nitrogen loss through anaerobic ammonium oxidation coupled...

European Journal of Soil Science, July 2018, 69, 732–741 doi: 10.1111/ejss.12552

Nitrogen loss through anaerobic ammonium oxidationcoupled with iron reduction in a mangrove wetland

Q . S . G u a n , W . Z . C a o , M . W a n g, G . J . W u, F . F . W a n g, C . J i a n g, Y . R . T a o & Y . G a oState Key Laboratory of Marine Environmental Science, Key Laboratory of the Ministry of Education for Coastal Wetland Ecosystems,College of the Environment and Ecology, Xiamen University, Xiang’an South Road, Xiamen 361102, China

Summary

Anaerobic ammonium oxidation coupled with iron(III) reduction (Feammox) with dinitrogen, nitrite or nitrateas end-products is the most recently discovered nitrogen cycling process. This reaction has been observedin tropical forest soils, paddy soils and intertidal wetlands. However, Feammox has not been measured inmangrove wetlands. In this study, sediment slurry incubation experiments were combined with isotope tracingand acetylene inhibition techniques. Feammox was detected in mangrove sediments and ‘bare flats’ (mud flatswithout mangrove), with potential rates of 0.48 (±0.03 SE) mg N kg−1 day−1 (accounting for 6.4% of the totalnitrogen loss through N2) and 0.38 (±0.02 SE) mg N kg−1 day−1 (accounting for 6.7% of the total nitrogen lossthrough N2), respectively. Microbially reducible iron(III) was added, which significantly (P < 0.01) increased theFeammox rate in contrast to no addition of iron(III). It was estimated that a loss of 12.33 t N year−1 was associatedwith Feammox in mangrove sediments of the Jiulong River Estuary, accounting for 0.04% of the total externalinorganic nitrogen transported into the estuary. Overall, these findings demonstrate that Feammox can act as anitrogen loss mechanism in mangroves.

Highlights

• Feammox was investigated by ammonium 15N labelled isotopic tracing technique.• Feammox rates in mangrove were larger than those in mud flats without mangrove.• The nitrogen loss contribution of Feammox in mangrove was less than in other ecosystems.• Tidal fluctuations and the large TOC would accelerate the Feammox process.

Introduction

Two important microbial processes, denitrification and anaerobicammonium oxidation (anammox), are considered to be involvedin nitrogen (N) loss from terrestrial and marine ecosystems (Dals-gaard et al., 2003; Galloway et al., 2008; Fernandes et al., 2012;Shen et al., 2016) through the generation of nitrous oxide or dini-trogen gas (Galloway et al., 2008; Seitzinger, 2008; Canfield et al.,2010). Recently, another microbial process, anaerobic ammoniumoxidation coupled with the reduction of ferric iron (Fe(III)), knownas Feammox (Sawayama, 2006), which can produce dinitrogen gas(N2) (Equation (1)) (Yang et al., 2012; Ding et al., 2014; Li et al.,2015), NO2

− (Equation (2)) (Clement et al., 2005; Sawayama,2006) or NO3

− (Equation (3)) (Yang et al., 2012; Li et al., 2015),has been identified in many ecosystems, including tropical forest

Correspondence: W. Z. Cao. E-mail: [email protected] 12 May 2017; revised version accepted 12 January 2018

soils (Yang et al., 2012), paddy soils (Ding et al., 2014) and inter-tidal wetlands of the Yangtze Estuary (Li et al., 2015):

3Fe (OH)3 + 5H+ + NH+4 → 3Fe+2 + 9H2O + 0.5N2. (1)

6Fe (OH)3 + 10H+ + NH+4 → 6Fe+2 + 16H2O + NO−

2 . (2)

8Fe (OH)3 + 14H+ + NH+4 → 8Fe+2 + 21H2O + NO−

3 . (3)

Mangroves are highly productive ecosystems growing at theinterface between land and sea along much of the tropical andsubtropical coastlines and estuaries (Valiela et al., 2001; Hatha &Chacko, 2010); they are typically saline, anoxic and are frequentlywaterlogged (Reef et al., 2010). Reducing conditions are strong inmangrove sediments (Ovalle et al., 1990; Marchand et al., 2004)

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Feammox in a mangrove wetland 733

because they are anoxic and waterlogged, which is conduciveto the reduction of Fe(III). Mangrove ecosystems are rich inorganic matter because of the large amount of carbon allocatedto roots and the small rates of decomposition imposed by anoxicconditions (Nedwell, 1994). Denitrifying bacteria are abundant inmangrove sediments (Alongi, 1994; Fernandes et al., 2012), andrates of denitrification can be large, which makes ammonium themost common form of nitrogen observed in mangrove sediments.Dissimilatory nitrate reduction to ammonium (DNRA) also playsan important role in converting nitrate to ammonium, which resultsin its accumulation (Cao et al., 2016). Moreover, iron content inmangrove sediments is appreciable (Chandan, 2007; Krishnan &Bharathi, 2009). Mangrove sediments switch between oxic andanoxic conditions because they are intertidal, which affects nitrogenavailability and the redox state of iron. The controlling factorsof Feammox in mangrove sediments have an advantage over theother ecosystems because of abundant ammonium and the activereduction of Fe(III). However, no reports on the occurrence ofFeammox in mangrove forests have been provided before, and wehypothesized that mangrove sediments probably support Feammox.

The primary objective of this study was to examine the occurrenceof Feammox in mangrove forests in southeast China. The relativecontribution of Feammox to nitrogen loss through N2 was alsoidentified by combining 15N-labelled NH4

+-based isotopic tracingand acetylene (C2H2) inhibition techniques.

Materials and methods

Site description and sediment sampling

The mangrove forest nature reserve of the Jiulong River Estuary isin Fujian Province, southeast China. Agricultural nitrogen from theJiulong River catchment is the largest source of nitrogen (Cao et al.,2005). The climate is subtropical oceanic monsoon, the averageannual temperature is 21∘C, the annual rainfall is 1371.3 mmand the annual average relative humidity is 86%. This area isa typical estuarine mangrove forest with a semidiurnal tide andmean tidal range of 4 m. In 1 month, the forest is inundated byhigh tides for only 6–8 days (Jin et al., 2013). The research site(121∘59′E, 31∘30′N) is in Fugong along the southern coastlineof the Jiulong River Estuary (Figure 1), and the main speciesis Kandelia candel (L.) Druce. Tides are semi-diurnal and theduration of the inundation is 1.4 hours day−1 on the mangrovesediments and 9 hours day−1 on the ‘bare flats’ (i.e. mudflatswithout mangrove) (Cao et al., 2017). At neap tides bare flats andmangrove sediments are exposed. These two types of sedimentswere selected for sampling in November 2016. Three replicates ofsediment samples (0–5-cm depth) were collected from an area of3 m2 of homogeneous sediments in both mangrove sediments andbare flats using Plexiglas corers. The samples were then transferredto a portable refrigerator and transported to the laboratory within2 hours. After returning to the laboratory, each sediment core wasimmediately divided into two fractions. The first fraction wasincubated immediately to determine the Feammox rates using

isotope tracer incubations, whereas the second fraction was storedat 4∘C for sediment characteristic measurements.

Isotope tracer incubations

Isotope experiments were carried out in an anaerobic gloveboxfilled with helium (He) to measure Feammox and rates of Fe(III)reduction (Yang et al., 2012; Ding et al., 2014; Li et al., 2015).Briefly, anoxic artificial seawater was prepared through boilingultrapure water before adding sea salt, and then purged withvery pure He for 1 hour. The salinity of the artificial seawaterwas 15‰, the same as the estuary water. Then, sediment slurrieswere prepared by adding anoxic artificial seawater to the freshsediments at a ratio of 1:1 (500 ml water and 500 g sediment) andpreincubated anaerobically in the glove box for 12 hours to removeindigenous nitrite, nitrate and oxygen. After the preincubation,4 ml of the slurries were transferred to 12.6-ml Labco Exetainervials (Labco Limited, Lampeter, UK). Five treatments with threereplicates per treatment were established: (i) control (sterile anoxicdeionized water instead of 15NH4Cl), (ii) addition of 15NH4Cl (15Nat 98%, Sigma–Aldrich, St. Louis, MO, USA), (iii) addition of15NH4Cl and C2H2 (15NH4 +C2H2), (iv) addition of 15NH4Cl andFe(III) (15NH4 + Fe(III)) and (v) addition of 15NH4Cl and Fe(III)and C2H2 (15NH4 + Fe(III)+C2H2). The final concentration of15NH4Cl-N was 28 mg kg−1 (dry weight) achieved by injecting100 μl of ultrapure He-purged stock solution, which was basedon a realistic concentration of ammonium in the sediments. Theeffects of Fe(III) concentration on Feammox were identified bytreatments with the addition of Fe(III). For both the 15NH4 +Fe(III)and 15NH4 + Fe(III)+C2H2 treatments, we added sufficient Fe(III)(ferric chloride) to activate Feammox to form NO2

− with a sto-ichiometric molar ratio of 6:1 Fe(III) to 15NH4

+. For the C2H2

treatments, the headspace gas in each vial was removed andreplaced by C2H2 to reach 30% (2.6 ml) in the headspace. Weused C2H2 to distinguish between the direct production of N2 byFeammox and denitrification of the NO3

− and NO2− produced by

Feammox (Yang et al., 2012), or to determine whether both wereproduced. Acetylene (C2H2) has been reported to not only blockthe reduction of N2O to N2 in denitrification (Qin et al., 2012),but also to block anammox (Jensen et al., 2007). Therefore, theonly 30N2 produced was considered to be N2 produced directly byFeammox in the treatments of 15NH4

+ and C2H2.All vials were shaken vigorously to homogenize the treatment

solutions at an interval of 3–4 hours. After 24 hours of incuba-tion, 1-ml samples of gas were collected immediately using gastightsyringes and then injected into 12-ml pre-evacuated Labco Exe-tainer vials sealed with butyl rubber septa (Labco Limited, HighWycombe, UK). Before sampling, each vial was shaken vigorouslyto equilibrate the gas between the dissolved and gaseous phases.The 15N enrichment of N2 in the gas samples was determined witha GasBench II-Delta V Advantage isotope ratio mass spectrometer(IRMS, Thermo Fisher, Bremen, Germany), and the 15N enrichmentin N2O was determined by IRMS coupled with a pre-concentrationunit. The rates of production of 29N2 and 30N2 and 45N2O and 46N2O

© 2018 British Society of Soil Science, European Journal of Soil Science, 69, 732–741

734 Q. S. Guan et al.

Figure 1 Sampling sites in mangroves of the Jiulong River Estuary.

were calculated from the change in 29N2 and 30N2, and 45N2O and46N2O concentrations in the vial headspace after 24 hours.

Potential rates of Feammox were estimated conservatively fromthe differences in 30N2 production in the vial headspace with andwithout the addition of 15NH4

+ as follows:

F =((

C1 − C′1

)− C0

)× V

t × m,

where F is rate of Feammox (mg N kg−1 day−1), C1 is 30N2-Nconcentration after 1 day of incubation with the addition of 15NH4

(mg l−1), C1′ is 30N2-N concentration after 1 day incubation without

addition of 15NH4 (mg l−1), C0 is 30N2-N concentration at thebeginning of incubation with the addition of 15NH4 (mg l−1), V isthe volume of headspace, t is total incubation time (1 day) and m isthe dry weight of sediment (kg).

After gas sampling, the slurries were subsampled to analyseHCl-extractable Fe(II) (Lovley & Phillips, 1987). Briefly, 1 g ofsoil slurry sample was extracted with 10 ml 0.5 m HCl for 2 hoursat room temperature, and the extracted Fe(II) was determined bythe ferrozine method. Rates of reduction of microbially reducibleFe(III) (considered as hydroxylamine-reducible Fe(III)) were

calculated from the change in Fe(II) concentrations during theincubations.

Measurement of the denitrification and anammox potentialrates

To identify the relative contributions of Feammox to nitrogen lossby N2, potential denitrification and anammox were determined bythe 15N isotope pairing technique. The entire incubation experimentwas performed in an ultra-high-purity helium (He)-filled anaerobicglovebox. Briefly, slurries were prepared by adding sterile anoxicartificial seawater (salinity, 15‰) to the sediments at a ratio of 1:1(500 ml water and 500 g sediment). Then, 4 ml of mixed slurrieswere transferred into He-flushed 12.6-ml glass vials sealed withbutyl rubber septa. The vials were preincubated in the dark for12 hours to eliminate initial nitrate, nitrite and oxygen at the in situtemperature. After preincubation, three treatments were carried out:(i) 15NH4

+, (ii) 15NH4+ + 14NO3

− and (iii) 15NO3− (15N at 98%,

Sigma–Aldrich). The final concentration of 15N in each vial was100 μm. Incubation of the slurries was stopped by injecting 200 μlof saturated HgCl solution at intervals of 0, 4, 8 and 16 hours.The potential rates of anammox and denitrification, and their



respective contributions to total N2 production, were calculatedfrom the production of 29N2 and 30N2, as described by Thamdrup& Dalsgaard (2002).

Analysis of sediment characteristics

The sediment water contents were determined from the weight lostfrom a known amount of wet sediments that had been dried at 60∘Cto a constant value. The pH of sediment was measured with deion-ized water at a ratio of 1:2.5 (10 g:25 ml). The total organic carbon(TOC) in the sediments was determined with an elemental analyser(vario EL cube, Elementar, Hanau, Germany) after leaching with1 m HCl to remove carbonate. The KCl-extractable NH4

+, NO3−

and NO2− in the sediments were extracted with a 2 m KCl solution

and determined with a continuous-flow nutrient autoanalyser (AA3AutoAnalyzer, Bran+Luebbe GmbH, Norderstedt, Germany). Thetotal extractable Fe and Fe(II) in the sediments were determined bythe ferrozine method (Lovley & Phillips, 1987). Briefly, 2-g sam-ples were extracted from 50-ml mixtures of 0.5 m HCl for 2 hoursto determine the total extractable Fe(II) and total extractable Fe bythe same procedure, except that the extractant was 0.25 m hydrox-ylamine hydrochloride in 0.25 m HCl. The amount of microbiallyreducible Fe(III) (considered as hydroxylamine-reducible Fe(III))was calculated as the difference between the total extractable Feand Fe(II).

Statistical analyses

We carried out t-tests to compare the chemical characteristics ofthe sediments between mangrove sediments and bare flats, and therates of Fe(III) reduction between treatments. We also performedan analysis of variance (anova) to compare the rates of 30N2

and 29N2. We checked normality by the Shapiro–Wilk test andhomoscedasticity of residuals by the Levene test; none of thevariables required transformation. We also computed Pearson’scorrelation coefficients to reveal the relations between the rates ofreduction of Fe(III) and 29N2 and 30N2. Standard statistical testswere performed with SPSS 20.0 statistical software (SPSS Inc.,Chicago, IL, USA).

Results

Chemical characteristics of the sediments

The water contents of the bare flat areas were larger than thosein the mangrove sediments. The bulk densities of the bare flatareas were slightly smaller than those in the mangrove sediments.The sediment pH values were 7.70 (bare flats) and 7.49 (man-grove sediments). The TOC of mangrove sediments (2.16%) wassignificantly (P< 0.01) larger than that in the bare flats (1.37%).Mangrove trees, as they grow older, produce a dense mat of deadroots and boles in sediments, resulting in larger TOC content inmangrove sediments than in the bare flats. The NH4

+ concentrationswere significantly larger (P< 0.0.1) than the NOx

− concentrationsin both the bare flats and mangrove sediments. The Fe(II) contents

(HCl extractable) accounted for 94% of total Fe in the bare flatsand for 86% in the mangrove sediments. The concentrations ofFe(III) (microbially reducible Fe(III)) in the mangrove sediments(1.04 (±0.10 SE) g Fe kg−1), which is the pivotal constituent ofFeammox, were significantly (P< 0.0.1) larger than those in thebare flats (0.57 (± 0.02 SE) g Fe kg−1).

Production of 29N2 and 30N2, and 45N2O and 46N2O fromanoxic incubations

The potential rates of 29N2 and 30N2 production in bare flatsand mangrove sediments were determined by 15N-labelledammonium-based isotope tracing and acetylene inhibition tech-niques (Figure 2). Significant production of 30N2 was detected intreatments with the addition of 15NH4

+, but no 30N2 was detected inthe control (without 15NH4

+). This result demonstrated the occur-rence of Feammox in mangrove sediments and bare flats because N2

is produced directly from Feammox or Feammox-produced NO2−

or NO3− followed by anammox or denitrification; these are the only

potential sources of 30N2 under anoxic conditions (Table 1) (Yanget al., 2012). To analyse the effect of Fe(III) content on Feammox,we had treatments with the addition of Fe(III). The results show(Figure 2a,b; Table 2) that the production of 30N2 and 29N2 in thetreatments with added Fe(III) was significantly larger (P< 0.01)than that in the treatments without added Fe(III) (Table 2). The ratesof 30N2 and 29N2 production in the mangrove sediments were largerthan those in the bare flats (Figure 2a,b; Table 2). Potential rates ofFeammox are conservative estimates calculated from the produc-tion of 30N2 alone (Yang et al., 2012). For the 15NH4

+ treatments,the rates of 30N2 production (Feammox rates) in the bare flats(0.38 (±0.02 SE) mg N kg−1 day−1) were significantly (P< 0.05)smaller than those in the mangrove sediments (0.48 (±0.03 SE)mg N kg−1 day−1) (Figure 2a). Furthermore, the presence of C2H2

(15NH4+ + C2H2) caused a decrease, of 0.07 mg N kg−1 day−1

in the bare flats and 0.12 mg N kg−1 day−1 in the mangrove sedi-ments, in the rates of production of 30N2 (Table 2). Dinitrogen gasproduced directly from Feammox accounted for 82% of the 30N2

loss with the 15NH4+ treatment in the bare flats and 75% in the

mangrove sediments. During the isotope-tracing incubations, therates of 29N2 production showed similar patterns of variation tothose of the 30N2 rates of production. The production of 29N2 in allthe treatments was greater than 30N2 (Figure 2a,b), mainly becauseof the utilization of background 14NH4

+ in combination with added15NH4

+ in Feammox to produce 29N2, consumption of consequentNO2

− after Feammox by anammox to 29N2 and consumption ofconsequent NO2

− or NO3− after Feammox by denitrification to

29N2 (Table 1).The 46N2O and 45N2O that accumulated in the headspace in

the presence of C2H2, and the rates of production of 46N2Owere larger than 45N2O (Figure 2c,d). We estimated the potentialrates of 15NO2

− or 15NO3− based on 46N2O production in the

presence of C2H2 (no 46N2O accumulated without C2H2). One moleof 15NH4

+ is needed to generate 1 mole of 15NO2− or 15NO3

−

according to the stoichiometry of Feammox to the NO2− or NO3

−



Figure 2 Rates of production of 29N2 and 30N2 and 45N2O and 46N2O for the bare flats and mangrove sediments: (a) rate of 30N2 production, (b) rate of 29N2

production, (c) rate of 46N2O production and (d) rate of 45N2O production. nd, not detectable. Values are the means (n= 3).

Table 1 Chemical characteristics of sediments

Site pH

Water

contents/%

Bulk

density/g cm−3

Microbially

reducible

Fe(III)/g kg−1

HCl-extractable

Fe(II)/g kg−1

Total

Fe/g kg−1 TOC/% NH4+-N NO3-N/mg kg −1 NO2-N

Bare flats 7.70 ± 0.01 57.79 ± 0.83 0.91 ± 0.01 0.57 ± 0.02 8.91 ± 0.07 9.48 ± 0.05 1.37 ± 0.21 27.02 ± 1.37 0.56 ± 0.16 0.028 ± 0.008

Mangrove

sediments

7.49 ± 0.01 50.01 ± 0.79 0.95 ± 0.01 1.04 ± 0.10 6.62 ± 0.13 7.66 ± 0.07 2.16 ± 0.13 21.56 ± 0.89 0.84 ± 0.32 0.014 ± 0.008

Values are mean±SE (n= 3).

pathway (Equation (2) or (3)). Then, it is assumed that 2 molesof 15NO2

− or 15NO3− are needed to generate 1 mole of 46N2O

through denitrification. Based on the total rates of production of46N2O (Figure 2c), approximately 0.05 mg N kg−1 day−1 in the bareflats and 0.11 mg N kg−1 day−1 in the mangrove sediments wereoxidized by Fe(III) to NO3

− or NO2− or both, and then reduced

to N2O through denitrification, which accounted for 13% (bareflats) and 23% (mangrove sediments) of the 30N2 produced. Thisproportion supported the results calculated from the difference in30N2 production with and without C2H2.

Moreover, the decrease in the rates of production of 30N2 were0.07 mg N kg−1 day−1 in the bare flats and 0.12 mg N kg−1 day−1

in mangrove sediments in the 15NH4+ + C2H2 treatment compared

with the 15NH4+ treatment, and overall this was larger than the rates

of production of 46N2O. This could be due to the partial dissolutionof 46N2O dissolved into the liquid phase.

Rates of anammox and denitrification

The rates of anammox and denitrification were calculated fromthe production of 29N2 and 30N2 (Figure 3). Rates of anammoxand denitrification were 1.44 mg N kg−1 day−1 in the bare flatsand 1.65 mg N kg−1 day−1 in mangrove sediments, and 3.5 mg Nkg−1 day−1 in the bare flats and 3.16 mg N kg−1 day−1 in mangrovesediments, respectively (Figure 4).

Rates of reduction of Fe(III) in isotopic tracer incubations

In this study, the rates of Fe(III) reduction in mangrove sedi-ments were larger than those in the bare flats (Figure 5). In boththe bare flats and mangrove sediments, the rates of Fe(III) reduc-tion were significantly enhanced (P< 0.01) in the 15NH4

+ and15NH4

+ +C2H2 treatments compared with the controls. Moreover,the rates of Fe(III) reduction were significantly enhanced (P< 0.01)



Table 2 Analysis of variance for the rates of production of 29N2 and 30N2, 46N2O and 45N2O

Source of variation Degrees of freedom Sum of squares Mean square F P

30N2 Sediment 1 0.053 0.053 47.25 < 0.001Fe(III) 1 0.112 0.112 99.64 < 0.001C2H2 1 0.066 0.066 58.40 < 0.001Sediment • Fe(III) 1 0.001 0.001 1.203 0.289Sediment • C2H2 1 0.002 0.002 1.999 0.177Fe(III) • C2H2 1 0.001 0.001 0.975 0.338Sediment • Fe(III) • C2H2 1 1.742 × 10−5 1.742 × 10−5 0.015 0.905Error 16 0.018 0.001Total 23 0.253

29N2 Sediment 1 0.048 0.048 54.38 < 0.001Fe(III) 1 0.093 0.093 104.2 < 0.001C2H2 1 0.037 0.037 42.04 < 0.001Sediment • Fe(III) 1 0.002 0.002 2.459 0.136Sediment • C2H2 1 1.500 × 10−4 1.500 × 10−4 0.174 0.682Fe(III) • C2H2 1 0.001 0.001 0.733 0.405Sediment • Fe(III) • C2H2 1 1.800 × 10−4 1.800 × 10−4 0.210 0.653Error 16 0.140 0.009Total 23 0.321

46N2O Sediment 1 0.013 0.013 52.37 < 0.001Fe(III) 1 0.002 0.002 7.528 0.025Sediment • Fe(III) 1 8.008 × 10−5 8.008 × 10−5 0.326 0.584Error 8 0.002 2.457 × 10−4

Total 11 0.01729N2 Sediment 1 0.048 0.048 38.94 < 0.001

Fe(III) 1 0.004 0.004 3.120 0.115Sediment • Fe(III) 1 1.633 × 10−5 1.633 × 10−5 0.013 0.911Error 8 0.010 0.001Total 11 0.061

in the 15NH4+ + Fe(III) and 15NH4

+ + Fe(III)+C2H2 treatmentsrelative to the other treatments (Figure 5). These results indicatethat a portion of the sedimentary indigenous Fe(III) was reducedto Fe(II) and that the addition of 15NH4

+ or Fe(III) facilitated thereduction of Fe(III). In the 15NH4

+ treatment, the rates of Fe(III)reduction in bare flats and mangrove sediments were 0.32 (± 0.08SE) and 0.39 (± 0.08 SE) g Fe kg−1 day−1, respectively. In the15NH4

+ + Fe(III) treatment, the rates of Fe(III) reduction were sig-nificantly enhanced by 18% in mangrove sediments and 25% inbare flats (P< 0.01) compared with the 15NH4

+ treatment. However,the comparisons between 15NH4

+ and 15NH4++C2H2 treatments,

and 15NH4++Fe(III) and 15NH4

+ + Fe(III)+C2H2 treatments, indi-cated that the rates of Fe(III) reduction did not decrease significantly(P> 0.05).

Discussion

Occurrence of Feammox in mangrove systems

The isotopic experiments demonstrated that Feammox occursin mangrove forests. All sediment slurries were preincubatedanoxically to remove indigenous O2 and NOx

−. Strict anaerobic

procedures were maintained throughout the experiment, and aero-bic nitrification was negligible in this study. Therefore, N2 produceddirectly from Feammox or Feammox-generated NO2

− or NO3−

followed by anammox or denitrification are the only potential path-ways for the production of 30N2 (Yang et al., 2012; Li et al., 2015).The potential rates of Feammox in our study were comparable tothose observed in tropical forest soils (approximately 0.32 mg Nkg−1 day−1) by Yang et al. (2012), in intertidal wetlands of theYangtze Estuary (0.24–0.36 mg N kg−1 day−1) by Li et al. (2015)and in paddy soils (0.17–0.59 mg N kg−1 day−1) by Ding et al.

(2014). Mangroves are highly productive, with mean estimates ofnet primary productivity ranging from 2 to 50 Mg C ha−1 year−1

(Alongi, 2009), rivalling some of the most productive old-growthtropical forests (Clark et al., 2001). Large concentrations of organicmatter have been reported to increase the release of structural Fefrom clay minerals and result in the formation of Fe(III) oxides,which would promote Feammox reactions (Ding et al., 2014). TheTOC and rates of Feammox in the mangrove sediments werelarger than those in the bare flats, which is consistent with thisinterpretation. Nevertheless, in the Yangtze Estuary study (Li et al.,2015), no significant relation was observed between Feammoxand TOC.



Figure 3 Production of 29N2 and 30N2 in incubations for mangrove sediments and bare flats: (a) incubations with 15NH4+, (b) incubations with 15NH4

+ and15NO3

− and (c) incubations with 15NO3−.

Figure 4 Rates of potential anammox and denitrification in bare flats andmangrove sediments. Error bars show the standard errors.

The regular tidal fluctuations change the physical and chemicalproperties in the sediments and, in particular, affect the oxygenconcentration of sediments, which can alter microbial nitrogentransformation in intertidal wetlands (Reef et al., 2010; Santoset al., 2012). The flood time in the bare flats is longer than that in the

Figure 5 The rates of Fe(III) reduction measured through isotope tracerincubations. Values are the means (n= 3).

mangrove sediments, and the Fe(II) concentrations in the bare flatswere larger than those in the mangrove sediments. These differencescould be explained by the effect of tidal fluctuations on Fe oxidationand reduction reactions. In this study, the NH4

+ concentrationswere larger than NOX

− concentrations, which provided abundant



Table 3 Possible processes for 29N2 and 30N2 and 45N2O and 46N2O generation from 15NH4+ under anaerobic conditions

Product Nitrogen substrate 1 Nitrogen substrate 2 Process

30N2 Added 15NH4+ Added 15NH4

+ Feammox to N2

Added 15NH4+ Feammox-generated 15NO2

− and 15NO3− Anammox

Feammox-generated 15NO2− and 15NO3

− Feammox-generated 15NO2− and 15NO3

− Denitrification29N2 Added 15NH4

+ Background 14NH4+ Feammox to N2

Added 15NH4+ Feammox-generated 14NO2

− and 14NO3− Anammox


− Background 14NH4+ Anammox



− Denitrification46N2O Feammox-generated 15NO2

− and 15NO3− Feammox-generated 15NO2

− and 15NO3− Denitrification (C2H2)

45N2O Feammox-generated 15NO2− and 15NO3


− Denitrification (C2H2)

Taken from Ding et al. (2014) Environmental Science and Technology 48, 10641–10647.

Figure 6 Relations between the rates of Fe(III) reduction and production of 30N2 and 29N2: (a) relation between the rates of Fe(III) reduction and productionof 30N2 and (b) relation between the rates of Fe(III) reduction and production of 29N2. The correlations, r, between the reduction of Fe(III) and production of30N2 and 29N2 are given in the figure with the associated P-values.

substrate for Feammox (Table 3). Our previous studies have shown

that dissimilatory nitrate reduction to ammonium (DNRA) played

an important role in converting nitrate to ammonium, resulting in

the accumulation of NH4+ (Cao et al., 2016). Moreover, large rates

of denitrification deplete the nitrate and nitrite pools (Alongi, 1994),

and large rates of ammonification and N fixation also contribute

to the production of ammonium (Alongi et al., 2002). The Fe(II)

concentrations were significantly larger (P< 0.01) than those of

Fe(III) in the bare flats and mangrove sediments (Table 3). In

the mangrove ecosystem, there is a negative correlation between

pH and Eh (Marchand et al., 2004). In this study, the pH was

7.7 for bare flats and 7.5 for mangrove sediments, indicating

small values of Eh and the reduction of Fe(III) in mangrove

sediments and bare flats. Therefore, the higher pH and consequently

small Eh of mangrove sediments and bare flats could favour the

Feammox process.

Iron reduction associated with Feammox

The reduction of Fe(III) could be associated with the production of30N2 and 29N2 in the presence of 15NH4

+. In this study, the ratesof Fe(III) reduction in the 15NH4

+ treatments (without acetylene)were strongly correlated with the rates of production of both 30N2

(r = 0.932, P< 0.01) and 29N2 (r = 0.884, P< 0.01) (Figure 6), indi-cating further that Feammox played an important role in anaerobicammonium oxidation in mangrove wetlands. Moreover, we deter-mined the proportion of Fe(III) reduction attributed to Feammox inthe 15NH4

+ treatment in bare flats and mangrove sediments usingthe theoretical ratio of 3–6 mol Fe(III) reduced per mole of NH4

+

oxidized according to thermodynamic calculations (Ding et al.,2014). In the bare flats 1.43–2.86% and in the mangrove sediments1.48–2.96% of Fe(III) reductions were associated with Feammox.Ding et al. (2014) explained that the largest proportion of Fe(III)reduction associated with Feammox had the smallest TOC, whereas



Table 4 The rates of Feammox and ecosystem nitrogen loss in different types of soil

Types Feammox rates/mg N kg−1 day−1 Ecosystem nitrogen loss through Feammox/t N km−2 year−1

This study (mangrove) Bare flat: 0.38 (±0.02 SE)Mangrove: 0.48 (±0.03 SE)

Mangrove sediment: 6.26 (0–5-cm depth) or 12.33 t N year−1

Tropical forest soils (Yang et al., 2012) 0.32 0.1–0.4 (0–10-cm depth)Paddy soils (Ding et al., 2014) 0.17–0.59 0.78–6.1 (0–10-cm depth)Yangtze Estuary (Li et al., 2015) 0.24–0.36 11.5–18 (0–5-cm depth)

the smallest proportion had the largest TOC. In the present study,there was no significant relation between Fe(III) reduction associ-ated with Feammox and TOC.

Remarkably, there was no significant decrease in Fe(III) reductionin the 15NH4

+ + C2H2 treatment compared with the 15NH4+ treat-

ment or in the 15NH4+ + Fe(III)+C2H2 treatment compared with

the 15NH4+ + Fe(III) treatment (Figure 5), indicating that the addi-

tion of acetylene did not decrease Fe(III) reduction significantly aspreviously reported (Ding et al., 2014). We consider that the addi-tion of acetylene would block the reduction of N2O to N2 in deni-trification (Qin et al., 2012). However, acetylene could not inhibitprocesses described by Equations (1) or (2) and Equation (3), andproduction of NO2

− and NO3− in Equations (2) and (3) provided

nitrogen for denitrification only. Therefore, acetylene did not inhibitthe Feammox process and thus did not block the reduction of Fe(III)associated with Feammox. This result might be explained by thefact that the rate of Fe(III) reduction did not decrease significantlyafter the additions of acetylene.

Contribution of Feammox to nitrogen loss

The total nitrogen loss (the sum of denitrification, anammox andFeammox) by Feammox accounted for 6.7% in the bare flatsand 6.4% in the mangrove sediments, and the difference in thisproportion was not significant. In the intertidal wetland of theYangtze Estuary (Li et al., 2015), the contribution of Feammox tototal nitrogen loss through N2 was 14–34%, which was larger thanthat in this study. This could be because the rates of denitrificationand anammox in mangrove sediments were larger than those in theYangtze Estuary, where the rates of anammox and denitrificationranged from 0.12 to 0.22 mg N kg−1 day−1 and from 0.41 to 1.68 mgN kg−1 day−1, respectively.

We also estimated the potential nitrogen loss by N2 through Feam-mox in the mangroves in the Jiulong River Estuary based on theN2 produced directly from Feammox in the laboratory incubationand bulk density (Table 3). Yan et al. (2012a) estimated the poten-tial nitrogen loss by Feammox at 12.33 t N year−1 from 1.97 km2

of mangrove forest (without bare flats) in mangrove sediments(0–5 cm depth) of the Jiulong River, accounting for approximately0.04% of the total external inorganic nitrogen transported annuallyinto the Jiulong River Estuary (34.3× 103 t N year−1; Yan et al.,2012b). Similar to denitrification and anammox, Feammox is likelyto be very variable in space and time because of spatial and temporalheterogeneity in substrate availability, redox potential and pH. The

potential nitrogen loss by Feammox in the wetland system couldbe larger than that in terrestrial ecosystems (Table 4). In mangroveareas tidal fluctuations affect the oxygen concentrations in sedi-ments. Denitrification could be overestimated in the field comparedwith anaerobic experiments in the laboratory; thus, the contributionof Feammox to N2 loss could be underestimated. Moreover, nitrifi-cation is a dominant process in aerobic environments (Seitzinger,2008) and competes for ammonium with Feammox to producenitrite and nitrate. This might largely inhibit the Feammox process.

Feammox could potentially be an important pathway for N lossin mangrove sediments and change our current global estimates oftotal N loss. However, uncertainties remain about the Feammoxprocess in mangrove sediments because slurry incubations in ananoxic glove box in this study might have overestimated in situFeammox. Therefore, more in situ studies are needed to determinequantitatively the rates of Feammox in mangrove sediments.

Conclusions

We have demonstrated the occurrence of Feammox in mangroveecosystems for the first time. The potential nitrogen loss throughN2 linked to Feammox was 12.33 t N year−1. Tidal fluctuationsand the large TOC content in mangrove ecosystems are importantcontrolling factors that regulate nitrogen loss through Feammox andlead to greater nitrogen loss. This study provides new insights intoFe-driven microbial N transformations and nitrogen cycling, andcould change our estimates of total nitrogen loss from terrestrialenvironments.

Acknowledgements

The authors gratefully acknowledge the funding for this study fromthe National Key R&D Program of China (2016YFC0502901) andthe National Natural Science Foundation of China (41771500).

References

Alongi, D.M. 1994. The role of bacteria in nutrient recycling in tropicalmangrove and other coastal benthic ecosystems. Hydrobiologia, 285,19–32.

Alongi, D.M. 2009. The Energetics of Mangrove Forests. Springer, Dor-drecht.

Alongi, D., Trott, L., Wattayakorn, G. & Clough, B. 2002. Below-groundnitrogen cycling in relation to net canopy production in mangrove forestsof southern Thailand. Marine Biology, 140, 855–864.



Canfield, D.E., Glazer, A.N. & Falkowski, P.G. 2010. The evolution andfuture of Earth’s nitrogen cycle. Science, 330, 192–196.

Cao, W.Z., Hong, H.S. & Yue, S.P. 2005. Modelling agricultural nitrogencontributions to the Jiulong River estuary and coastal water. Global andPlanetary Change, 47, 111–121.

Cao, W.Z., Yang, J.X., Li, Y., Liu, B.L., Wang, F.F. & Chang, C.T.2016. Dissimilatory nitrate reduction to ammonium conserves nitrogen inanthropogenically affected subtropical mangrove sediments in SoutheastChina. Marine Pollution Bulletin, 110, 155–161.

Cao, W., Guan, Q., Li, Y., Wang, M. & Liu, B. 2017. The contribution ofdenitrification and anaerobic ammonium oxidation to N2 production inmangrove sediments in Southeast China. Journal of Soils and Sediments,17, 1–10.

Chandan, G.S. 2007. Bacterial contribution to mitigation of iron andmanganese in mangrove sediments. Marine Pollution Bulletin, 54,1427–1433.

Clark, D.A., Brown, S., Kichlighter, D.W., Chambers, J.Q., Thomlinson,J.R. & Ni J.& Holland E.A. 2001. Net primary production in tropicalforests: an evaluation and synthesis of existing field data. EcologicalApplications, 11, 371–384.

Clement, J.C., Shrestha, J., Ehrenfeld, J.G. & Jaffe, P.R. 2005. Ammoniumoxidation coupled to dissimilatory reduction of iron under anaerobicconditions in wetland soils. Soil Biology & Biochemistry, 37, 2323–2328.

Dalsgaard, T., Canfield, D.E., Petersen, J., Thamdrup, B. &Acuña-González, J. 2003. N2 production by the anammox reactionin the anoxic water column of Golfo Dulce, Costa Rica. Nature, 422,606–608.

Ding, L.J., An, X.L., Li, S., Zhang, G.L. & Zhu, Y.G. 2014. Nitrogen lossthrough anaerobic ammonium oxidation coupled to iron reduction frompaddy soils in a chronosequence. Environmental Science & Technology,48, 10641–10647.

Fernandes, S.O., Michotey, V.D., Guasco, S., Bonin, P.C. & Bharathi,P.A.L. 2012. Denitrification prevails over anammox in tropical mangrovesediments (Goa, India). Marine Environmental Research, 74, 9–19.

Galloway, J.N., Townsend, A.R., Erisman, J.W., Bekunda, M., Cai, Z.,Freney, J.R. et al. 2008. Transformation of the nitrogen cycle: recenttrends, questions, and potential solutions. Science, 320, 889–892.

Hatha, A.A.M. & Chacko, J. 2010. World atlas of mangroves. International

Journal of Environmental Studies, 69, 304.Jensen, M.M., Thamdrup, B. & Dalsgaard, T. 2007. Effects of specific

inhibitors on anammox and denitrification in marine sediments. Applied

and Environmental Microbiology, 73, 3151–3158.Jin, L., Lu, C.Y., Ye, Y. & Ye, G.F. 2013. Soil respiration in a subtropical

mangrove wetland in the Jiulong River Estuary, China. Pedosphere, 23,678–685.

Krishnan, K.P. & Bharathi, P.A.L. 2009. Organic carbon and iron modulatenitrification rates in mangrove swamps of Goa, south west coast of India.Estuarine, Coastal and Shelf Science, 84, 419–426.

Li, X.F., Hou, L.J., Liu, M., Zheng, Y.L., Yin, G.Y., Lin, X.B. et al. 2015.Evidence of nitrogen loss from anaerobic ammonium oxidation coupledwith ferric iron reduction in an intertidal wetland. Environmental Science

& Technology, 49, 11560–11568.Lovley, D.R. & Phillips, E.J.P. 1987. Rapid assay for microbially reducible

ferric iron in aquatic sediments. Applied and Environmental Microbiol-

ogy, 53, 1536–1540.Marchand, C., Baltzer, F., Lallier-Vergès, E. & Albéric, P. 2004. Pore-water

chemistry in mangrove sediments: relationship with species composi-tion and developmental stages (French Guiana). Marine Geology, 208,361–381.

Nedwell, D.B. 1994. Dynamic nature of the turnover of organic carbon,nitrogen and sulfur in the sediments of a Jamaican mangrove forest.Marine Ecology Progress, 110, 223–231.

Ovalle, A.R.C., Rezende, C.E., Lacerda, L.D. & Silva, C.A.R. 1990. Factorsaffecting the hydrochemistry of a mangrove tidal creek, sepetiba bay,Brazil. Estuarine, Coastal and Shelf Science, 31, 639–650.

Qin, S.P., Hu, C.S. & Oenema, O. 2012. Quantifying the underestimationof soil denitrification potential as determined by the acetylene inhibitionmethod. Soil Biology & Biochemistry, 47, 14–17.

Reef, R., Feller, I.C. & Lovelock, C.E. 2010. Nutrition of mangroves. TreePhysiology, 30, 1148–1160.

Santos, I.R., Cook, P.L.M., Rogers, L., Weys, J.D. & Eyre, B.D. 2012. The“salt wedge pump”: convection-driven pore-water exchange as a sourceof dissolved organic and inorganic carbon and nitrogen to an estuary.Limnology and Oceanography, 57, 1415–1426.

Sawayama, S. 2006. Possibility of anoxic ferric ammonium oxidation.Journal of Bioscience and Bioengineering, 101, 70–72.

Seitzinger, S. 2008. Nitrogen cycle–out of reach. Nature, 452, 162–163.Shen, L.D., Zheng, P.H. & Ma, S.J. 2016. Nitrogen loss through anaero-

bic ammonium oxidation in agricultural drainage ditches. Biology and

Fertility of Soils, 52, 127–136.Thamdrup, B. & Dalsgaard, T. 2002. Production of N2 through anaerobic

ammonium oxidation coupled to nitrate reduction in marine sediments.Applied and Environmental Microbiology, 68, 1312–1318.

Valiela, I., Bowen, J.L. & York, J.K. 2001. Mangrove forests: one ofthe world’s threatened major tropical environments. Bioscience, 51,807–815.

Yan, J., Zhang, C.Y., Luo, Y.M., Wang, W.Q., Zhang, W.Z. & Shang, S.L.2012a. Assesing the areal variation of mangrove coverage forests in theJiulongjiang estuary of Fujian China with remote sensing data. Journal

of Xiamen University (Natural Science), 51, 426–433.Yan, X.L., Zhai, W.D., Hong, H.S., Yan, L., Guo, W.D. & Xiao, H. 2012b.

Distribution, fluxes and decadal changes of nutrients in the Jiulong Riverestuary, Southwest Taiwan Strait. Science Bulletin, 57, 2307–2318.

Yang, W.H., Weber, K.A. & Silver, W.L. 2012. Nitrogen loss from soilthrough anaerobic ammonium oxidation coupled to iron reduction. Nature

Geoscience, 5, 538–541.


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