AEM Accepts, published online ahead of print on 26 September 2014 Appl. Environ. Microbiol. doi:10.1128/AEM.02379-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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Evidence for the co-occurrence of nitrite-dependent anaerobic ammonium and

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methane oxidation processes in a flooded paddy field

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Li-dong Shen1, Shuai Liu1, Qian Huang1, Xu Lian1, Zhan-fei He1, Sha Geng1,

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Ren-cun Jin2, Yun-feng He1, Li-ping Lou1, Xiang-yang Xu1, Ping Zheng1, Bao-lan

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Hu1,*

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1

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China

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310036, China

Department of Environmental Engineering, Zhejiang University, Hangzhou 310058,

Department of Environmental Science, Hangzhou Normal University, Hangzhou

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*For correspondence

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Bao-lan Hu

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Department of Environmental Engineering,

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Zhejiang University

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Hangzhou, 310058, China

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Tel.: 0086 571 88982340

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Fax: 0086 571 88982819

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E-mail: [email protected]

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Running title: Co-occurrence of anammox and n-damo

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Journal section: microbial ecology

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ABSTRACT: Anaerobic ammonium oxidation (anammox) and nitrite-dependent

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anaerobic methane oxidation (n-damo) are two of the most recent discoveries in the

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microbial nitrogen cycle. In the present study, we provided direct evidence for the

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co-occurrence of the anammox and n-damo processes in a flooded paddy field in

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southeastern China. Stable isotope experiments showed that the potential anammox

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rates ranged between 5.6 and 22.7 nmol N2 g-1 (dry weight) d-1, and the potential

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n-damo rates varied from 0.2 to 2.1 nmol CO2 g-1 (dry weight) d-1 in different layers

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of soil cores. Quantitative PCR showed that the abundance of anammox bacteria

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ranged between 1.0 × 105 and 2.0 × 106 copies g-1 (dry weight) in different layers of

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soil cores and the abundance of n-damo bacteria varied from 3.8 × 105 to 6.1 × 106

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copies g-1 (dry weight). Phylogenetic analyses of the recovered 16S rRNA gene

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sequences showed that anammox bacteria affiliated to Candidatus Brocadia and

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Candidatus Kuenenia and n-damo bacteria related to Candidatus Methylomirabilis

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oxyfera were present in the soil cores. It is estimated that a total loss of 50.7 g N m-2

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per year could be linked to the anammox process, which is at intermediate levels for

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the nitrogen flux ranges of aerobic ammonium oxidation and denitrification reported

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in wetland soils. In addition, it is estimated that a total of 0.14 g CH4 m-2 per year

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could be oxidised via the n-damo process, while this rate is at the lower end of aerobic

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methane oxidation rates reported in wetland soils.

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KEYWORDS: anammox; n-damo; activity; nitrogen cycle; methane cycle; flooded

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paddy field

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INTRODUCTION

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Microbially mediated anaerobic ammonium oxidation (anammox), which was

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predicted in Broda (1) based on thermodynamic calculations, was first confirmed in

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the 1990s in a denitrifying pilot plant (2). Thermodynamically, it was believed that

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microorganism capable of using nitrite as an electron acceptor for anaerobic methane

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oxidation could also exist in nature (3). The nitrite-dependent anaerobic methane

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oxidation (n-damo) was first confirmed in 2006 in an enrichment culture (4).

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Currently, five genera of anammox bacteria (Candidatus Brocadia, Candidatus

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Kuenenia, Candidatus Scalindua, Candidatus Anammoxoglobus and Candidatus

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Jettenia) which form a monophyletic order of bacteria, the Brocadiales (5), have been

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enriched and described. At present, it is believed that the anammox process is

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responsible for 50% of dinitrogen gas (N2) production in marine ecosystems (6-8).

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Although a limited number of recent studies has reported the presence of anammox

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bacteria and the occurrence of the anammox process in freshwater wetlands (9-11),

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the overall importance of this process in wetland systems is still unclear owing to a

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lack of data.

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The n-damo process is catalysed by “Candidatus Methylomirabilis oxyfera” (12),

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which is affiliated to the NC10 phylum. This process constitutes a unique association

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between the two major global nutrient cycles of carbon and nitrogen (4) and might

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serve as an important and overlooked sink of the greenhouse gas methane (13). Until

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now, however, the distribution of n-damo bacteria and the occurrence of the n-damo

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process in environments are not well known. Two recent studies have reported the 3

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presence of n-damo bacteria in the sediments of two freshwater lake ecosystems, Lake

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Constance in Germany (14) and Lake Biwa in Japan (15), and the activity of the

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n-damo process was confirmed in Lake Constance using radiotracer experiments.

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Wang et al. (16) and Zhou et al. (17) provided molecular evidence for the presence of

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n-damo bacteria in paddy fields. Furthermore, Shen et al. (18, 19) reported the

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presence of n-damo bacteria in the sediments of the Qiantang River and the Jiaojiang

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Estuary in China. The distribution of n-damo bacteria was also confirmed in the

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sediments of South China Sea (20). Recently, Hu et al. (21) and Shen et al. (22)

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reported the presence of n-damo bacteria and the occurrence of the n-damo process in

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wetlands.

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Among different types of wetlands (23), paddy fields are one of the most important

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nitrogen sinks, represent one of the most important sources of the greenhouse gas

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methane, and are responsible for 10-25% of global methane emissions (24).

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Furthermore, the paddy fields are characterised by cultivation patterns including water

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logging, which cause anoxic soil conditions (16). The anoxic soil conditions

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theoretically provide suitable habitats for both anammox bacteria and n-damo bacteria.

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In addition, the application of nitrogen-rich fertilisers further makes the paddy fields

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suitable habitats for these two groups of bacteria.

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The primary objectives of the present study were to investigate the distribution,

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diversity and significance of anammox bacteria and n-damo bacteria in a flooded

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paddy field (at a depth of 0-100 cm). Previous studies have indicated that the

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anammox bacteria were mainly present in the surface paddy soils, while the n-damo 4

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bacteria were mainly present in the deep paddy soils (16). To further ascertain the

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vertical distribution characteristics of these two groups of bacteria, two representative

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surface soil layers (0-10 and 20-30 cm) and two representative deep soil layers (50-60

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and 90-100 cm) were analysed in the current study. The distribution and diversity of

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anammox bacteria and n-damo bacteria were studied based on 16S rRNA gene clone

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library analyses, and the abundance of these bacteria was quantified by quantitative

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PCR (qPCR). The potential rates of the anammox and n-damo processes were

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determined using 15N and 13C stable isotope labelling experiments, respectively.

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MATERIALS AND METHODS

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Site description and sampling

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The flooded paddy field selected for this study is located in Zhejiang Province and

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represents a typical agricultural region of subtropical southeastern China, which has

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been planted in a rice rotation with a long history of fertilisation. The total rate of

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nitrogen fertilisation is approximately 300-350 g N m-2 per year. The paddy filed has

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been used for investigation of n-damo bacteria in a previous study (21), while a

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different sampling site was selected in the current study. A total of five soil cores were

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collected from the paddy field in September 2012 using a stainless steel ring sampler

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(5 cm in diameter and 100 cm in length). The cores were sliced at 10-cm intervals and

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mixed in the field for each depth to form one composite sample. The samples were

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immediately placed in sterile containers, sealed, and transported to the laboratory on

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ice within 12 h. The collected soil samples were subsequently divided into three parts.

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The first part was incubated to determine the potential anammox activity and n-damo 5

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activity immediately after arrival at the laboratory, the second part was stored

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anaerobically at 4 °C for subsequent physicochemical analyses, and the third part was

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stored at -80 °C for later molecular analyses.

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Physicochemical analyses

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The pH and temperature of the intact soil were determined in situ using an IQ150 pH

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meter (IQ Scientific Instruments Inc., Carlsbad, CA, USA). Soil ammonium and

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nitrate were extracted using 2 M KCl, as previously described (25, 26). The extracted

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NH4+ was determined through the method of salicylate acid (27). The NOx- was

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determined by reduction of NO3- to NO2- via cadmium reduction and measured

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through the method of N-(1-Naphthyl)ethylenediamine dihydrochloride (28), and the

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NO2- and NO3- were not differentiated in the current study. Because the methods used

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for determination of soil ammonium and nitrate did not exclude interference from

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humic substances, the current methods may overestimate the lower end of the

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concentration values of soil NH4+ and NOx-. The soil OrgC content was determined

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using the K2Cr2O7 oxidation method (25), and the soil TN content was determined

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using the FOSS Kjeltec™2300 analyser (FOSS Group, Höganäs, Sweden). The water

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content of soils was determined by oven drying overnight at temperature of 110 °C.

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The below-ground gas samples were gathered at 10 cm intervals through soil gas

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samplers as previously described (21). The methane was determined using an Agilent

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6890N gas chromatograph (Agilent) as previously described (21). All the above

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analyses were performed in triplicate on the soil samples or gas samples.

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Isotope tracer experiments 6

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Soil samples were transferred to He-flushed 75-mL glass vials together with

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He-purged deionised water. The soil slurries were pre-incubated under anaerobic

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conditions for at least 30 h to remove the residual NOx- and oxygen in the slurries. The

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slurries were subsequently divided into six treatment groups: (i)

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99.6%), (ii) 15NH4+ + NO2-, (iii) 15NO2- (15N at 99.6%), (iv) 13CH4 (13C at 99.9%), (v)

135

13

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ranged from 67.8-150.0 µmol kg-1 dry soil in treatments (i), (ii), (iii) and (v). Three

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independent experiments per sample were performed for each treatment group.

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Immediately after the pre-incubation step, 2 mL of headspace gas was removed and

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replaced with an equal volume of 13CH4, resulting in a final concentration of 4.5 × 103

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μmol L-1 in the headspace of each vial in treatments (iv), (v) and (vi). The production

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of 29N2, 30N2 and 13CO2 was measured directly from the headspace of each vial with

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GS-MS (Agilent 7890/5975C inert MSD; Agilent, United States) as previously

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described (21, 29, 30). The potential anammox rates could be calculated by the linear

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regression of the concentration of

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or

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study contained relatively high concentrations of NH4+. As a result, the background

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NH4+ in the slurries cannot be exhausted under anoxic conditions after the

148

pre-incubation. Thus the potential rates would be underestimated based on the slurries

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amended with 15NH4+ + NO2- because the background NH4+ could react with NO2- for

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production of 28N2. On the other hand, the background NO2-/NO3- in the slurries can

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be exhausted by denitrification and anammox under anoxic conditions. Actually, the

15

NH4+ (15N at

CH4 + NO2- and (vi) 13CH4 + SO42-. The final concentrations of NH4+ or NOx- were

15

29

N2 produced from slurries amended with

15

NO2-

NH4+ + NO2-. But the soil samples (especially for the surface soils) used in this

7

15

NH4+ + NO2- were

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potential anammox rates obtained from slurries amended

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approximately 85-97% of the rates obtained from slurries amended with only 15NO2-

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in our pre-experiment. Therefore, the concentration of

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amended with

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rates. The potential n-damo rates were calculated by the linear regression of the

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concentration of

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headspace of the vial over time. The coefficients of determination (R2) for linear

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regression of the

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0.9 for most data sets.

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DNA extraction and PCR amplification

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Soil DNA was extracted using a Power Soil DNA kit (Mo Bio Laboratories, Carlsbad,

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California, USA) according to the manufacturer’s instructions. Approximately 0.3 g

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of homogenised soil was used for the DNA isolation. The quality of the extracted

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DNA was evaluated on 1% agarose gel, and the DNA concentration was measured

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with a NanoDrop spectrophotometer (ND-1000; Isogen Life Science, the

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Netherlands).

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The 16S rRNA genes of anammox bacteria were amplified using a nested PCR

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protocol, as previously described (31). In the first round of PCR, the forward primer

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pla46f (32) and the reverse primer 1545r (33) were used. In the second round, the

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PCR reaction was conducted using the anammox bacterial specific primers Amx368f

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(34) and Amx820r (35). A nested PCR protocol was also used to amplify the 16S

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rRNA genes of n-damo bacteria, as previously described (36). In the first round of

15

29

N2 produced from slurries

NO2- was finally used for determination of the potential anammox

13

CO2 produced from slurries amended with

29

N2 and

13

13

CH4 + NO2- in the

CO2 concentrations change over time were greater than

8

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PCR, the n-damo bacterial specific forward primer 202F (30) and the general bacterial

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reverse primer 1545R (33) were used. In the second round, the PCR reaction was

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performed using primers qP1F and qP2R (30), which are specific for n-damo bacteria.

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The detailed information of the primers used in this study is shown in Table 1.

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Cloning and sequencing

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The PCR products were cloned using the pMD19-T vector (TaKaRa, Bio Inc., Shiga,

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Japan) according to the manufacturer’s instructions. Randomly selected positive

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clones for each sample were subjected to sequencing (Life Technology, Shanghai,

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China).

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Phylogenetic analysis

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The recovered 16S rRNA gene sequences were aligned with the MUSCLE algorithm

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(37) and imported into the MEGA 5 software (38), where the alignment was manually

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checked and trimmed. Phylogenetic analysis of the sequences was performed by

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Mega 5 software using the neighbour-joining method (38), and a BLAST search was

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performed to search for related sequences in GenBank. The evolutionary distances

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were computed using the Maximum Composite Likelihood method. The robustness of

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the tree topology was tested with a bootstrap analysis (1000 replicates), and bootstrap

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values > 70 (700 replicates) are shown at the branches.

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Quantitative PCR

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Hydrazine synthase (hzs) is a very important enzyme in anammox metabolism,

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responsible for the synthesis of hydrazine from nitric oxide and ammonium (39, 40).

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In this study, the primer set hzsA_1597f-hzsA_1857r targeting subunit α of the hzs 9

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genes of anammox bacteria was used to determine the abundance of anammox

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bacteria, as previously described (41). The abundance of n-damo bacteria was

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estimated by quantifying their 16S rRNA genes using the primer set qp1f-qp1r, as

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previously described (30). The standard curves were constructed from a series of

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10-fold dilutions of a known copy number of plasmid DNA containing the target

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genes. Negative controls in which the DNA template was replaced by nuclease-free

202

water were also performed. Triplicate qPCR analyses were performed for each sample.

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Single peaks were observed in the melting curves for both qPCR assays, and the

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amplifying efficiency was greater than 90% for both qPCR assays. In addition, the

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specificity of the primer sets on the anammox bacteria and n-damo bacteria was

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further confirmed by sequencing the qPCR products from several soil samples.

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Phylogenetic analysis showed that the sequences recovered from primer set

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hzsA_1597f-hzsA_1857r were all very closely related to the hzsA genes of anammox

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bacteria (Fig. S1). Similarly, phylogenetic analysis of the qPCR products from primer

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set qp1f-qp1r were all closely related to the 16S rRNA gene of M. oxyfera (Fig. S2).

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Statistical analyses

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The OTUs (operational taxonomic units) for the determination of the 16S rRNA gene

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diversity of anammox bacteria and n-damo bacteria were defined using 3%

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differences in the nucleotide sequences, using the furthest-neighbour algorithm in the

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DOTUR programme (42). The Chao1 estimator and the Shannon index were also

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generated using the DOTUR programme.

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Nucleotide sequence accession numbers 10

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The 16S rRNA gene sequences reported in this study have been deposited in the

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GenBank database under accession numbers KF754815-KF754836 (anammox 16S

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rRNA), KM403486-KM403495 (anammox hzsA) and KF754837-KF754861 (n-damo

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16S rRNA).

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RESULTS

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Physicochemical analyses of the collected core samples

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The vertical distribution profiles of the soil NH4+, NOx-, CH4, pH, temperature, total

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nitrogen (TN) and organic carbon (OrgC) at 10-cm intervals are shown in Fig. 1. The

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NH4+ content peaked at the layer depth of 10-20 cm and then decreased with depth

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from 2270.7 ± 122.6 to 9.0 ± 0.4 µmol kg-1 dry soil. The content of NOx- peaked at the

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0-10 cm layer and then decreased with depth from 745.1 ± 45.5 to 7.9 ± 0.4 µmol kg-1

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dry soil. The simultaneous decrease of both NH4+ and NOx- with depth may indicate

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the occurrence of anammox and denitrification. As opposed to NOx-, the CH4

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concentration in soil gas showed an increasing trend with depth from 13.1 ± 0.7 to 6.3

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± 0.7 × 103 μmol L-1. The co-existence of NOx- and methane may suggest that the

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paddy soil could provide a suitable habitat for the n-damo bacteria. Soil samples

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collected from four representative layers (0-10 cm, 20-30 cm, 50-60 cm and 90-100

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cm) were selected for further molecular analyses and activity tests.

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Phylogenetic analyses of anammox bacteria and n-damo bacteria

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Phylogenetic analysis of the recovered 16S rRNA gene sequences of anammox

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bacteria showed that these sequences were grouped into three distinct clusters (Fig. 2).

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The sequences of the Brocadia cluster, which were recovered from the layer of 90-100 11

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cm, showed 95.0-96.2% identity to the 16S rRNA gene of Candidatus Brocadia

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anammoxidans. This cluster was most closely related to the sequences obtained from

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the Baiyangdian lake sediments (10), with 99% identity. The sequences of the

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Kuenenia cluster, which were recovered from the layer of 0-10 cm, showed

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94.8-97.5% identity to the 16S rRNA gene of Candidatus Kuenenia stuttgartiensis.

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The closest relatives of this cluster were the sequences retrieved from Qiantang River

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sediments (43) with 98% identity. Furthermore, a new anammox cluster formed in the

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phylogenetic tree (Fig. 2), which was distantly related to the 16S rRNA gene of

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Candidatus Kuenenia, with 92.6-95.0% identity. This cluster was most closely related,

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with 99% identity, to the clones obtained from another paddy field also located in

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southeastern China (9).

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Phylogenetic analysis of the recovered 16S rRNA gene sequences of n-damo bacteria

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showed that the recovered sequences were grouped into three separate clusters (Fig.

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3), which were assigned to two groups of n-damo bacteria, group A and group B,

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according to Ettwig et al. (30). The sequences of cluster I, which were primarily

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recovered from the 50-60 cm and 90-100 cm layers, showed 95.8-96.9% identity to

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the 16S rRNA gene of M. oxyfera. The closest relatives of this cluster, with 98%

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identity, were the clones recovered from another paddy field (16). The sequences of

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clusters II and III, which were primarily recovered from the 0-10 cm and 20-30 cm

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layers, only showed 91.6-92.1% and 90.1-90.9% identities to the 16S rRNA gene of

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M. oxyfera, respectively. These two clusters were also most closely related to clones

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recovered from another paddy field (16), with 98% identity.

12

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Genetic diversity analyses of anammox bacteria and n-damo bacteria

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The diversity levels of the 16S rRNA genes of anammox bacteria and n-damo bacteria

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in each sample were determined based on the number of OTUs, the Shannon index

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and the Schao1 estimators (Table S1). A total of 7 and 11 OTUs of the 16S rRNA genes

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of anammox bacteria and n-damo bacteria were observed, respectively. Similar

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diversity of anammox bacterial 16S rRNA genes was observed between different

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layers of soil cores (Table S1). The diversity of the 16S rRNA genes of n-damo

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bacteria was also very similar between the different layers of soil cores (Table S1). It

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could be observed that the diversity of the n-damo bacteria was higher than that of the

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anammox bacteria at each layer (Table S1).

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Quantitative analyses of anammox bacteria and n-damo bacteria

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The qPCR results further confirmed the co-existence of anammox bacteria and

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n-damo bacteria in different layers of soil cores. The abundance of anammox bacteria

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ranged between 1.0 ± 0.04 × 105 and 2.0 ± 0.14 × 106 copies g-1 (dry weight) assuming

276

that the anammox bacteria contain one copy of the hzsCBA gene cluster, as previously

277

reported (39, 40). Different abundances of anammox bacteria were observed at the

278

different layers of soil cores, with the highest abundance at the 0-10 cm layer (Fig. 4a).

279

Different abundances of n-damo bacteria were also observed at different layers of soil

280

cores, with the highest abundance (6.1 ± 0.25 × 106 copies g-1 (dry weight)) at the

281

90-100 cm layer and the lowest abundance (3.8 ± 0.12 × 105 copies g-1 (dry weight))

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at the 20-30 cm layer (Fig. 4a).

283

Activity analyses of the anammox process and n-damo process 13

284

Stable isotope experiments confirmed the co-occurrence of anammox and n-damo

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processes in the examined paddy field (Fig. 5). The results showed that the potential

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anammox rates ranged between 5.6 ± 0.6 and 22.7 ± 1.0 nmol N2 g-1 (dry weight) d-1,

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which contributed 8.7-29.8% to soil N2 production. Different potential anammox rates

288

were observed at the different layers of soil cores, with the higher potential anammox

289

rates at the layers of 0-10 cm and 20-30 cm (Fig. 4b). The cell specific anammox rates

290

ranged from 9.5 to 36.2 fmol N per cell per day. The potential n-damo rates ranged

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between 0.2 ± 0.01 and 2.1 ± 0.08 nmol CO2 g-1 (dry weight) d-1 in the examined core

292

samples. No n-damo activity could be detected at the layer of 0-10 cm, while obvious

293

n-damo activities were observed at the layers of 20-30 cm, 50-60 cm and 90-100 cm

294

(Fig. 4b). The cell specific n-damo rates ranged from 0.3 to 0.4 fmol CO2 cell-1 d-1.

295

DISCUSSION

296

Distribution and diversity of anammox bacteria and n-damo bacteria

297

Multiple co-occurring anammox populations were found together, and a higher level

298

of n-damo bacterial diversity was also observed in the current study. Soil is a highly

299

heterogeneous

300

micro-environments for different species of anammox bacteria and n-damo bacteria. It

301

was found that only Candidatus Kuenenia was detected at the 0-10 cm soil layer,

302

while only Candidatus Brocadia was detected at the 90-100 cm soil layer (Fig. 2). All

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the sequences retrieved from the 20-30 cm and 50-60 cm layers were affiliated to the

304

new cluster (Fig. 2). The vertical variation in the community structures of anammox

305

bacteria was more or less similar to the results reported by Zhu et al. (9). For the

environment,

and

the

14

paddy

field

may

provide

diverse

306

n-damo bacteria, the group A members, which were reported to be the dominant

307

bacteria responsible for the n-damo process (30, 36, 44-46) were only detected at the

308

soil layers of 50-60 cm and 90-100 cm (Fig. 3). The group A members were also

309

primarily present in the deep layer of the reported wetland systems (below 40-50 cm

310

layer; 16, 21, 22).

311

Abundance of anammox bacteria and n-damo bacteria

312

The abundance of anammox bacteria (1.0 ± 0.04 × 105 - 2.0 ± 0.14 × 106 copies g-1

313

(dry weight)) observed in this study was lower than the values reported in freshwater

314

river sediments (106 -107copies g-1 sediment; 43), while within the range of another

315

paddy field (105-107 copies g-1 soil; 9). The abundance of n-damo bacteria (3.8 ± 0.12

316

× 105 - 6.1 ± 0.25 × 106 copies g-1 soil) in the examined paddy field was similar to the

317

values reported for lake sediments (105-106 copies g-1 sediment; 15), river sediments

318

(106-107 copies g-1 sediment; 18) and wetland systems (106-107 copies g-1 soil; 21, 45).

319

The abundance of anammox bacteria showed a decreasing trend from the 0-10 cm

320

layer to the 90-100 cm layer (Fig. 4a), as previously described (9). In contrast, the

321

abundance of n-damo bacteria showed an increasing trend from the 0-10 cm layer to

322

the 90-100 cm layer (Fig. 4a). Previous studies also suggested that n-damo bacteria

323

were most abundant in deep wetland soils (16, 17, 21, 45).

324

Activities and roles of the anammox process and n-damo process

325

The potential anammox rates (5.6 ± 0.6 - 22.7 ± 1.0 nmol N2 g-1 (dry weight) d-1)

326

measured in this paddy field were in the same range as those reported in most marine

327

and freshwater environments (11, 47, 48), but lower than the values reported in

15

328

land-freshwater interfaces of Baiyangdian Lake (84-240 nmol N2 g-1 d-1; 10). The

329

contribution (8.7-29.8%) of anammox to soil N2 production in the examined paddy

330

field was similar to the values of another paddy field (4-37%; 9). The potential

331

n-damo rates (0.2 ± 0.01 - 2.1 ± 0.08 nmol CO2 g-1 (dry weight) d-1) measured in this

332

paddy field were in the same range as those reported in lake sediments (1.8-3.6 nmol

333

CO2 mL-1 d-1; 14) and wetland soils (0.2-14.5 nmol CO2 g-1 d-1; 17, 21, 22).

334

The co-occurrence of the anammox and n-damo processes was confirmed in different

335

layers of the examined paddy field by incubation experiments. It should be noted that

336

a high concentration of in situ NOx- (228.6-745.1µmol kg-1 dry soil) was detected in

337

the upper layer (0-30 cm) of the examined paddy field, but a relatively lower

338

concentration of NOx- (7.9-101.7 µmol kg-1 dry soil) was observed below the layer of

339

50 cm (Fig. 1). The occurrence of the anammox and n-damo processes at the deep

340

layer may be limited by the availability of NOx- under in situ environments. As a

341

result, the incubation experiments could overestimate the in situ rates of the anammox

342

and n-damo processes at the layers of 50-60 cm and 90-100 cm because a

343

concentration of 67.8-150.0 µmol NO2- kg-1 dry soil was added in the slurries. To

344

make a conservative estimate for nitrogen flux by the anammox process occurring in

345

situ environments, only the potential anammox rates at the layers of 0-10 cm and

346

20-30 cm were used. Therefore, it can be calculated that the nitrogen flux by the

347

anammox process in the examined paddy field was approximately 50.7 g N m-2 per

348

year based on the reported mean density of paddy soil (1.24 g cm-3; 49). The aerobic

349

ammonium oxidation rates ranged from 3.7 to 784.8 g N m-2 per year in the reported

16

350

wetland soils (50, 51), and the nitrogen loss by denitrification ranged from 1.1 to

351

372.3 g N m-2 per year in the reported wetland soils (50, 52). Thus the nitrogen flux

352

(50.7 g N m-2 per year) by the anammox process is at intermediate levels for the

353

nitrogen flux ranges of aerobic ammonium oxidation and denitrification reported in

354

wetland soils. Similarly, only the potential n-damo rates at the layer of 20-30 cm were

355

used to make a conservative estimate for methane oxidation by the n-damo process in

356

the examined paddy field, and it is estimated that approximately 0.14 g CH4 m-2 per

357

year could be oxidised via the n-damo process. The methane oxidation rate by the

358

n-damo process is at the lower end of aerobic methane oxidation rates reported in

359

wetland soils (53).

360

Higher potential anammox rates were observed at the layers of 0-10 cm and 20-30 cm,

361

while higher potential n-damo rates were observed at the layers of 50-60 cm and

362

90-100 cm (Fig. 4b). In the surface soil layer, higher NH4+ concentration and NOx-

363

concentration were observed in the examined paddy field (Fig. 1), which could

364

stimulate the occurrence of the anammox process, as previously reported (9). Previous

365

studies also indicated that anammox activities in surface soil/sediments were greater

366

than those in deep soil/sediments (9, 10, 54, 55). It was found that group A of n-damo

367

bacteria were primarily present in the deep layers (Fig. 3) where a higher abundance

368

of n-damo bacteria was observed (Fig. 4a). These findings can explain the higher

369

potential n-damo rates measured at the deep layers. Generally, the microbial process

370

using NOx- as an electron acceptor would be limited by its availability in the deep soil

371

layer because a major part of NOx- could be consumed in the upper soil layer.

17

372

However, a certain concentration of NOx- (7.9-101.7 µmol kg-1 dry soil) was observed

373

in the deep layer of the examined paddy field (Fig. 1). The presence of NOx- in the

374

deep layer may be because of NOx- leaching from the upper layer (22). The examined

375

paddy field was frequently irrigated because it has been planted in a rice rotation. In

376

addition, the paddy field has a long history of fertilisation, and the total rate of

377

nitrogen fertilisation is approximately 300-350 g N m-2 per year. The NOx- leaching

378

has been shown to depend on rates of irrigation and nitrogen fertilisation and

379

increases with rates of irrigation and fertilisation (56-58).

380

ACKNOWLEDGEMENTS

381

We thank the Natural Science Foundation (No. 51108408 and No. 31170458) and the

382

Shanghai Tongji Gao Tingyao Environmental Science and Technology Development

383

Foundation.

384

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578

leaching of winter wheat grown in lysimeters as affected by fertilizers and

579

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580

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581

nitrate leaching losses under intensive crop production with Ochric Aquic

582

Cambosols in North China Plain. Environ. Int. 31: 904-912.

583 584 585 586 587 588 589 590 27

591

FIGURE LEGENDS

592

Fig. 1 Vertical distribution of soil NH4+ (a), NOx- (b), CH4 (c), pH (d), temperature (e),

593

total nitrogen (TN) (f), and organic carbon (OrgC) (g) in core samples collected from

594

the paddy field.

595

Fig. 2 Neighbour-joining phylogenetic tree showing the phylogenetic affiliations of

596

the anammox bacterial 16S rRNA gene sequences in core samples collected from the

597

paddy field. The bootstrap values included 1000 replicates, and the scale bar

598

represents 2% sequence divergence. The identifiers S10, S30, S60 and S100 represent

599

core samples collected from layers of 0-10 cm, 20-30 cm, 50-60 cm and 90-100 cm,

600

respectively. The numbers in the brackets indicate the number of clones in each

601

cluster out of the total number of clones sequenced.

602

Fig. 3 Neighbour-joining phylogenetic tree showing the phylogenetic affiliations of

603

the n-damo bacterial 16S rRNA gene sequences in core samples collected from the

604

paddy field. The bootstrap values included 1000 replicates, and the scale bar

605

represents 2% sequence divergence. The numbers in the brackets indicate the number

606

of clones in each cluster out of the total number of clones sequenced.

607

Fig. 4 The copy numbers of anammox bacterial hzsA genes and n-damo bacterial 16S

608

rRNA genes (a) and the potential anammox rates and n-damo rates (b) in core samples

609

collected from different layers of the paddy field.

610

Fig. 5 Examples of concentrations of 29N2/30N2 produced from core samples (collected

611

from 90-100 cm depth) amended with

612

and examples of concentrations of 13CO2 produced from core samples (collected from

15

NH4+ (a),

28

15

NH4+ + NO2- (b) and

NO2- (c),

15

613

90-100 cm depth) amended with 13CH4 (d), 13CH4 + NO2- (e) and 13CH4 + SO4- (f).

29

Table 1 PCR primers used in this study Primers

Sequence 5′-3′

Specificity

Pla46f

GGATTAGGCATGCAAGTC

Planctomycetes

1545r

CAKAAAGGAGGTGATCC

Bacteria

Amx368f

TTCGCAATGCCCGAAAGG

Anammox

Amx820r

AAAACCCCTCTACTTAGTGCCC

Anammox

202f

GACCAAAGGGGGCGAGCG

N-damo

1545r

CAKAAAGGAGGTGATCC

Bacteria

qP1f

GGGCTTGACATCCCACGAACCTG

N-damo

qP2r

CTCAGCGACTTCGAGTACAG

N-damo

qP1f

GGGCTTGACATCCCACGAACCTG

N-damo

qP1r

CGCCTTCCTCCAGCTTGACGC

N-damo

hzsA_1597f

WTYGGKTATCARTATGTAG

Anammox

hzsA_1857r

AAABGGYGAATCATARTGGC

Anammox

ND-not determined

Amplification length (bp)

Reference (32)

ND

(33) (34)

477

(35) (30)

ND

(33) (30)

459-460

(30) (30)

200

(30) (41)

261

(41)