. Microbial community coupled with the alternations between anaerobic and

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aerobic conditions further changes the degree of biogeochemical transformation of various

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N-compounds in riparian zones.

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Understanding the N cycle and its driving factors is essential for evaluating potentially feasible

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and adaptable N-mitigation measures. The traditional nitrification-denitrification processes have

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been studied for over a hundred years2,14,15. In addition to nitrogen gas (N2), N2O is also produced 4


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during denitrification from nitrite (NO2-) and NO3- ions16, which indicates that the water quality

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problem is just simply being converted into an atmospheric pollution problem17. Anaerobic

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ammonium oxidation (anammox) is an alternative pathway to denitrification, which oxidizes

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ammonium (NH4+) directly to N2, without emitting N2O18,19. The widespread distribution of

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anammox has significantly contributed to N-loss in aquatic ecosystems, including marine20, inland

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water21,22 and aquifer systems23,24. Our understanding of these processes has significantly changed

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our view of the global N cycle. A previous study in a lake system also showed that land-freshwater

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interfaces in riparian zone are hotspots of anammox process8. Here, we hypothesize that the

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land-water interfaces in riparian zone with significant oxidized and reduced N interactions and

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fluxes are not only hotspots of anammox activity but also of the entire N cycle. At present, some

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researchers have shown that riparian zones had higher denitrification activity than the adjacent

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areas25-27. However, several studies also showed the opposite28,29. Moreover, the complex

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interactions among biogeochemical mechanisms, especially the relationship between N2O emission

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and anammox processes, remain little understood.

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Riparian ecosystems are sites of important biogeochemical processes affecting the composition

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and structure of the costal biota and adjacent aquatic systems. Based on the current understanding of

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microbe-mediated N cycling and the hydrological and biogeochemical features of riparian zones,

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several integrated ecological approaches to simulate N-cycle hotspots have been proposed. These

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include implementing water flooding, enhancing water level fluctuation, extending hydraulic

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retention time (HRT), and recruiting biodiversity. The goals of these approaches are as follows: (i)

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flooding the dry soil with stimulated water-logging to enrich bacteria and to increase the microbial

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biomass30,31; (ii) regulating water level fluctuations to form fluctuating riparian zones and improve 5



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the interface effect to enhance the oxidation and reduction in N interactions and fluxes8,9,32,; (iii)

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extending the HRT and reducing the corresponding hydraulic shear force, thus improving the

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exchange of various N-compounds such as NH4+ and NO3- (or NO2-) 33. All of them are simple and

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feasible approaches for real-world operations.

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Given this background, the objectives of this study were (i) to simulate the artificial microbial

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N-cycle hotspots in the plant-bed/ditch system of a constructed wetland, and quantify the

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contribution of artificial microbial N-cycle hotspots in the overall N-cycle and mitigation of N2O

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emission, based on our proposed practical engineering measures; and (ii) to reveal the underlying

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mechanisms and provide a more complete overview of the N-cycle processes related to anammox

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using molecular methods and 15N isotope tracing techniques.

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Materials and methods Study site description and sample collection

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Based on the above approaches, the first large-scale drinking water source protection wetland

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in China –‘Shijiuyang Constructed Wetland’ (SJY CW) was completed in Zhejiang Province, China

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in July 2009, and took about 2-3 years to reach a steady state (Fig. 1 a; Supplementary Fig. S1 a b c).

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The final area of the CW is 110 ha, and more than 60 ha is submerged area, providing sufficient

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water-logging to stimulate N-cycle microbiology enrichment. According to the plan view and the

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side slope of the SJY CW, the ditch fringe area evolves to a size of 12,130 m2, and the ditch and

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channel central areas are 40,388 m2 and 300,637 m2, respectively (Supplementary Table S1). The

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water flow pathways extend for approximately 10 km. A pump station system was used to regulate

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the water level fluctuations of 30-40 cm twice per day, forming fluctuating riparian zones and 6


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improving the interface effect. Among the functioning zones, the plant-bed/ditch systems are the

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core reaction sites of the CW. These areas simulate the natural landscape structure in Baiyangdian

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Lake, where anammox hotspots have been found at the land-freshwater interfaces (Supplementary

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Fig. S1 d)9. The plant-bed/ditch systems are composed of plant beds and ditches (10 m in width),

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connected with blocking equipment (impervious gravel choke plugs) on alternate ends of the plant

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beds. Constructed root channels (25 m in width) meander through the plant-bed/ditch systems. At

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the top of the plant bed, reed (Phragmites australis (Cav.) Trin. ex Steud.) has been transplanted as

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the dominant species; its rhizosphere provides a strong water purification function in the traditional

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biogeochemical34,35 and anammox process10. Blocking equipment was also used to extend the HRT

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by forming a circuitous flow in the reed-bed/ditch systems. In addition, approximately 35% of

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inflow water flowed through the plant-bed/ditch systems, forming an area approximately twice the

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size of the channel (65% of the inflow water passed through). These mechanisms effectively

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decreased the water flow conditions from 0.1–0.9 m s−1 (channel) to 0.01–0.02 m s−1

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(plant-bed/ditch systems), extending the corresponding HRT from 0.8 to 2.97 d, and reducing the

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corresponding hydraulic shear force.

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To conduct this study, simulated systems were continuously operated for five years. Sediment

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samples from the channel, ditch center, and ditch fringe (three sample types from a total 16 sample

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sites) were collected in February 2014 to investigate the microbial N-cycle processes (Fig. 1 a, b).

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Ten parallel sediment core samples (25 cm depth and 5.5 cm i.d.; Gravity column sediment sampler,

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New Landmark, Beijing) and three parallel sediment slurry samples (25 cm depth; Peterson grab,

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New Landmark, Beijing) were collected at each sampling site. The collected samples were sealed

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and transported to the laboratory at 0–4°C. The core samples were incubated at the in-situ 7


Environmental Science & Technology

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Microbial N-cycle hotspots in CW with N2O mitigation

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temperature immediately after arrival to determine anammox, nitrification and denitrification rates

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to verify the microbial N-cycle hotspots. The slurry samples were used to reveal the underlying

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mechanisms and provide a more complete overview of the N-cycle processes using molecular

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methods and

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temperature immediately after arrival to determine anammox, NH4+ oxidation, NO2- oxidation, NO3-

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reduction, denitrification, and DNRA rates. Another subsample was sieved through 2.0 mm for

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chemical analysis; other subsamples were stored at −80°C for subsequent molecular analysis.

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Analytical procedures for sediments characteristics

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N isotope tracing techniques. One subsample was incubated at the in-situ

The sediment chemical and physical properties were measured according to Bao (2000)

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.

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Briefly, sediment NH4+, NO2- and NO3- concentrations were measured using a SEAL

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Auto-Analyzer 3 HR (Seal Analytical, UK) after extraction with 2M KCl (1:5 wt/vol). The

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detection limits were 0.015, 0.015 and 0.03 mg kg−1, respectively. The pH was measured using a

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DELTA 320 pH Analyzer (Mettler Toledo, USA) after shaking a dry sediment water (1:5 wt/vol)

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suspension for 30 min. The sediment moisture content (MC) was analyzed by oven-drying 2 g of

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fresh sediment at 108°C until a constant weight was reached. The sediment total nitrogen (TN),

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total carbon (TC), and total sulfur (TS) concentrations were determined using a VarioEL III

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Analyzer (Elementar Analysen System GmbH, Germany); detection limits were 0.05 mg kg−1, 0.2

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mg kg−1, and 0.25 mg kg−1, respectively. The N-compound concentration and pH value of inflow

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water were determined using the equivalent equipment for sediment. The dissolved oxygen

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concentration in surface sediments was measured in situ using an OXY Meter S/N 4164 with

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stainless electrode sensor (Unisense, Aarhus, Denmark), according to the reference37. All

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measurements were done in triplicate to ensure quality assurance/quality control (QA/QC). 8


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DNA extraction and Quantitative PCR assay

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DNA was extracted from freeze-dried sediment (approximately 0.33 g) using a FastDNA SPIN

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Kit for Soil (MP Biomedicals, Solon, OH, USA) according to the manufacturer’s protocol. The

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DNA concentration and quantity was determined using a NanoDrop 1000 spectrophotometer

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(Thermo Fisher Scientific, Schwerte, Germany).

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Quantitative PCR (qPCR) assays were conducted using the fluorescent dye SYBR-Green

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approach on an ABI 7500 Sequence detection system (Applied Biosystems, Foster City, CA). The

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abundances of anammox bacteria, nitrifier (AOB & AOA), denitrifier, DNRA, and total bacteria

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were quantified targeting the corresponding specific genes. The standard curves for qPCR were

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constructed using ten-fold serial dilutions of the corresponding genes with plasmid DNAs of known

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concentrations. A melting curve analysis was performed to confirm the specificity of the

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PCR-amplification. Amplification efficiencies ranged from 90 to 110%; the correlation coefficients

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(R2) were greater than 0.98; and the detection limit was 1.00×103 copies g-1. All tests were

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performed in triplicate. Details about the primer sets, thermal profiles, and experimental procedures

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are provided in Supplementary Table S1.

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Illumina hzsB amplicon sequencing and analysis

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In order to eliminate heterogeneity, the extracted DNA samples were mixed into three

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representative DNA sets to conduct high-throughput sequencing analysis on channel, ditch center,

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and ditch fringe samples. The hzsB gene (approximately 292 nucleotides) was amplified with the

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barcoded primer pairs 449F and 742R38 (Supplementary Table S1). The PCR products were

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sequenced on the Hiseq 2500 platform (Illumina, San Diego, CA, USA) at Novogene (Beijing,

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Environmental Science & Technology

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Microbial N-cycle hotspots in CW with N2O mitigation

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China) using a PE250 strategy (2 × 250 bp). Effective amino acid sequences were obtained using

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the quantitative insights into microbial ecology (QIIME) pipeline39. Operational taxonomic units

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(OTUs) and other α-diversity indexes were clustered at a 90% similarity using self-written Perl

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scripts, QIIME, and Mothur40. Phylogenetic trees with Heatmap analysis for representative OTUs

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were constructed using QIIME. All the sequence reads were deposited into GenBank database under

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the accession number SRR5404320 (Channel), SRR5403759 (Ditch center), and SRR5404262

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(Ditch fringe).

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Measuring anammox, denitrification and nitrification rates in sediments cores with

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technique

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N-tracer

The anammox, denitrification, and nitrification rates in this study were obtained by incubating 15

N-tracer technique41. The anammox, denitrification and

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intact sediment cores using an

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nitrification rates were detected in the intact sediment cores (100 cm length, 10.0 cm i.d., Plexiglas

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core tubes), collected from all the sampling sites. Cores were placed in an open tank filled with in

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situ water and maintained at the in situ temperature as that at the test site. Small Teflon coated

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magnets were placed and rotated above the sediment surface, to ensure the homogenous mixing of

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the water column. After 12 h pre-incubation to decrease the background 14NO3--N concentration, a

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stock solution of Na15NO3 [15N at 99.19%] for anammox and denitrification, and (15NH4)2SO4 [15N

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at 99.16%] for nitrification, were added to the water in the open incubation tank. In addition, to

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determine the nitrification rate, intact sediment cores were treated in the presence or absence of

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dicyandiamide (approximately 5 mmol DCD kg-1 sediment) which was used to block autotrophic

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ammonium oxidation 42. Gastight lids were then secured on all cores and incubation started. Three

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amended cores were sacrificed at 0, 3, 6, 12 and 24 h, by collecting 12 mL of slurry from each into 10


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a gastight vial (Exetainer, Labco, UK) containing 200 µL of 7 mol L−1 ZnCl2. N2 analysis was done

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to determine anammox and denitrification rates. The produced

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the nitrification rate, converting

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samples were further reduced to

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without any headspace. The

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N2 or

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NO3– to

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15

15

NO3− was analyzed to determine

NO2– at a pH of 8–9 with spongy cadmium. The

N2 using sulfamic acid. Samples in vials were then capped,

N2 produced using the process above were measured using isotope ratio mass

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spectrometers (IRMS, Gasbench II-MAT253, Bremen, Germany). IRMS precision for this study

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was

Ditch System of a

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