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Evidence of Nitrogen Loss from Anaerobic Ammonium Oxidation Coupled with Ferric Iron Reduction in an Intertidal Wetland Xiaofei Li, Lijun Hou, Min Liu, Yanling Zheng, Guoyu Yin, Xianbiao Lin, Lv Cheng, Ye Li, and Xiaoting Hu Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b03419 • Publication Date (Web): 11 Sep 2015 Downloaded from http://pubs.acs.org on September 12, 2015

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Environmental Science & Technology

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Evidence of Nitrogen Loss from Anaerobic Ammonium Oxidation

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Coupled with Ferric Iron Reduction in an Intertidal Wetland

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Xiaofei Li,†,§ Lijun Hou,∗,‡,§ Min Liu,*,† Yanling Zheng,‡ Guoyu Yin,‡ Xianbiao Lin,†

5

Lv Cheng,† Ye Li,† and Xiaoting Hu†

6 7

†

8

Zhongshan Road, Shanghai, 200062, China

9

‡

College of Geographical Sciences, East China Normal University, 3663 North

State Key Laboratory of Estuarine and Coastal Research, East China Normal

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University, 3663 North Zhongshan Road, Shanghai, 200062, China.

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∗Corresponding Authors:

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E-mail addresses: [email protected] (L.J.H.), and [email protected]

13

(M.L.); Phone: 86-21-62231669; fax: 86-21-62546441

14 15

§

Xiaofei Li and Lijun Hou contributed equally to this work.

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ACS Paragon Plus Environment


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ABSTRACT

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Anaerobic ammonium oxidation coupled with nitrite reduction is an important

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microbial pathway of nitrogen removal in intertidal wetlands. However, little is

20

known about the role of anaerobic ammonium oxidation coupled with ferric iron

21

reduction (termed Feammox) in intertidal nitrogen cycling. In this study, sediment

22

slurry incubation experiments were combined with an isotope-tracing technique to

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examine the dynamics of Feammox and its association with tidal fluctuations in the

24

intertidal wetland of the Yangtze Estuary. Feammox was detected in the intertidal

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wetland sediments, with potential rates of 0.24-0.36 mg N kg−1 d−1. The Feammox

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rates in the sediments were generally higher during spring tides than during neap tides.

27

The tidal fluctuations affected the growth of iron-reducing bacteria and reduction of

28

ferric iron, which mediated Feammox activity and the associated nitrogen loss from

29

intertidal wetlands to the atmosphere. An estimated loss of 11.5−18 t N km−2 year−1

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was linked to Feammox, accounting for approximately 3.1−4.9% of the total external

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inorganic nitrogen transported into the Yangtze Estuary wetland each year. Overall,

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the co-occurrence of ferric iron reduction and ammonium oxidation suggests that

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Feammox can act as an ammonium removal mechanism in intertidal wetlands.

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■ INTRODUCTION

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Nitrogen (N) pollution in aquatic ecosystems has become a more significant

36

environmental issue at regional and global scales,1−3 mainly due to the excessive use

37

of N fertilizers in agricultural activities4,5 and to the release of N from fossil fuel

38

combustion.2,4 Specifically, the substantial amount of N released into aquatic

39

environments is considered an important driver of water pollution (e.g., coastal

40

eutrophication, hypoxia, and harmful algal blooms).2,3,6-8 Shifts in microbial

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communities and diversity in response to N inputs9 also likely influence N

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transformations.10 Therefore, increasing concerns regarding the pathways of N cycling

43

in aquatic environments have been raised for decades and can be used to help reveal N

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budgets in natural ecosystems and provide a thorough understanding of global N

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biogeochemistry.1,2 Of the N transformation processes, canonical denitrification,

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which is the heterotrophic reduction of nitrate to N2, has traditionally been considered

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the only important mechanism of N loss from aquatic ecosystems.1,2,11 However, in

48

recent years, anaerobic ammonium oxidation (anammox), which oxidizes ammonium

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into dinitrogen gas by reducing nitrite,12,13 has been found to contribute to the removal

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of N from freshwater, estuaries and coastal marine environments.12−17 More recently,

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anaerobic ammonium oxidation coupled with Fe(III) reduction (termed Feammox),

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which can produce N2,5,18 NO3−5,18 or NO2−,5,18,19,20 has been identified in many

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ecosystems including riparian sediments,19,20 tropical forest soils,18 and paddy soils.5

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Because the production of N2 by Feammox is more energetically favorable than the

55

generation of NO2− or NO3−,18 this process is considered a potentially significant 3 / 36



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contributor to N removal.5,18 However, no reports regarding the occurrence of

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Feammox or its contribution to total N losses are currently available for intertidal

58

wetlands.

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Intertidal wetlands are key transitional zones between land and marine

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ecosystems, play an important role in global biogeochemical cycles, and provide

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extensive ecosystem services.21−24 However, intertidal wetlands are usually subjected

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to periodic water inundation and atmospheric exposure due to tidal fluctuations.27 In

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such environmental systems, sediments endure periodic desiccation , especially during

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neap tides, which can result in the oxidation of Fe(II) to Fe(III) and of ammonium to

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nitrite and nitrate. After desiccation, the rewetting of sediments during spring tides

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can contribute to Fe(III) reduction and to the generation of sedimentary ammonium

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through organic matter mineralization or ion exchange.25,26 In addition, changes

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between dry and wet processes can alter the sediment structure and sedimentary

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microenvironments.27 Overall, alternant processes, such as the results of tidal

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fluctuations, can cause a series of interrelated biotic and abiotic changes that can

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affect biogeochemical reactions in intertidal wetlands.27,28 Considering the

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environmental characteristics of intertidal wetlands,8,27 we hypothesize that the

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periodic dry and wet processes induced by spring and neap tidal fluctuations can

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facilitate the microbial process of Feammox in these environments.

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In this study, we provide geochemical evidence for Feammox in surface

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sediments from the intertidal wetland of the Yangtze Estuary. The main objective of

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this study is to examine the occurrence of Feammox and the associations of Feammox 4 / 36


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dynamics with sedimentary inorganic nitrogen changes and Fe redox reactions under

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tidal fluctuations. The abundance of iron-reducing bacteria (FeRB) which regulate

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Fe(III) reduction was measured. Furthermore, the contribution of Feammox to the

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total N loss from the intertidal wetland was identified. This work provides new

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insights regarding nitrogen biogeochemical cycles coupled with Fe redox reactions in

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intertidal wetlands.

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■ MATERIALS AND METHODS

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Site Description and Sediment Sampling. The Yangtze Estuary is located in the

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center of China’s eastern coast. Extensive intertidal mudflats have developed along

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the Yangtze estuarine and coastal zone due to the deposition of substantial amounts of

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suspended sediment from the Yangtze River Basin.29,30 In this work, the research site

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(121°59′E, 31°30′N) was located in the Chongming Eastern Intertidal Wetland which

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is the largest and most extensively developed mudflat in the mouth of the Yangtze

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Estuary (Figure S1, Supporting Information). Because of semi-lunar tidal fluctuations,

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the inundation duration in this intertidal wetland can be maintained for approximately

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3-5 d during spring tide. In contrast, this area is continuously exposed to air for

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approximately 10-12 d during intermediate and neap tides. Thus, these sediments in

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the intertidal wetland experience periodic dry and wet processes during semi-lunar

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tidal cycles, and this intertidal wetland was consequently selected as the case-study

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site. Fieldwork was conducted at the end of the spring and neap tides for two

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successive semi-lunar tidal cycles in August and December 2014, respectively. At 5 / 36



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each sampling, six replicates of sediment samples (0–5 cm depth) were collected from

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3 m2 of homogenous intertidal mudflat using plexiglass corers. After collection, the

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sediment core samples were packed into cleaned polyethylene bags and sealed

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without air before transporting on ice to the laboratory within 2 h. After returning to

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the laboratory, each sediment core was immediately homogenized under helium and

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divided into three fractions. The first fraction was immediately incubated to determine

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the potential nitrogen transformation rates using isotope tracer incubations, the second

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fraction was stored at –80 oC for molecular analyses, and the third fraction was stored

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at 4 oC for sediment characteristics measurements, including the sediment water

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content, pH, redox potential, total organic carbon content, sedimentary inorganic

110

nitrogen content, reactive Fe concentrations.

111 112

Isotopic Tracer Incubations. Sediment slurry incubations combined with nitrogen

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isotope-tracing technique were conducted in an anaerobic glove box to measure

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Feammox and Fe(III) reduction rates using the procedure from Ding et al.5 and Yang

115

et al.18 with slight modifications. Briefly, slurries were made by adding sterile anoxic

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artificial seawater (salinity: 10‰) to sediments at a ratio of 2:1 (v/w) and adequately

117

homogenized using a magnetic stirrer before anaerobic preincubation in the dark at

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nearly in situ temperatures (30 °C for August and 10 °C for December) for

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approximately 12 h to eliminate background nitrite, nitrate, and oxygen. After

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preincubation, 12 g of the homogenized slurries were transferred into 100-mL serum

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vials under helium before immediately sealing the vials with butyl rubber septa and 6 / 36


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crimp-capping with aluminum caps. These vials were divided into the three following

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treatments: (1) sterile anoxic artificial seawater instead of

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15

125

MA, USA,

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for both

127

purity He-purged stock solutions of 15NH4Cl through the septa to achieve a dry weight

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of 45 mg N kg-1, which was based on the annual inorganic N (IN) load into the

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Yangtze Estuary wetlands.21 For the

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gas in each vial was replaced with C2H2. All vials were vigorously shaken to

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homogenize the treatment solutions before incubation. After 24 h of incubation, 12

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mL of gas samples were collected immediately using gastight syringes and then

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injected into 12 mL pre-evacuated Labco Exetainer vials sealed with butyl-rubber

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septa (Labco Limited, High Wycombe, Buckinghamshire, UK). The

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of N2 in the gas samples was determined using an isotope ratio mass spectrometry

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(IRMS, Thermo Finnigan Delta V Advantage, Bremen, Germany), and a more

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detailed description is provided in the Supporting Information. The potential

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Feammox rates were conservatively quantified using the difference in 30N2 production

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with and without 15NH4+ addition.5 Furthermore, after the gas samples were collected

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to determinate the Feammox rates, the incubated slurries were also immediately

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subsampled to quantify the Fe(III) reduction rates based on changes in the Fe(II)

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concentration during the incubations.5

15

NH4Cl (control); (2)

NH4Cl addition (15N at 99.0%, Cambridge Isotope Laboratories, Inc., Tewksbury,

15

15

NH4+); and (3)

15

NH4Cl and C2H2 addition (15NH4+ + C2H2). The vials

NH4+ and 15NH4+ + C2H2 treatments were spiked with 1.0 mL of ultrahigh

15

NH4+ + C2H2 treatments, 30 mL of headspace

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15

N enrichment


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Analysis of Sediment Characteristics. The sediment water content was determined

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from the amount of weight lost from a known amount of wet sediment that had been

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dried at 60 °C to a constant value. The redox potential (Eh) of the sediment was

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measured using a combined Ag/AgCl and Pt (s) electrode connected to a

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potentiometer, and Zobell solution was used for calibration.27 The sediment pH was

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measured using a Mettler-Toledo pH meter after mixing the sediment with deionized

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water free of CO2 at a ratio (w/v) of 1:2.5.21 The total organic carbon (TOC) in

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sediments was determined using a thermal combustion furnace analyzer (Elementar

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vario MaxCNOHS analyzer, Germany) after leaching with 1 M HCl to remove

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carbonate.21 The exchangeable NH4+, NO3- and NO2- in the sediments were extracted

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using a 2 M KCl solution and determined via a continuous-flow nutrient autoanalyzer

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(SAN plus, Skalar Analytical B.V., Breda, The Netherlands) with detection limits of

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0.5 µM for NH4+ and 0.1 µM for NO3- and NO2-.21 The total extractable Fe and Fe(II)

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in the sediments were determined using the procedure described by Lovley et al.31

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with slight modifications. Briefly, 1.0 g samples were extracted in 10 mL mixtures of

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0.5 M HCl and 0.25 M hydroxylamine hydrochloride for 2 h to determine the total

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extractable Fe and Fe(II), respectively, using the ferrozine method.31 The amount of

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microbially reducible Fe(III) (considered as hydroxylamine-reducible Fe(III)) was

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calculated as the difference between the total extractable Fe and Fe(II).5,31

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DNA Extraction and PCR Amplification. The total genomic DNA of the sediment

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was extracted from 0.25 g of sediment and sampled using Power Soil DNA Isolation 8 / 36


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kits (MoBio, USA) according to the manufacturer’s directions.7,21 The purities and

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concentrations of DNA were measured on a Nanodrop-2000 Spectrophotometer

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(Thermo, USA). Quantitative PCR (qPCR) of the iron-reducing bacteria (FeRB) was

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performed using the specific primer sets and qPCR conditions reported by

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Somenahally et al.32 FeRB was determined by targeting Geobacteraceae spp. and

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Shewanella spp., which are important microbial communities involved in iron

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reduction under anaerobic conditions.32 The Geobacteraceae fragments in the

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extracted DNA were amplified using the Geo564F (5′- AAG CGT TGT TCG GAW

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TTA T -3′) and Geo840R (5′- GGC ACT GCA GG GGT CAA T A -3′) primers. The

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Shewanella fragments were amplified using the She 120F (5′- GCC TAG GGA TCT

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GCC CAG TCG -3′) and She 220R (5′- CTA GGT TCA TCC AAT CGC G -3′)

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primers. In addition, qPCR was conducted using an ABI 7500 Sequence Detection

178

System (Applied Biosystems, Canada) and the SYBR green qPCR method.7

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Additional detailed descriptions of the qPCR amplification for FeRB are provided in

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the Supporting Information.

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Statistical Analyses. All statistical analyses were performed using SPSS version 17.0

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for Windows (SPSS Inc., Chicago, IL, USA). Statistical analyses for comparing the

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obtained data during spring and neap tides were conducted using a one-way analysis

185

of variance (ANOVA) followed by a LSD test. Pearson’s correlation analysis was

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performed to reveal the relationships among the environmental factors, Fe(III)

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reduction rates, FeRB abundance and nitrogen transformation rates. 9 / 36



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■ RESULTS

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Dynamics of the Sediment Characteristics. Changes in the sediment characteristics

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during the neap and spring tidal fluctuations are shown in Figure 1. The sediment

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water contents were significantly higher during spring tides (66.4-73.3%) than during

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neap tides (25.4-31.3%) (P < 0.01). In contrast, the sediment Eh was significantly

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lower during spring tides (62.7-72.4 mV) than during neap tides (387.6-484.3 mV)

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(P < 0.01); the sediment pH was slightly lower during spring tides (8.32-8.46) than

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during neap tides (8.48-8.75), but no significant seasonal difference was observed (P >

196

0.05). The sediment TOC contents during the spring tides (0.51-0.76%) were

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comparable to those during the neap tides (0.58-0.81%) and were significantly lower

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(0.51-0.59%) during the summer than during the winter (0.72-0.81%) (P < 0.05). The

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sediment NH4+ concentrations generally increased during the spring tides (3.26-3.73

200

mg N kg-1) and significantly decreased during the neap tides (2.25-2.63 mg N kg-1) (P

201

< 0.01). Compared with NH4+, opposite changes in the sediment NOx- concentrations

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were observed during the periodic tidal fluctuations. The Fe(II) contents in the

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sediments varied from 0.54 to 1.03 g Fe kg-1 during the summer and from 0.74 to 1.04

204

g Fe kg-1 during the winter, and the

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kg-1 during the summer and from 0.57 to 0.87 g Fe kg-1 during the winter. Specifically,

206

the Fe(II) contents increased significantly during the spring tides as the Fe(III)

207

contents significantly decreased relative to the neap tides (P < 0.01).

Fe(III) contents varied from 0.57 to 1.07 g Fe

208 209

15

N2 Production Measured Through Isotopic Tracer Incubations. The potential

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30

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neap tides were determined using

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acetylene inhibition techniques (Figure 2). Significant 30N2 contents were detected in

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the treatments with both 15NH4+ and 15NH4+ + C2H2, but no 30N2 was detected in the

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control treatments (without 15NH4+ addition). This result demonstrates the occurrence

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of Feammox in the intertidal sediments because the N2 resulting directly from

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Feammox and/or Feammox-produced NO3− or NO2−, which resulted from

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denitrification or anammox after Feammox, are the only potential sources of

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under anoxic conditions.5,18 The mean 30N2 production rates in the 15NH4+ treatments

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were significantly greater than those in the 15NH4+ + C2H2 treatments, regardless of

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seasonal changes or tidal fluctuations (P < 0.05; Figure 2c,d). In the

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treatments, the mean 30N2 production rates during the spring tides ranged from 0.35 to

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0.36 mg N kg−1 d−1 in the summer and from 0.32 to 0.33 mg N kg−1 d−1 in the winter,

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which were significantly higher (P < 0.05) than the rates observed during the neap

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tides (0.26–0.27 mg N kg−1 d−1 in summer and 0.24–0.25 mg N kg−1 d−1 in winter). In

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contrast, the presence of C2H2 resulted in a decrease of 0.02−0.06 mg N kg−1 d−1 in

226

the

227

showed similar seasonal changes to those observed in the

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formation of 29N2 in the sediments under the tidal fluctuations was approximately

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2-fold greater than that of

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treated with both 15NH4+ and 15NH4+ + C2H2, the 29N2 production rates showed similar

231

variations as the 30N2 production rates.

N2 and

30

29

N2 production rates in intertidal sediments influenced by the spring and

N2 production rates. The

30

30

15

N-labeled ammonium-based isotope tracing and

N2 production rates in the

15

15

30

N2

NH4+

NH4+ + C2H2 treatments 15

NH4+ treatments. The

N2 (Figure 2a,b). During the isotope-tracing incubations

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Fe(III) Reduction Rates in Isotopic Tracer Incubations. In this study, the Fe(III)

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reduction rates were significantly enhanced in the

234

treatments, compared with the rates observed in the control treatments (P < 0.05;

235

Figure 3). Additionally, the increase in Fe(III) reduction rates was greater in the

236

15

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indicate that a portion of the sedimentary indigenous Fe(III) was reduced to Fe(II) and

238

that the addition of

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were generally higher during the summer than during the winter (P < 0.05), likely

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because of the seasonal differences in temperature, sediment microbial communities,

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and/or sediment compositions. Moreover, in the 15NH4+ and 15NH4+ + C2H2 treatments,

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the Fe(III) reduction rates in the neap tidal sediments were greater during the summer

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(0.21–0.24 g Fe kg-1 d-1) than during the winter (0.19−0.21 g Fe kg-1 d-1) and

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increased by 31.0−52.5% and 18.4−36.3%, respectively, compared with the control

245

treatments (P < 0.05). In the spring tidal sediments, the Fe(III) reduction rates during

246

the summer (0.23–0.29 g Fe kg-1 d-1) were significantly higher than those during the

247

winter (0.20–0.23 g Fe kg-1 d-1).

NH4+ treatments than in the

15

15

15

NH4+ and

15

NH4+ + C2H2

NH4+ + C2H2 treatments (P < 0.05). These results

NH4+ facilitated Fe(III) reduction. The Fe(III) reduction rates

248 249

Abundance of Iron-reducing Bacteria in Intertidal Sediments. The gene copy

250

numbers of iron-reducing bacteria in the sediments under the tidal fluctuations were

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measured to demonstrate their activity, which may regulate Feammox (Figure 4). The

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abundance of Geobacteraceae spp. serves a significant role in iron reduction, varying

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from 1.0 × 108 to 1.4 × 108 copies g-1 during neap tides and increasing significantly 12 / 36


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from 1.9 × 108 to 2.3 × 108 copies g-1 during spring tides (P < 0.05). Significant

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seasonal variations in the abundance of Geobacteraceae spp. occurred during the neap

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tides (P < 0.05), and no significant seasonal difference in Geobacteraceae spp.

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abundance was observed during the spring tides. The Shewanella spp. abundances

258

during neap tides ranged from 1.5 × 107 to 1.9 × 107 copies g-1 and were lower than

259

the abundances (2.2 × 107 –3.7 × 107 copies g-1) observed during spring tides (Figure

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4). Furthermore, significant seasonal differences in the abundances of both

261

Geobacteraceae spp. and Shewanella spp. were observed during spring tides (P
0.05).

263 264

■ DISCUSSION

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Occurrence of Feammox in Intertidal Wetlands. Tidal fluctuations can result in

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periodic changes in sediment characteristics, which can alter the microbial nitrogen

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transformation processes in the intertidal wetlands.33 Nitrification, the sequential

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oxidation of NH4+ to NO2- and then to NO3-, is a dominant microbial process in

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aerobic environments.35 However, under anaerobic conditions, the accumulation of

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NH4+ derived from organic nitrogen mineralization increases,33 and NOx- is reduced

271

by anammox and denitrification.5 In this study, significant differences in the

272

sedimentary NOx- and NH4+ concentrations were detected between the spring and

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neap tidal sediments (P < 0.05) (Figure 1), which indicates the likely occurrence of

274

tide-mediated microbial nitrogen transformations. In addition, Fe oxidation/reduction

275

reactions can occur with tidal fluctuations, resulting in changes in the Fe(III) and/or 13 / 36



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Fe(II) concentrations in intertidal wetlands.36 Fe(III), which serves as an electron

277

acceptor, is widely available in intertidal wetlands and plays an important role in

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anaerobic organic matter oxidation37 and microbial nitrogen cycling.19 In this study,

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the Fe(II) concentrations were significantly higher during the spring tides than during

280

the neap tides. In contrast, Fe(III) concentrations were higher during the neap tides

281

than during the spring tides. The changes in reactive Fe concentrations under these

282

tidal fluctuations indicate that tidal action is an important factor regulating the Fe

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redox reactions in intertidal wetlands. Overall, these concurrent changes in

284

sedimentary IN and reactive Fe concentrations induced by tidal fluctuations may be

285

important for Feammox in intertidal wetlands. Our isotopic experiments further demonstrated that Feammox occurred in the

286 287

intertidal sediments, because significant 30N2 production (Figure 2c, d) and high Fe(III)

288

reduction (Figure 3) occurred following the addition of 15NH4+ in the treatments with

289

15

290

observed between the Fe(III) reduction and 30N2 production rates (P < 0.01) (Figure 5),

291

which supports the occurrence of Feammox in the intertidal sediments. Before the

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isotopic tracer incubations, the sediment slurries were anoxically incubated to

293

eliminate initial NOx- and O2 (Figure S2, Supporting Information). In addition,

294

experimental procedures were conducted in an anaerobic glove box filled with high

295

purity helium. Therefore, N2 directly from Feammox and/or Feammox-produced

296

NO3− or NO2−, which are produced by denitrification or anammox following

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Feammox, are the only potential pathways for 30N2 generation.5,18

NH4+ and

15

NH4+ + C2H2. Furthermore, a significant and positive correlation was

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In this study, the potential Feammox rates estimated from 30N2 production in the

298 299

15

NH4+ treatments5,18 ranged from 0.24 to 0.36 mg N kg-1 d-1 (Figure 2c, d), which are

300

comparable to the Feammox rates observed in tropical forest soils (approximately

301

0.32 mg N kg-1 d-1) by Yang et al.18 and in paddy soils (0.17-0.59 mg N kg-1 d-1) by

302

Ding et al.5 Additionally, the estimated Feammox rates were significantly higher

303

during the spring tides (0.32–0.36 mg N kg−1 d−1) than during the neap tides

304

(0.24–0.27 mg N kg−1 d−1), which implies that tidal actions favor the occurrence of

305

Feammox. Previous studies have revealed that Feammox reactions strongly depends

306

on microbially reducible Fe(III) levels in soils.5,18,19,20 Likewise, a significant

307

correlation was observed between the Feammox rates and Fe(III) concentrations in the

308

present study (r = 0.81, P < 0.01), which indicates that the Feammox dynamics

309

(Figure 2c, d) are likely attributable to the accumulation and/or reduction of Fe-oxides

310

induced by tidal fluctuations. The Feammox process could also be affected by pH and

311

Eh5,18,19 because pH and Eh are important factors regulating the reactivity of Fe oxide

312

minerals and Fe redox reactions.32,36,39 However, no significant relationship was

313

observed between sediment pH and Feammox rates in this study (r = −0.29, P > 0.05),

314

indicating that sediment pH may have a slight influence on Feammox reactions in

315

intertidal wetlands. In contrast, the sediment Eh was significantly correlated with the

316

Feammox rates (r = −0.87, P < 0.01). This relationship indicates that changes in

317

sediment Eh may effectively control the conversion of Fe, which would drive the

318

occurrence of Feammox. In addition, high concentrations of organic matter have been

319

reported to increase the release of structural Fe from clay minerals and result in the 15 / 36



320

formation of Fe(III) oxides, which would promote the Feammox reactions.5

321

Nevertheless, in this study, no significant relationship was observed between

322

Feammox and TOC (r = 0.34, P > 0.05), which suggests that the availability of

323

organic matter is not an important factor responsible for Feammox in intertidal

324

wetlands. Therefore, these results suggest that environments with high Fe(III)-oxide

325

availability and low or fluctuating redox conditions are favorable for the occurrence

326

of the microbial Feammox process.5,18,19,20

327 328

Links Among Fe(III) Reduction Rates, 15N2 Production Rates and Iron-reducing

329

Bacterial Abundance. Fe(III) reduction rates were significantly enhanced in the

330

15

331

treatments (0.16−0.18 g Fe kg-1 d-1) (P < 0.05; Figure 3), which indicates that the

332

addition of NH4+ can facilitate Fe(III) reduction.5 Furthermore, the Fe(III) reduction

333

rates varied significantly between the neap and spring tides, providing further

334

evidence for the importance of tidal fluctuations in regulating the Fe redox reaction in

335

intertidal sediments.34,35 Fe(III) availability and reduction rates can control the

336

microbial Feammox process,5,18 mainly because Fe(III) can serve as an electron

337

acceptor for organic matter degradation and ammonium oxidation under anaerobic

338

conditions19,38 and stimulate the growth of iron-reducing bacteria.39 Therefore, Fe(III)

339

reduction could be associated with 30N2 and 29N2 production in the presence of 15NH4+.

340

In this study, the Fe(III) reduction rates within the 15NH4+-amended incubations were

341

significantly correlated with both 30N2 (r = 0.44, P < 0.01) and 29N2 (r = 0.69, P < 0.01)

NH4+ and 15NH4+ + C2H2 treatments (0.19−0.29 g Fe kg-1 d-1) relative to the control

16 / 36


Page 16 of 36

Page 17 of 36


342

production rates (Figure 5), further indicating that Feammox plays a significant role in

343

anaerobic ammonium oxidation in intertidal wetlands. Considering the production of

344

29

345

of the 15NH4+ added to the neap and spring tidal sediments, respectively.

N2 and 30N2, anaerobic ammonium oxidation consumed 1.21−2.26% and 1.25−2.75%

346

Iron-reducing bacteria (FeRB) can affect Feammox by controlling Fe(III)

347

reduction in anaerobic environments.32,39 The FeRB targeting Geobacteraceae spp.

348

and Shewanella spp. that serve as Fe(III) reducers40,41 may be directly involved in

349

ammonium oxidation.19,20 In this study, the abundances of Geobacteraceae spp. and

350

Shewanella spp. were quantified to reveal the microbially physiological mechanisms

351

underlying the interactions between Fe(III) reduction and Feammox. The Fe(III)

352

reduction rates within the

353

the bacterial abundances of Geobacteraceae (r = 0.52, P < 0.01) and Shewanella (r =

354

0.46, P < 0.01) (Table S1, Supporting Information), as observed for the production of

355

30

356

were significantly correlated with the bacterial abundances of Geobacteraceae (r =

357

0.59, P < 0.01) and Shewanella (r = 0.57, P < 0.01) (Table S1, Supporting

358

Information). These relationships likely provide genetic evidence of the importance of

359

microbially reducible Fe(III) in the occurrence of Feammox. The

360

rates were positively correlated with the

361

Table S1, Supporting Information). This correlation likely indicates the concurrence

362

of many microbial processes, including the use of indigenous

363

15

15

NH4+-amended incubations were significantly related to

N2 and 29N2 vs. reduction of Fe(III) (Figure 5). In addition, the 30N2 production rates

29

30

N2 production

N2 production rates (r = 0.66, P < 0.01,

14

NH4+ with added

NH4+ by Feammox to form N2, Feammox to NO3− and/or NO2− followed by

17 / 36



364

denitrification to N2, and Feammox to NO2− followed by anammox.5,18 This

365

conclusion may also be supported by the weak correlations that were observed

366

between the 29N2 and 30N2 production rates and denitrification and anammox bacterial

367

abundances in this study (Figure S3, Supporting Information).

368

Compared with the neap tides, the high abundances of Geobacteraceae and

369

Shewanella bacteria during the spring tides indicate that the tidal fluctuations

370

significantly influenced the growth of iron-reducing bacteria and impacted Feammox.

371

The abundance of Geobacteraceae bacteria during the neap tides was higher during

372

the summer than during the winter (P < 0.01), with no seasonal differences during the

373

spring tides (P > 0.05). However, the abundance of Shewanella bacteria was higher

374

during the spring tides than during the neap tides, and significant seasonal changes

375

were observed during the spring tides (P < 0.05; Figure 4). These results indicate that

376

FeRB had various physiological characteristics in response to the tidal fluctuations,

377

which further affected the Feammox activity.32,39 However, some other microbial

378

processes, such as anaerobic organic matter and methane oxidation,42,43 are also

379

potential contributors to Fe(III) reduction,5,37,42 which may promote FeRB growth and

380

metabolic activity.

381 382

Contribution of Feammox to Nitrogen Loss. To obtain the N2 produced directly

383

from Feammox, acetylene was added in the isotopic tracer incubations to directly

384

separate the generation of N2 via Feammox and/or denitrification of NO3− and NO2−

385

produced from Feammox.5,18,44 Previous studies have indicated that acetylene not only

386

restrains the reduction of N2O produced from denitrification to N245 but also inhibits 18 / 36


Page 18 of 36

Page 19 of 36


387

anammox.5,44 In this study, the concentration of acetylene in the headspace of the

388

serum vials reached up to 30% (v/v, about 12.78 mM), which could completely

389

suppress N2O reduction45 and anammox.44 Therefore, the N2 produced directly from

390

Feammox was considered the only microbial pathway of

391

treatments of

392

15

393

approximately 6.8−21.5% of the 30N2 produced during the incubations was attributed

394

to anammox and/or denitrification of Feammox-produced NO2− and NO3−.5 Therefore,

395

the N2 directly resulting from Feammox was estimated to account for 78.5 to 93.2%

396

of the total 30N2 production during the incubations in which only 15NH4+ was added.

397

Based on the potential N2O production rates in the presence of acetylene (Figure S4,

398

Supporting Information), approximately 0.02−0.08 mg N kg-1 d-1 was oxidized by

399

Fe(III) to NO3− and/or NO2− and then reduced to N2O through denitrification,5,18 which

400

accounted for 8.3−21.4% of the 30N2 produced within the incubations. This proportion

401

could also support the differences in

402

addition.

15

30

N2 generation in the

NH4+ + C2H2.5,18 Compared to the incubations treated only with

NH4+, the decrease in

30

N2 production due to acetylene addition indicates that

30

N2 generation with and without acetylene

403

To identify the relative contributions of Feammox to nitrogen loss, the potential

404

anammox and denitrification rates under the spring and neap tidal fluctuations were

405

determined in the present study (Supplementary Methods, Supporting Information).

406

The measured anammox and denitrification rates in the intertidal sediments ranged

407

from 0.12 to 0.22 mg N kg-1 d-1 and from 0.41 to 1.68 mg N kg-1 d-1, respectively

408

(Figure S5, Supporting Information). Based on the anammox and denitrification rates, 19 / 36



409

Feammox accounted for 14–34% of the total nitrogen loss (sum of denitrification,

410

anammox and Feammox) through NH4+ oxidation coupled with Fe(III) reduction

411

(Figure S6, Supporting Information).5,18 The remainder of the nitrogen loss was

412

attributed to both denitrification and anammox. Additionally, Feammox resulted in

413

slightly greater contributions to nitrogen loss during spring tides (16–34%) than

414

during neap tides (14-30%), likely because relatively high Feammox rates occurred

415

during the spring tides (Figure 2). Furthermore, although the Feammox rates in the

416

intertidal sediments were slightly different in winter and summer, a significant

417

seasonal difference in the contributions of Feammox to the total nitrogen loss was

418

observed (P < 0.05). The relatively high Feammox contribution to nitrogen loss in

419

winter sediments was likely attributed to the low denitrification rates that occurred

420

during this season. Overall, these results suggested that Feammox plays an important

421

role in NH4+ removal from intertidal wetlands.

422

Based on the potential rates of Feammox measured during the incubation

423

experiments, the potential nitrogen loss via Feammox from the intertidal sediments

424

was estimated at 11.5−18 t N km−2 year−1, assuming that the dry sediment bulk

425

density of the study area was approximately 2.68 g cm−3.3 This amount of nitrogen

426

removal via Feammox represented approximately 3.1−4.9% of the total external IN

427

transported annually into the Yangtze Estuary wetland (approximately 366.67 t N

428

km−2 year−1).21 However, the environmental importance of Feammox in intertidal

429

wetlands remains uncertain because anaerobic organic matter degradation and

430

anaerobic methane oxidation can also contribute to Fe(III) reduction.5,37,42,43 In this 20 / 36


Page 20 of 36

Page 21 of 36


431

study, only 1.58−3.16 % of Fe(III) reduction was attributed to Feammox during the

432

isotopic tracer incubations, based on a theoretical ratio of 3−6 moles of Fe(III)

433

reduced per mole of NH4+ oxidized.5,18

434

In summary, this study provides experimental evidence for the occurrence of

435

Feammox in intertidal wetlands influenced by spring and neap tidal fluctuations. Tidal

436

action can contribute to the growth of iron-reducing bacteria, which would accelerate

437

Feammox activity and lead to more N loss from intertidal wetlands to the atmosphere.

438

A loss of 11.5−18 t N km−2 year−1 was linked to Feammox and accounted for

439

approximately 3.1−4.9% of the total external IN load transported annually into the

440

Yangtze Estuary wetland. Tidal fluctuations are an important driver shaping

441

sedimentary microenvironments and can regulate N loss through Feammox and

442

provide new insights into Fe-driven microbial N transformations in intertidal

443

ecosystems.

444 445

■ ASSOCIATED CONTENT

446

Supporting Information

447

This material is available free of charge via the Internet at http://pubs.acs.org.

448 449

■ AUTHOR INFORMATION

450

Corresponding Author

451

*Phone:

452

[email protected]. (M.L.) E-mail: [email protected].

86-21-62231669;

fax:

86-21-62546441;

21 / 36


(L.J.H.)

e-mail:


453

Notes

454

The authors declare no competing financial interests.

455 456

■ ACKNOWLEDGMENTS

457

This work was together funded by the National Natural Science Foundation of China

458

(Nos. 41322002, 41130525, 41271114 and 41071135), the Program for New Century

459

Excellent Talents in University (NCET), and the State Key Laboratory of Estuarine

460

and Coastal Research. Great thanks are given to Wayne Gardner and anonymous

461

reviewers for their constructive suggestions on earlier versions of this manuscript.

462 463

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596 597 598 599 600 601 602 603 604 605 606 28 / 36


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Figure captions

607 608

Figure 1. Dynamics of water content, Eh, pH, TOC, exchanged inorganic nitrogen,

609

Fe(II) and Fe(III) in sediments collected at the two successive spring and neap tides in

610

summer and winter, respectively. NOx- = NO3- + NO2-. The different small letters

611

above the column denote statistically significant (P < 0.05) differences between the

612

spring and neap tides in summer and winter. Error bars represent standard errors (n =

613

6).

614

Figure 2. Mean

615

C2H2 treatments for the sediments influenced by tidal fluctuations. The different

616

capital and small letters above the column denote statistically significant (P < 0.05)

617

differences between the spring and neap tides in the

618

treatments, respectively. Error bars represent standard errors (n = 6). nd = not

619

detectable within a detection limit of 0.006 mg 15N kg−1 d−1.

620

Figure 3. Fe(III) reduction rates measured through isotope tracer incubations. The

621

different small letters above the column denote statistically significant (P < 0.05)

622

differences between the spring and neap tides. Error bars represent standard errors (n

623

= 6).

624

Figure 4. The gene copy numbers of Geobacteraceae spp. and Shewanella spp.

625

bacteria in the sediments influenced by tidal fluctuations. The different capital and

626

small letters above the column denote statistically significant (P < 0.05) differences

627

between the spring and neap tides in summer and winter, respectively. The asterisks

628

above the horizontal line denote statistically significant (P < 0.05) differences in

30

N2 and

29

N2 production rates in the control,

15

15

NH4+ and

NH4+ and

29 / 36


15

15

NH4+ +

NH4+ + C2H2


629

seasonal variations. Error bars represent standard errors (n = 6).

630

Figure 5. Pearson’s correlations of iron reduction rates with both 30N2 (a) and 29N2 (b)

631

production rates in the 15NH4+ treatment.

30 / 36


Page 30 of 36

Page 31 of 36


632

b b

b

b

20 0

10.0

Sediment pH

a 400 300 200

0

Neap-tide 1 Spring-tide 1 Neap-tide 2 Spring-tide 2 a

a

a

a

a

a

a

a

2.1 1.4

-1

-1

b

b

0.4 0.2 Neap-tide 1 Spring-tide 1 Neap-tide 2 Spring-tide 2 Tidal cycles

Neap-tide 1 Spring-tide 1 Neap-tide 2 Spring-tide 2 a

2.0

b a

1.5

a b

b

0.5

Neap-tide 1 Spring-tide 1 Neap-tide 2 Spring-tide 2 a a

1.0

a

a

0.8 b

b

b

b

0.6 0.4 0.2 0.0

Neap-tide 1 Spring-tide 1 Neap-tide 2 Spring-tide 2 Tidal cycles

Figure 1.

634

b

1.0

1.2

a

b

a

0.2

0.0

Fe(III) concentration (g kg )

-1

Fe(II) concentration (g kg )

b

0.8

0.0

633

a

a

a

a

a

Neap-tide 1 Spring-tide 1 Neap-tide 2 Spring-tide 2

1.0

a

0.4

0.0

-

0.7

a

0.8 0.6

b

a

a

2.5 NOx concentration (mg kg )

Neap-tide 1 Spring-tide 1 Neap-tide 2 Spring-tide 2 a 4.2 a a a 3.5 b b 2.8 b b

+

-1

NH4 concentration (mg kg )

0.0

a

b

b


a

2.5

0.6

b

1.0

5.0

1.2

a

a

100

7.5

0.0

a

500

Sediment TOC content (%)

Sediment water content (%)

a

60 40

600

a a

Sediment Eh (mV)

Summer Winter a

80

635 636 637 638 639 640 31 / 36



Page 32 of 36

641

-1

B b

b

nd

nd

nd

nd

(c)

-1

0.8 0.6 A

0.4 B

A

a

B

b

a

b

0.2 0.0 nd

nd

nd

nd


0.4

+

A

a

b

a

b

0.2 nd

nd

nd

nd


Winter

(d)

0.8 0.6 0.4

A B

0.2 0.0

nd

a

B

b nd

nd

A

a

b nd

Neap-tide 1 Spring-tide 1 Neap-tide 2 Spring-tide 2 Tidal cycles

Figure 2.

643

NH4 + C2H2 B

Tidal cycles

642

15

A

1.0 Summer

NH4

B

0.0


+

15

0.6

-1

0.0

(b)

Control

0.8

29

0.2

N2 production rate (mg N kg d )

-1

a

B

0.4

-1


A

a

0.6

1.0

30

A

30

-1 29

0.8

Winter

-1

(a)

N2 productin rate (mg N kg d )

1.0

Summer

-1


1.0

644 645 646 647 648 649 650 651 652 653 654 32 / 36


Page 33 of 36


655 0.40 Summer 0.32 0.24

a

b

b a

b

b b a

b

0.16

-1

-1

Fe(III) reduction rate (g Fe kg d )

a

a b

0.08 0.00 Neap-tide 1

Spring-tide 1

Neap-tide 2

0.40

Spring-tide 2 Control

Winter

15

+

NH4

0.32

15

0.24 0.16

b b

b

a b

a

b

+

NH4 + C2H2 a a

a b

a

0.08 0.00 Neap-tide 1

656

Spring-tide1 Neap-tide 2 Tidal cycles

Spring-tide 2

Figure 3.

657 658 659 660 661 662 663 664 665 666 667 668 669 670 33 / 36



Page 34 of 36

671

2.4

4.2

Summer Winter A

a

b

-1

2.0

Bacterial abundance (10 copies g dry sediment)

Geobacteraceae spp. A

-1

Bacterial abundance (10 copies g dry sediment)

2.8

* 1.6

c

* B

B

0.8 0.4

0.0

672

3.6

*

*

a

3.0 B b

2.4

B

1.8

B

c c

1.2 0.6 0.0



Tidal cycles

Tidal cycles

Figure 4.

673

A

7

8

c

1.2

Shewanella spp.

34 / 36


Page 35 of 36


674 0.40 0.9

(b)

0.8

-1

0.32 0.30 0.28 0.26 0.24

r = 0.69 P < 0.0001

-1

0.34

(a)


0.36

r = 0.44 P < 0.005

29

30

-1

-1


0.38

0.7 0.6 0.5 0.4 0.3

0.22 0.2 0.18 0.21 0.24 0.27 0.30 0.33 -1 -1 Fe(III) reduction rate (g Fe kg d )

675

0.18 0.21 0.24 0.27 0.30 0.33 -1 -1 Fe(III) reduction rate (g Fe kg d )

Figure 5.

676 677

35 / 36



678 679

TOC Art

680

681

36 / 36


Page 36 of 36

Evidence of Nitrogen Loss from Anaerobic ... - ACS Publications

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