ENVIRONMENTAL BIOTECHNOLOGY

Differential responses of ammonia/ammonium-oxidizingmicroorganisms in mangrove sediment to amendmentof acetate and leaf litter

Yong-Feng Wang & Xiao-Yan Li & Ji-Dong Gu

Received: 16 September 2013 /Accepted: 3 October 2013# Springer-Verlag Berlin Heidelberg 2013

Abstract The effects of acetate and leaf litter powder onammonia/ammonium-oxidizing microorganisms (AOMs) inmangrove sediment were investigated in a laboratoryincubation study for a period of 60 days. The results showedthat different AOMs responded differently to the addition ofacetate and leaf litter. A higher diversity of anaerobicammonium-oxidizing (anammox) bacteria was observedwhen acetate or leaf litter was added than the control.However, acetate and leaf litter generally inhibited the growthof anammox bacteria despite that leaf litter promoted theirgrowth in the first 5 days. The inhibitory effects on anammoxbacteria were more pronounced by acetate than by leaf litter.Neither acetate nor leaf litter affected ammonia-oxidizingarchaea (AOA) community structures, but promoted theirgrowth. For ammonia-oxidizing bacteria (AOB), the additionof acetate or leaf litter resulted in changes of communitystructures and promoted their growth in the early phase ofthe incubation. In addition, the promoting effects by leaf litteron AOB growth were more obvious than acetate. These

results indicated that organic substances affect AOMcommunity structures and abundances. The study suggeststhat leaf litter has an important influence on the communitystructures and abundances of AOMs in mangrove sedimentand affects the nitrogen cycle in such ecosystem.

Keywords AOB . AOA .Anammox bacteria . Slurryincubation . Acetate . Leaf litter

Introduction

Nitrification is a two-step conversion of ammonia (NH3) intonitrate (NO3

−) via nitrite (NO2−), connecting nitrogen (N2)

fixation and denitrification in the biogeochemical nitrogencycle. Ammonia oxidation is the first and rate-limiting stepin nitrification. The first discovered contributor to this processabout one century ago is a group of ammonia-oxidizingbacteria (AOB) (Winogradsky 1890). Recent phylogeneticstudies showed that all cultured AOB belong to twomonophyletic lineages within β- and γ-Proteobacteria (Headet al. 1993; Purkhold et al. 2000), including Nitrosomonas(β),Nitrosospira (β), andNitrosococcus (γ). Through surveyof 16S rRNA gene and small α-subunit of ammoniamonooxygenase gene (amoA ), species in β-Proteobacteriaappeared to dominate the terrestrial and freshwater environments(Head et al. 1993; Nold et al. 2000; Whitby et al. 1999). Incontrast, γ-Proteobacteria species were only found in marineenvironments (Purkhold et al. 2000). Species of Nitrosospiragenerally have high affinity for ammonia and tend to dominatein environments with low concentrations of ammonia;however, members ofNitrosomonas usually have low affinityfor ammonia and prefer environments with highconcentrations of ammonia (Chain et al. 2003; Okano et al.2004). The niche differentiation of these microorganismsmight be due to the different physiological traits of the genera

Y.<F. WangLaboratory of Microbial Ecology, Guangdong Academy of Forestry,No. 233, Guangshan 1st Road, Guangzhou, People’s Republic ofChina

Y.<F. Wang : J.<D. Gu (*)Laboratory of Environmental Microbiology and Toxicology, Schoolof Biological Sciences, The University of Hong Kong, PokfulamRoad, Hong Kong SAR, Hong Kong, People’s Republic of Chinae-mail: jdgu@hku.hk

X.<Y. LiDepartment of Civil Engineering, The University of Hong Kong,PokfulamRoad, HongKong SAR, HongKong, People’s Republic ofChina

J.<D. GuThe Swire Institute of Marine Science, The University of HongKong, Shek O, Cape d’Aguilar, Hong Kong SAR, Hong Kong,People’s Republic of China

Appl Microbiol BiotechnolDOI 10.1007/s00253-013-5318-7

over time of evolution (Chain et al. 2003; Stein et al. 2007).However, there are some exceptions to this rule. For example,the affinity for ammonia by Nitrosospira strains L115 and40K1 was lower than Nitrosomonas europaea (Jiang andBakken 1999), and both Nitrosomonas ureae andNitrosomonas oligotropha have very high affinities forammonia (Koops and Pommerening-Röser 2001). Hence, it isproposed that the adaptation and functioning of AOB aredetermined by the species or ecotypes rather than the genera(Ke and Lu 2012).

Until recently, the newly discovered ammonia-oxidizingarchaea (AOA) updated our knowledge about ammoniaoxidizers (Könneke et al. 2005). Based on the discovery ofthe unique amoA gene on an archaeal-associated scaffold,Venter et al. (2004) suggested that some archaea may becapable of catalyzing chemoautotrophic ammonia oxidation.Later, amoA genes were confirmed to exist in the Archaea(Schleper et al. 2005) and the first isolated ammonia-oxidizingarchaea was classified into Crenarchaeota (Könneke et al.2005). Later, AOA were reclassified into the phylumThaumarchaeota, which are mesophilic rather thanhyperthemophilic and branch deeply in the archaea domain(Brochier-Armanet et al. 2008; Pester et al. 2011). AOA havebeen demonstrated to be widely distributed and abundant innatural environments (Agogue et al. 2008; Alves et al. 2013;Cao et al. 2013; Francis et al. 2005; Könneke et al. 2005; Keet al. 2013; Leininger et al. 2006). Some studies have shownthat AOA were even more abundant than AOB in someenvironments, e.g., soils and rhizosphere (Chen et al. 2008;Leininger et al. 2006). Compared to AOB, AOA have a higheraffinity for ammonia and frequently dominate environmentswith low concentration of ammonia. For example, the half-saturation constant (Km) of Candidatus Nitrosopumilusmaritimus SCM1 is only 133 nM ∑NH3+NH4

+, and thethreshold substrate concentration is no more than 10 nM∑NH3+NH4

+ (Martens-Habbena et al. 2009). However, Km

values of AOB are generally between 46 and 1,780 μM∑NH3+NH4

+, and their threshold substrate concentration is100-fold higher (>1 μM near neutral pH) than AOA (Koperet al. 2010; Martens-Habbena et al. 2009). The high affinity ofAOA for ammonia allows for their observed competence overAOB and heterotrophic microbes in natural environments.

In addition to the newly discovered AOA, one group ofbacteria in the Planctomycetales which are capable ofoxidizing ammonium coupling with nitrite as the electronacceptor into dinitrogen gas (the process is called anammox)draws large attention (Mulder et al. 1995). The first enrichedanammox bacterium, named Candidatus Brocadiaanammoxidans, belongs to the Planctomycetales (Strouset al. 1999). To date, five genera have been established: Ca .Brocadia, Ca . Kuenenia, Ca . Scalindua (Schmid et al. 2003),Ca . Anammoxoglobus (Kartal et al. 2007), and Ca . Jettenia(Quan et al. 2008), all of which fall into the Planctomycetales.

However, no pure cultures of anammox bacteria have yet beenobtained by now even though many efforts have been made.In addition to the applications to inorganic N removal insewage and wastewater treatment (Hu et al. 2013; Kartalet al. 2010; Yang et al. 2013), anammox bacteria have beendemonstrated to exist in marine environments, includingmarine water column and sediments (Dang et al. 2010;Hong et al. 2011; Kuypers et al. 2005; Lam et al. 2009;Thamdrup 2013), coastal and estuary sediments (Cao et al.2011b; Dale et al. 2009; Li et al. 2011c, d; Han and Gu 2013),and even polar marine sediments and sea ice (Rysgaard andGlud 2004). Anammox bacteria were also detected infreshwater and terrestrial environments such as lakes(Hamersley et al. 2009; Schubert et al. 2006), rivers (Zhanget al. 2007), ground water (Clark et al. 2008), reservoir (Hanand Gu 2013; Han et al. 2013) terrestrial soils (Penton et al.2006), agricultural soils (Long et al. 2013), rice paddy fields(Wang and Gu 2012), hot springs (Jaeschke et al. 2009), andeven oil fields (Li et al. 2010). It was estimated that anammoxbacteria might be responsible for more than 50 % of globalnitrogen losses from the oceans (Brandes et al. 2007; Kuyperset al. 2003). Anammox bacteria have a high affinity for thesubstrates NH4

+ and NO2−, with the Km values of NH4

+ andNO2

– lower than 5 μM. However, the metabolic activity ofanammox bacteria is comparatively low (15–80 μmol of N2

g−1 DW cells min−1), which may be a key reason for their lowgrowth rates (reviewed by Jetten et al. 2009).

As described above, AOB, AOA, and anammox bacteria areall ammonia/ammonium-oxidizing microorganisms (AOMs),acquiring energy by oxidizing ammonia/ammonium to supporttheir life. However, some of them might also utilize othersources of energy, e.g., simple organic substances. Generally,all cultured AOB are strictly chemolithoautotrophic (Kowalchukand Stephen 2001), but they could also utilize hydrogen ororganic substances under condition of oxygen limitation whenammonia are available (Bock et al. 1995; Stüven et al. 1992).AOA might be able to utilize small molecular organicsubstances and some of them are probably even heterotrophic(Pester et al. 2011). Selective groups of anammox bacteriacould also utilize organic substances (Jetten et al. 2009). Apartfrom affecting AOMs directly, organic matter could drivenitrogen cycle by providing ammonium in oxygen minimumzone (Kalvelage et al. 2013). Different AOMs frequentlycoexist in soils and sediments despite that they may havepreference for different niches (Wang and Gu 2013). Recentstudies have shown that all kinds of AOMs were detected inthe mangrove wetland of Mai Po Natural Reserve insubtropical Hong Kong (Cao et al. 2011a, b, c, 2012; Liet al. 2011a, b, d; Wang et al. 2013). They have beendemonstrated to have high diversity and abundance in thiswetland. AOB and AOA in this wetland also showed obviousseasonality: lower abundances in summer than winter (Wanget al. 2013). Mangrove ecosystem supports abundant life and

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possesses great diversity (Smith et al. 1991). Plants are themost important primary producers in wetlands and theycontribute a large quantity of leaf litters into the surroundingsediments annually, which may affect AOM community.However, to the best of our knowledge, no reports have beenpublished on the effects of leaf litter on the activity of AOMsin mangrove sediments. In the present study, we designed anexperiment to delineate the influences of leaf litter, as well asanother simpler organic substance acetate, on AOMs in amangrove wetland. In this experiment, slurries were preparedwith the sediments collected freshly from the mangrovewetland of Mai Po Nature Reserve. The slurries wererespectively amended with acetate or leaf litter powder, whichrepresented simple and complex organic substances. Theslurries were incubated at 20 °C for a period of 60 days.During the incubation, samples were taken. Polymerase chainreaction and denaturing gradient gel electrophoresis (PCR-DGGE) and quantitative real-time PCR (qPCR) were used torespectively trace the changes of the community structuresand the abundances of AOMs during the incubation.

Materials and methods

Description of sampling

Mai Po Nature Reserve, which was designated as a Ramsarsite in 1995, is a well-preserved wetland in Hong Kong. Thepivotal part of Mai Po Nature Reserve is a mangrove wetland,dominated by Kandelia obovata . Previous studies haveshown that the sediment of the mangrove wetland containeddiverse and abundant AOMs (Cao et al. 2011c; Li et al. 2011b,d; Wang et al. 2013). In the present study, 15 kg of mangrovesediment to a depth of 20 cm and 10 L of overlying seawaterwere taken from the mangrove wetland (22°29′N, 114°2′E) onDecember 21, 2010 for preparing slurries of this study. Twokilograms of shed leaves on the surface of the sediment in themangrove wetland were collected for preparing slurriesamended with leaf litter powder.

Preparation of slurries and incubation methods

In the laboratory, 15 kg of sediment was stirred thoroughlyimmediately after being taken back from the field in a glovebox to prevent excessive oxidation of the sediment. Thehomogenized sediment had a density of 1.4 kg L−1. Then,2.8 kg of the sediment and 2 L of the overlying seawater weremixed thoroughly in a sterile container, which was washedwithMilli-Q water trice before use. The slurry was then sieved(0.5 mmmesh) to remove plant detritus and large gravels. Thewhole process was carried out under anaerobic conditions tominimize the exposure to oxygen in ambient atmosphere. For

convenience, the sieved slurry was named “original slurry.”All the following slurries were treated with original slurry.

The controls were set up by transferring 100 mL of theoriginal slurry to each set of ten 125 mL Erlenmeyer flasks.The flasks were then wrapped with aluminum foil before thestart of incubation. Acetate treatment was prepared bydissolving 17 g of CH3COONa⋅3H2O in 1 L of the originalslurry and mixed thoroughly to achieve 3 g C L−1 of acetate inthe slurry. Similar to the control, 10 of 125 mL Erlenmeyerflasks were prepared. To each flask, 100 mL of the preparedslurry containing acetate was transferred. The flasks were thenwrapped with aluminum foil before incubation. Leaf litterpowder treatment was made with the collected leaves. Theleaves were autoclaved under dry heat and dried at 80 °C for48 h till constant weights. The dried leaves were then groundinto powder and sieved (0.5 mm mesh). Because the averagecarbon content of leaf litter is approximately 40 % (Singh andMudgal 2000), 7.5 g of leaf litter powder was mixed with 1 Lof the original slurry thoroughly to achieve ~3 g C L−1 of leaflitter powder in slurry. In this treatment, 10 of 125 mlErlenmeyer flasks were prepared and each flask contained100 mL of the treated slurry. The flasks were then wrappedwith aluminum foil before the start of incubation.

The prepared slurries in the flasks were incubated at 20 °Cin a dark cabinet for a total duration of 60 days. For eachtreatment, two samples/flasks were taken randomly and mixedthoroughly as one sample on days 0, 5, 10, 20, 40, and 60 foreach treatment. For each sample, 20 g of the slurry were storedat −80 °C for subsequent molecular studies, and the remainingslurry was processed immediately for physiochemical analysis.

Physiochemical analyses

Aliquot fraction of the slurries was acquired by centrifugationat 4 °C, 5,000 rpm for 20 min using a Hitachi high-speedrefrigerated centrifuge CR21F (Japan). Ammonium-N andnitrite-N in the aliquot fraction were determined in duplicatewith the Lachat QuikChem 8000 Flow Injection Analyzer(Lachat Instruments Inc.). The analysis procedures were inaccordance with the manual of the instrument. Slurry dryweights were measured in triplicate after drying in an ovenat 105 °C for 24 h till constant weight was achieved. Organicmatter of each sample was measured in triplicate using loss onignition method (Heiri et al. 2001), in which organic matterwas oxidized at 500–550 °C for 2 h in Thermolyne MuffleFurnace (Type 47900).

Sediment DNA extraction

Total genomic DNA of each slurry sample was extracted induplicate using the PowerSoil® DNA isolation kit (MO BIOLaboratories, Inc., USA) according to the manual of themanufacturer. The extracted duplicate DNA was then

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combined as one sample and stored at −20 °C for subsequentmolecular analysis.

PCR amplification

The genes used for DGGE were amplified by a nested PCRprocedure available (Auguet et al. 2011; Nicol et al. 2008;Nicolaisen and Ramsing 2002; Park et al. 2008; Verhammeet al. 2011). Briefly, the partial sequence of the genes was firstamplified using PCR primers without a GC clamp. Theproducts were then checked by agarose gel electrophoresis,and the expected size bands were cut and purified. Thepurified products were then diluted and used as templates fora second round of PCR amplification using primers with a GCclamp (5′-CCGCCGCGCGGCGGGCGGGGCGGGGGCACGGGG-3′) (Muyzer et al. 1993). This method has beendemonstrated in our lab to be unbiased and effective if thenumber of PCR cycles and template quantity were wellcontrolled (unpublished data). The detailed protocols areindicated in the succeeding text.

The 16S rRNAgene fragments of anammox bacteria were firstamplified using Amx368F (5′-TTCGCAATGCCCGAAAGG-3′)and Amx820R (5′-AAAACCCCTCTACTTAGTGCCC-3′)(Schmid et al. 2000, 2003). The optimized PCR reaction mixturein a final volume of 50 μL contained the following: 1.5 μL oftemplate DNA (20 ng μL−1), 10 μL of 5× GoTaq Flexi buffer(Promega;HongKong), 4μL ofMgCl2 (25mM; Promega), 1μLof deoxyribonucleotide triphosphates (dNTPs; 10 mM of each;Promega), 1 μL of each forward and reverse primers (20 μM),0.25 μL of GoTaq Flexi polymerase (5 U μL−1; Promega), and5 μL of bovine serum albumin (BSA; 0.1 %). PCR conditionswere set as follows: 94 °C for 4 min; 30 cycles of 95 °C for 45 s,59 °C for 50 s, followed by 72 °C for 1 min; and finally 72 °C for15 min. PCR products were checked by electrophoresis in 1%agarose gel stained with GelRed™ (Biotium; Hayward) at 1:10,000 and then purified using Gel Advance™ Gel ExtractionSystem (Viogene-Bio Tek Co., Taiwan, ROC). The purifiedPCR product was then amplified with Amx368F-GC andAmx820R. The PCR reactionmixture contained in a final volumeof 50 μL the following: 2 μL of template DNA (0.1 ng μL−1),10 μL of 5× GoTaq Flexi buffer (Promega), 4 μL of MgCl2(25 mM), 1 μL of dNTPs (10 mM of each; Promega), 1 μL ofeach forward and reverse primers (20 μM), 0.25 μL of GoTaqFlexi polymerase (5UμL−1; Promega), and 5μLofBSA (0.1%).PCR conditions were set as follows: 94 °C for 4 min; 17 cycles of95 °C for 45 s, 63 °C for 50 s, followed by 72 °C for 1 min; andfinally 72 °C for 7 min. The PCR product was checked byelectrophoresis in 1 % agarose gel stained with GelRed (1/10,000). The PCR product was then subject to subsequent DGGEanalysis.

The archaeal amoA genes were first amplified using thePCR primers Arch-amoAF (5′-STAATGGTCTGGCTTAGACG-3′) and Arch-amoAR (5′-GCGGCCATCCATCTG

TATGT-3′). Based on the common procedures in themanufacturer's instructions and results of previous studies(Francis et al. 2005), the optimized PCR reaction mixture ina final volume of 50 μL contained the following: 1.5 μL ofDNA (20 ng μL−1), 10 μL of 5× GoTaq Flexi buffer(Promega), 3 μL of MgCl2 (25 mM; Promega), 1 μL ofdNTPs (10 mM of each; Promega), 1 μL of each forwardand reverse primers (20 μM), 0.25 μL of GoTaq Flexipolymerase (5 U μL−1; Promega), and 5 μL of BSA(0.1 %). PCR conditions were set as follows: 95 °C for5 min; 30 cycles of 94 °C for 45 s, 53 °C for 1 min, and72 °C for 1 min; and finally 72 °C for 15 min. PCR productswere checked by electrophoresis in 1 % agarose gel stainedwith GelRed (1/10,000) and then purified using Gel Advance.The purified PCR product was then amplified with Arch-amoAF-GC and Arch-amoAR. The optimized PCR reactionmixture contained in a final volume of 50 μL the following:2 μL of template DNA (0.1 ng μL−1), 10 μL of 5× GoTaqFlexi buffer (Promega), 3 μL of MgCl2 (25 mM), 1 μL ofdNTPs (10 mM of each; Promega), 1 μL of each forward andreverse primers (20 μM), 0.25 μL of GoTaq Flexi polymerase(5 U μL−1; Promega), and 5 μL of BSA (0.1 %). PCRconditions were set as follows: 95 °C for 5 min; 17 cycles of94 °C for 45 s, 58 °C for 1 min, followed by 72 °C for 1 min;and finally 72 °C for 15 min. The PCR product was checkedby electrophoresis in 1 % agarose gel stained with GelRed (1/10,000). The PCR product was then subject to subsequentDGGE analysis.

The bacterial amoA genes were amplified using the PCRprimers amoA-1F (5′-GGGGGTTTCTACTGGTGGT-3′) andamoA -2R (5′-CCCCTCKGSAAAGCCTTCTTC-3′). Theoptimized PCR reaction mixture in a final volume of 50 μLcontained the following: 1.5 μL of template DNA(20 ng μL−1), 10 μL of 5× GoTaq Flexi buffer (Promega),2.5 μL of MgCl2 (25 mM; Promega), 1 μL of dNTPs (10 mMof each; Promega), 1 μL of each forward and reverse primers(20 μM), 0.25 μL of GoTaq Flexi polymerase (5 U μL−1;Promega), and 5 μL of BSA (0.1 %). PCR conditions were setas follows: 94 °C for 3 min; 30 cycles of 94 °C for 45 s, 55 °Cfor 45 s, and 72 °C for 50 s; and finally 72 °C for 10 min. PCRproducts were checked by electrophoresis in 1 % agarose gelstained with GelRed (1/10,000) and then purified using GelAdvance. The purified PCR product was then amplified withamoA-1F-GC and amoA-2R. The optimized PCR reactionmixture contained in a final volume of 50 μL the following:2 μL of template DNA (0.1 ng μL−1), 10 μL of 5× GoTaqFlexi buffer (Promega), 2.5 μL of MgCl2 (25 mM), 1 μL ofdNTPs (10 mM of each; Promega), 1 μL of each forward andreverse primers (20 μM), 0.25 μL of GoTaq Flexi polymerase(5 U μL−1; Promega), and 5 μL of BSA (0.1 %). PCRconditions were set as follows: 94 °C for 3 min; 17 cycles of94 °C for 45 s, 60 °C for 45 s, followed by 72 °C for 50 s; andfinally 72 °C for 10 min. The PCR product was checked by

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electrophoresis in 1 % agarose gel stained with GelRed (1/10,000). The PCR product was then subject to subsequent DGGEanalysis.

DGGE analysis and band sequencing

PCR products were separated in polyacrylamide gels (6 %acrylamide–bisacrylamide [37.5:1; Bio-Rad]; 1.5 mm thick;16 cm×16 cm). The denaturant gradient ranging from 30 to50 % denaturant (100 % denaturant was 7 M urea and 40 %formamide in 1× Tris–acetate–EDTA (TAE) buffer) was foranammox bacteria and AOB and from 30 to 40 % for AOA.DGGE of PCR amplified products was performed with theDCode system (Bio-Rad) with 1× TAE at 60 °C, 120 V for 6 hfor anammox bacteria and AOB and at 60 °C, 90 V for 12 hfor AOA. Gels were stained with GelRed (1.5/10,000) for15 min and scanned with GelDoc EQ (Bio-Rad).

At least two bands of the same position in two differentlanes were carefully excised from DGGE gels. The excisedbands were washed thrice with Milli-Q water and thensuspended in 100 μL of Milli-Q water overnight, and thenre-amplified using PCR primers without GC clamp accordingto protocols described above. Products were gel-checked andpurified according to the methods described above. Eachpurified product was sequenced by a set of forward andreverse primers with ABI 3730xl DNA Analyzer (AppliedBiosystems) at Genome Research Centre of The University ofHong Kong.

Phylogenetic analysis

Sequences were analyzed against those in GenBank withBLAST (Altschul et al. 1990). After chimeric check andaligned, phylogenetic trees were constructed using MEGA,version 5.1 (Tamura et al. 2011). For anammox bacteria, 16SrRNA gene nucleotide sequences (477 nucleotides) were usedfor phylogenetic analysis. For AOA and AOB, putative aminoacid sequences of AmoA (198 and 150 amino acids,respectively) deduced from the nucleotide sequences obtainedwere used for phylogenetic analysis. Phylogenetic trees wereconstructed with the neighbor-joining method with 1,000bootstrapping to estimate the confidence of the tree topologies.

Quantitative real-time PCR analysis

The abundances of anammox bacterial 16S rRNA genes andarchaeal and bacterial amoA genes in samples weredetermined in triplicate with quantitative real-time PCRamplification using a FastStart Universal SYBRGreenMaster(Rox) Kit (Roche; Germany). Real-time qPCRwas performedin 96-well optical plates placed in the ABI PRISM® 7000Sequence Detection System (Applied Biosystems). Theprimer set composed of Amx368F and Amx820R was used

for the amplification of 16S rRNA genes of anammoxbacteria. The primer sets composed of Arch-amoAF andArch-amoAR and amoA-1F and amoA-2R were used for theamplification of the amoA genes of AOA and AOB,respectively. The final reaction volume was 20 μL, and thereaction composition and PCR cycling conditions were inaccordance with the manual.

The specificity of the PCR amplification was determinedby the melting curve and the gel electrophoresis. Cyclethresholds were determined by comparing with the standardcurves constructed using a 10-fold serial dilution (102–107

gene copies μL−1) of newly extracted plasmids containingcorresponding gene fragments. Relative copy numbers amongtarget groups were evaluated, and some replicates of apparentdiscrepancy were excluded in order to decrease standard error.The correlation coefficient R2 values were greater than 0.97for all of the standard curves.

Nucleotide sequence accession numbers

The anammox bacterial 16S rRNA gene sequencesdetermined in this study are available in GenBank underaccession numbers JX914646 to JX914654, AOA amoA genesequences under accession numbers JX914628 to JX914635,and AOB amoA gene sequences under accession numbersJX914636 to JX914645.

Results

Dynamics of organic matter, NH4+, and NO2

−

Organic carbon in the control treatment decreased slightlyfrom the initiation of the study (2.99 %) to the end of theincubation (2.86 %); however, it, after amendment withacetate and leaf litter powder, decreased significantly from3.44 to 2.57 % and 3.56 to 3.09 %, respectively (Fig. 1a, b).For acetate treatment, organic matter decreased rapidly in thefirst 5 days. Because of acetate addition, organic carbon ofacetate treatment was higher than the control in the initial daysof incubation. However, organic carbon content of acetatetreatment was even lower than the control after the first10 days (Fig. 1a), indicating the priming effects from theaddition of the utilizable carbon. In contrast, the organiccarbon of leaf litter powder treatment decreased rapidly inthe first 20 days (Fig. 1b). Unlike acetate treatment, organiccarbon of leaf litter amendment was always higher than thecontrol during the whole period of the incubation, indicatingthat not all added leaf carbon was consumed by organisms.Leaves contain organic substances with different chemicalstructures and degradability and were not easily utilized byheterotrophic microbes. Consequently, the effect of promoting

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the growth of heterogenic organisms by leaf litter powder wasslow and slight.

NH4+ in the control decreased sharply by more than 50 %

in the first 5 days of incubation and then decreased slowly inthe remaining of the incubation (Fig. 1c, d). The abruptdecrease in the first 5 days was probably due to the stirringof slurries which enhanced the contacts between microbes andsubstrates, resulting in the quick consumption of NH4

+. Forthe slurries amended with acetate or leaf litter powder, NH4

+

decreased sharply in the first 5 days, but the concentrationbegan to increase from the fifth day onward (Fig. 1c, d). Forslurries amended with acetate, NH4

+ increased to a very highlevel on day 40. This is because organic matter such as acetateand leaf powder after addition into the slurries promotedconsumption of more NH4

+ by microorganisms to build upthe biomass. But in the later days, the increased population ofmicroorganisms began to die due to the micro-environmentalchanges of the slurry system and limitation of resources.Hence, NH4

+ was released from the decomposition of thebiomass, leading to a gradual increase of NH4

+ in theremaining time of incubation. This trend was more obviousfor slurries amended with acetate because the more easilyutilized acetate had a priming effect on the growth ofmicroorganisms and then more NH4

+ was released when thedecomposition of the dead biomass started.

NO2− of the control increased from 0.26 μM on the initial

day to 0.70μMon day 60 of incubation (Fig. 1e, f). Comparedto the control, NO2

− of slurries with acetate addition increasedsignificantly during incubation (Fig. 1e). For the slurriesamended with leaf litter powder, NO2

− increased in the first5 days; however, the concentration decreased to a level similarto the control afterward (Fig. 1f). NO2

− is the product of bothammonia oxidation and denitrification. In this incubation,NO2

− probably mainly came from denitrification rather thanammonia oxidation. For the control, stirring slurries increasedthe interaction between denitrifying bacteria and substrates,resulting in more NO2

− during incubation. When acetate wasadded, denitrifying bacteria were boosted because acetatecould enhance the heterotrophic denitrifying bacteria andmore NO2

− was produced by the increased denitrifyingbacterial population. Leaf litter powder increased NO2

− onlyin the first 5 days because it could not be utilized as easily asacetate.

Changes of anammox bacteria community structureand abundance

For the slurries on the initial days, L1-ana, L2-ana, and L9-anawere the main DNA bands of the anammox bacteriacommunity detected (Fig. 2a). Almost no observable changesof the community structure were detected for the controlduring the course of incubation. For slurries amended withacetate, some new bands could be observed during the

incubation: L3-ana appeared on day 10; L3-ana and L4-anacould be observed on day 20; and L6-ana, L7-ana, and L8-anawere noticeable on day 40. For slurries amended with leaflitter powder, additional new bands also appeared: L3-ana andL4-ana emerged on day 5; L8-ana appeared on day 10; L7-anaand L8-ana appeared on day 40; and L5-ana was detectable onday 60. The results indicated that both acetate and leaf litterpowder had promoted more diverse anammox bacteria in theslurries. The phylogeny of the nucleotide sequences fromthese bands is shown in Fig. 2b. Bands from L1-ana throughL4-ana are related to Ca . Scalindua wagneri, L5-ana to Ca .Scalindua brodae, and L6-ana through L9-ana to Ca .Kuenenia.

The changes of anammox bacteria abundances during theincubation are shown in Fig. 3a and b. Generally, abundanceof anammox bacteria in the control increased gradually duringthe incubation, reaching the highest level on day 40 (Fig. 3a,and b). The increase was probably due to stirring of the slurry,which made anammox bacteria more accessible to substrates.For slurries amended with acetate, the abundance of anammoxbacteria was always lower than the control during the wholeperiod of the incubation (Fig. 3a). For the slurries amendedwith leaf litter powder, there was an obvious increase ofanammox bacteria on day 5; however, in the later incubation,the abundance of anammox bacteria was always lower thanthe control (Fig. 3b), indicating that acetate could inhibit thegrowth of anammox bacteria. Leaf powder could promote thegrowth in the first 5 days but began to show inhibitory effectson anammox bacteria afterward. The inhibitory effects ofacetate on anammox bacteria were more pronounced thanthe leaf litter powder.

Changes of AOA community structure and abundance

For the slurries on the initial day, there were eight major bandsfor the AOA community (Fig. 4a). For all the treatments (thecontrol, amendment with acetate or leaf litter powder), AOAcommunity structures did not show any obvious changesduring the whole period of the incubation, indicating that theaddition of acetate and leaf powder had no apparent effects onAOA community structures in this mangrove sediment. Thephylogeny of the bands is shown in Fig. 4b. The bands L1-aoaand L2-aoa in cluster 3 belong to sediment/soil clade, whilethe bands L3-aoa to L8-aoa in cluster 1 belong to sediment/water column clade.

The changes of AOA abundances during the incubation areshown in Fig. 3c and d. For the control, the quantity of AOAdecreased by more than 50 % in the first 10 days ofincubation. But in the later days of the incubation, AOA beganto increase, reaching the highest level on day 40. The cause forthe decrease in the first 10 days probably is that AOA needtime to adapt to the new conditions of the slurries. For theslurries amended with acetate and leaf powder, however,

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AOA increased significantly in the first 10 days compared tothe control. From day 20 onward, AOA began to decreasecompared to the control. The result indicates that both acetateand leaf litter powder could promote the growth of AOA for ashort term.

Changes of AOB community structure and abundance

For the slurries on the initial day, the bands L2-aob, L3-aob,and L7-aob were the main components of the AOBcommunity, and the minor bands L4-aob, L5-aob, and L9-aob were also detectable (Fig. 5a). For the control slurries, theAOB community had slight changes in the early phase of theincubation. For example, on day 5, L6-aob became adominant band in the community, but was absent in the initialdays. Although there were some changes during theincubation, the community structure recovered to the initiallevel in slurries on day 60. For slurries amended with acetate,L4-aob, L5-aob, and L6-aob were dominant on day 5. Ondays 10 and 20, the community structure recovered to theinitial level in slurries. However, on days 40 and 60, L4-aoband L5-aob became the dominant ones again. For slurriesamended with leaf litter powder, the community structureobviously changed during the incubation. On day 5, L8-aob

was dominant and L3-aob was absent. On day 10, L5-aob andL6-aob were dominant. On day 20, L1-aob, L5-aob, L9-aob,and L10-aob were major bands. On days 40 and 60, thecommunity structure became stable, with L4-aob, L5-aob,and L6-aob being dominant. Generally, acetate and leaf litterpowder could induce the changes of AOB communitystructures. L4-aob, L5-aob, and L6-aob became dominantfor extended incubation in the slurries amended with acetateor leaf litter powder. The initial dominant bands L2-aob, L3-aob, and L7-aobwere absent at the end of the incubation in theslurries amended with acetate or leaf litter powder. Thephylogeny of the bands is shown in Fig. 5b. As shown inthe phylogenetic tree, the bands on the initial day and duringthe incubation are widely distributed in different species. L1-aob belongs to cluster 4; L2-aob, L3-aob, and L4-aob belongto cluster 3; L5-aob and L6-aob belong to cluster 5; L7 is notclosely related to any known species; and L8-aob, L9-aob, andL10-aob belong to cluster 1.

The changes of AOB abundances during the incubation areshown in Fig. 3e and f. For the control, AOB increasedgradually during the incubation, and on day 40, the abundancereached the highest level of about 2-fold of the initial day. Thisphenomenon indicated that stirring slurry probably had apositive effect on the growth of AOB. For slurries amended

1.15

1.00 0.99 0.92 0.92 0.90

0.6

0.7

0.8

0.9

1.0

1.1

1.2

2.4%

2.6%

2.8%

3.0%

3.2%

3.4%

3.6%

0 5 10 20 40 60

Rat

io o

f org

anic

mat

ter

(ace

t/ct

rl)

Org

anic

mat

ter

(wet

slu

rry)

Time (day)

ctrl acet acet/ctrla

1.19 1.15 1.11

1.06 1.08 1.08

0.6

0.7

0.8

0.9

1.0

1.1

1.2

2.4%

2.6%

2.8%

3.0%

3.2%

3.4%

3.6%

0 5 10 20 40 60

Rat

io o

f org

anic

mat

ter

(lea

f/ct

rl)

Org

anic

mat

ter

( wet

slu

rry)

Time (day)

ctrl leaf leaf/ctrlb

1.00 0.34 0.53 2.07

26.06

36.25

0

10

20

30

40

50

0

100

200

300

400

0 5 10 20 40 60

Rat

io o

f NH

4+ (a

cet/

ctrl

)

NH

4+ of p

ore

wat

er (µ

M)

Time (day)

ctrl acet acet/ctrlc

1.00 0.00 0.11 0.92 3.46

9.28

0

10

20

30

40

50

0

100

200

300

400

0 5 10 20 40 60

Rat

io o

f NH

4+ (l

eaf/

ctrl

)

NH

4+ o

f po

re w

ater

(µM

)

Time (day)

ctrl leaf leaf/ctrld

1.00 3.94

10.48 9.28

14.42

24.56

0

5

10

15

20

25

30

0.00

5.00

10.00

15.00

20.00

0 5 10 20 40 60

Rat

io o

f NO

2- (a

cet/

ctrl

)

NO

2- of p

ore

wat

er (µ

M)

Time (day)

ctrl acet acet/ctrle

1.00

2.46

0.42

0.84 1.04

0.68

0.0

0.5

1.0

1.5

2.0

2.5

3.0

0.0

0.5

1.0

1.5

2.0

0 5 10 20 40 60

Rat

io o

f NO

2- (l

eaf/

ctrl

)

NO

2- o

f po

re w

ater

(µM

)

Time (day)

ctrl leaf leaf/ctrlf

Fig. 1 Dynamics of organic matter (a , b), NH4+ (c , d), and NO2

− (e , f) during the slurry incubation (mean ± SD, n =2)

Appl Microbiol Biotechnol

with acetate and leaf litter powder, AOB were intensivelyenhanced in the first 10 days compared to the control. The

effect of leaf litter powder (3.65-fold of the control on day 5)was more intensive than acetate. However, in the remaining

a

bS0-s-6(1) GQ331343

S1-l-17(6) GQ331341S1-s-12(2) GQ331342

S1-l-5(14) GQ331337S1-s-13(8) GQ331339

Clone from estuarial sediment GQ427391L5-ana

Clone from estuary JN010126Clone from river sediment DQ647431

S0-s-16(2) GQ331348S1-l-1(6) GQ331345S1-s-17(4) GQ331346

S0-s-26(1) GQ331352S0-s-7(4) GQ331336

Clone from estuary JN051559Clone from estuary JN051567

S0-s-1(5) GQ331335Ca. Scalindua profunda EU142947

S0-s-4(3) GQ331334S1-s-6(6) GQ331333

S0-s-29(2) GQ331358S0-s-5(4) GQ331356

S0-s-3(5) GQ331361S0-s-8(5) GQ331360

Ca. Scalindua sorokinii AY257181Ca. Scalindua brodae AY254883Clone from lake water FJ830382

Ca. Scalindua brodae EU142948Clone from sea water DQ368256

L4-anaL3-ana

Ca. Scalindua wagneri EU478692S1-s-1(8) GQ331354

Clone from estuarine sediment GQ427457Clone from river sediment HM537536

L1-anaL2-ana

Ca. BrocadiaCa. Anammoxoglobus

Ca. JetteniaL6-ana

Ca. Kuenenia stuttgartiensis AF375995L7-ana

Clone from marine sponge FJ652538Clone from Fish gut EU310470

Clone from tidal flat sediment JN010145Clone form estuarine sediment FJ490116Clone from estuarine sediment HM209495

L8-anaL9-ana

S1-l-4(4) GQ331353Clone from aquifer HM066573

Clone from SCS EU048620Clone from hypersaline water JF747676

Clone from water HM851665Planctomyces brasiliensis AJ231190

7797

99

82

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99

55

55

68

62

71 87

7267

9652

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Ca.

Scal

indu

a w

agne

ri

Ca.

Scal

indu

a br

odae

Ca.

Kue

neni

a

Ca.

Scal

indu

a

Unknown

Fig. 2 DGGE analyses of PCR-amplified anammox bacterial 16S rRNAgene fragments of the control, acetate, and leaf powder treatments duringthe slurry incubation (a) and phylogenetic tree based on nucleotidesequences of 16S rRNA gene fragments of anammox bacteria (477nucleotides) (b ). The phylogenetic tree was constructed with theneighbor-joining method with 1,000 bootstrapping to estimate the

confidence of the tree topologies. Bootstrap values (>50 %) are indicatedat the branch points. The scale bar represents 0.02 sequence divergence.The solid squares in red represent the DGGE band sequences of thepresent study. The solid and hollow diamonds in blue represent the clonesequences within and 10 m away from the vegetated area by anotherstudy in our laboratory

Appl Microbiol Biotechnol

days of incubation, both acetate and leaf litter powder seemedto have an inhibitory effect on AOB growth compared to thecontrol. Generally, the results indicated that both acetate andleaf litter powder could promote AOB growth in the earlyphase of the incubation, and the promotion effect of leaf litterpowder was more obvious.

Discussion

Effects of organic substances on anammox bacteria

Being a branch of the order Planctomycetales, anammoxbacteria are believed to be lithoautotrophic microorganisms,extracting energy from the biochemical process of combiningammonium and nitrite into dinitrogen gas (Kartal et al. 2010;Strous et al. 1999). However, increasing evidence showed thatanammox bacteria are not strictly chemolithoautotrophic. Inaddition to utilizing ammonium, anammox bacteria could alsoobtain energy by using simple organic substances such asformate, acetate, propionate, monomethylamine, anddimethylamine (Kartal et al. 2007, 2008). For example, Ca .

Brocadia fulgida and Ca . Anammoxoglobus propionicuswere, respectively, enriched through feeding with acetateand propionate when ammonium was provided at the sametime. In addition, Sabumon (2007) showed that anaerobicremoval of ammonium was practicable in the presence oforganic matter. In addition to the culture in the bioreactoramended with organic substances, metagenomics of both thefreshwater species Ca . Kuenenia stuttgartiensis and themarine species Ca . Scalindua profunda showed thatanammox bacteria had a versatile lifestyle including usingorganic substances (Strous et al. 2006; van de Vossenberget al. 2013).

Asmany evidences suggested that anammox bacteria couldutilize organic substances (Kartal et al. 2007, 2008), it mightbe presumed that leaf litters could benefit them in a naturalenvironment. However, our results did not support thisviewpoint. On the contrary, anammox bacteria were inhibitedto different degrees when the slurries were amended withacetate or leaf litter powder (Fig. 3). Furthermore, acetatehad a stronger inhibition on anammox bacteria than leaf litterpowder. The slurries were prepared from the sediment of themangrove wetland, which contained a variety of heterotrophic

1.00 0.79

0.83

0.22 0.19 0.36

-1.5

-1.0

-0.5

0.0

0.5

1.0

1.5

0.0E+0

2.0E+6

4.0E+6

6.0E+6

8.0E+6

1.0E+7

1.2E+7

0 5 10 20 40 60

Rat

io o

f 16S

rR

NA

gen

es

(ace

t/ct

rl)

16S

rR

NA

gen

es g

-1 w

et s

lurr

y

Time (day)

ctrl acet acet/ctrla

1.00

1.79

0.85 0.40 0.33

0.77

-3

-2

-1

0

1

2

3

0.0E+0

2.0E+6

4.0E+6

6.0E+6

8.0E+6

1.0E+7

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0 5 10 20 40 60

Rat

io o

f 16S

rR

NA

gen

es

(lea

f/ct

rl)

16S

rR

NA

gen

es g

-1 w

et s

lurr

y

Time (day)

ctrl leaf leaf/ctrlb

1.00 1.21 1.59 0.76

0.42 0.88

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-3

-1

1

3

5

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1E+7

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0 5 10 20 40 60

Rat

io o

f AO

B a

moA

gen

es

(ace

t/ct

rl)

AO

B a

mo

A g

enes

g-1

wet

slu

rry

Time (day)

ctrl acet acet/ctrle

1.00

3.65 3.06

0.68 0.59 0.93

-5

-3

-1

1

3

5

0E+0

1E+7

2E+7

3E+7

4E+7

0 5 10 20 40 60

Rat

io o

f AO

B a

moA

gen

es

(lea

f/ct

rl)

AO

B a

mo

A g

enes

g-1

wet

slu

rry

Time (day)

ctrl leaf leaf/ctrlf

1.00 2.22

3.60

0.88 0.33 0.58

-6

-4

-2

0

2

4

6

0E+0

2E+6

4E+6

6E+6

8E+6

1E+7

0 5 10 20 40 60 R

atio

of A

OA

am

oA g

enes

(a

cet/

ctrl

)

AO

A a

mo

A g

enes

g-1

wet

slu

rry

Time (day)

ctrl acet acet/ctrl

c

1.00

2.58 3.75

0.74 0.61 0.99

-6

-4

-2

0

2

4

6

0E+0

2E+6

4E+6

6E+6

8E+6

1E+7

0 5 10 20 40 60

Rat

io o

f AO

A a

moA

gen

es

(lea

f/ct

rl)

AO

A a

mo

A g

enes

g-1

wet

slu

rry

Time (day)

ctrl leaf leaf/ctrl

d

Fig. 3 Abundances of 16S rRNA genes (a , b) and archaeal (c , d) and bacterial amoA genes (e , f) in the control, acetate, and leaf powder treatmentsduring the slurry incubation (mean ± SD, n =3)

Appl Microbiol Biotechnol

bL3-aoa

L7-aoaS0-s-1 (3) GQ331517L6-aoa

L4-aoaS1-l-6 (17) GQ331393

L5-aoaL8-aoaS1-s-5 (5) GQ331441S1-s-4 (1) GQ331397

Clone from a constructed wetland JF743004S1-l-41 (1) GQ331394

Nitrosopumilus maritimus SCM1 EU239959S0-s-3 (7) GQ331518

S1-s-10 (3) GQ331443Marine water cluster A

Clone from sediment of Lake Taihu FJ951672LuffGrA from sponge EU049831DR13 from freshwater river GQ414591Clone from esturary sediment HM537372

Clone from estuary sediment HM537381S0-s-2 (5) GQ331519

S1-s-7 (14) GQ331446Clone from estuary sediment EU651269Clone from root of Littorella sp. EU309878

S0-l-4 (10) GQ331476S1-s-26 (1) GQ331445

Marine water cluster BCa. Nitrosotalea devanaterra JN227489N-PL5 from grassland soil HQ917134

S0-s-14 (1) GQ331521L2-aoaL1-aoa

S0-s-13 (18) GQ331520S1-l-1 (17) GQ331390S1-l-10 (10) GQ331391S1-s-1 (10) GQ331437S1-s-22 (1) GQ331413S0-s-17 (8) GQ331522

Clone from Fish River sediment FJ227736Clone from sediment of SCS HQ888990Clone from mangrove sediment HM754732Clone from marine sediment JF924662Clone from deep sea sediment EU885658

Clone from sediment of SCS HQ888993S0-l-1 (34) GQ331473

S1-l-46 (2) GQ331392S1-s-3 (1) GQ331439

Nitrososphaera gargensis GA15P03 EU281321Clone from WTTP sludge HM055568

Nitrososphaera viennensis EN76 FR773159Thermophilic AOA

100

100

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5099

72

87

97

92

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93

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50

8852

92

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ster

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ater

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umn

Soil/

Sedi

men

t

a

Appl Microbiol Biotechnol

microorganisms. When slurries were amended with acetate orleaf litter powder, heterotrophic microorganisms would utilizethem much more quickly than anammox bacteria. Because ofthat, anammox bacteria, outcompeted by these heterotrophicorganisms, were inhibited by the addition of organic matterthrough direct competition and also access to availableresource. Our results suggested that anammox bacteria couldnot benefit from, if not inhibited by, leaf litters in themangrove wetland. In addition to our research, some reportsalso showed that organic matter inhibited anammox bacteriain bioreactors. For example, anammox process was inhibitedin a traditional denitrification bioreactor when organic loadingwas increased (Molinuevo et al. 2009). As a result, whetheranammox bacteria could benefit from organics depends onhow strongly their coexisting microorganisms compete for thesame organics.

Interestingly, anammox bacteria in slurries amended withleaf materials had a surge on day 5 during the incubation,which was probably due to the physical effect of leaf litterpowder. Leaf litter powder could affect the structure of theslurry by offering a larger surface for anammox bacteria toadhere to and multiply. Because of that, leaf litter powdercould promote the growth of anammox bacteria. When theleaf litter powder was degraded after the first few days, thesurface effect was decreased and the organic substancesreleased from leaf litter powder could inhibit anammoxbacteria, which is why in the later incubation, anammoxbacteria were lower than the control. Acetate did not have aboosting effect on anammox bacteria because acetate wasdissolved instantly when added into slurries and, thus, didnot exist in particles for anammox bacteria to inhabit.

In addition to inducing the changes of abundances, acetateand leaf litter powder also resulted in a higher diversity ofanammox bacteria during the incubation (Fig. 2a), probablybecause some phylotypes of anammox bacteria benefitedfrom acetate or leaf litter powder. Anammox bacteria aredistributed in five genera and have diverse lifestyles asdiscussed above. Studies showed that anammox bacteria ofclosely correlated species may have distinct metabolisms. Forexample, Ca . Brocadia fulgida and Ca . Anammoxoglobuspropionicus were enriched from the same sludge under theconditions amended with acetate and propionate, respectively(Kartal et al. 2007, 2008). Because of the minor differences in

the mode of metabolism between different species, a selectivefraction of anammox bacteria might be enhanced in theslurries when acetate or leaf powder was added.

Effects of organic substances on AOA

As indicated in the Results above acetate and leaf litterpowder promoted the growth of AOA mostly in the first20 days compared to the control. This result is quite consistentwith the newly published finding: addition of Douglas firneedles to soils could lead to the changes in the abundancesof AOA while the community structure stayed invariable(Yarwood et al. 2013). Until now, there is no solid evidenceconfirming whether AOA are completely autotrophic ormixotrophic, or even heterotrophic. Nitrosopumilusmaritimus and Nitrososphaera gargensis , representatives ofmarine and soil species, respectively, have been shown to beautotrophic (Hatzenpichler et al. 2008; Könneke et al. 2005).Genomic analysis of N . maritimus also suggested that avariant of 3-hydroxypropionate/4-hydroxybutyrate (HP/HB)cycle that fixes CO2 exists in AOAmetabolism. However, thegenome of N . maritimus revealed the occurrence of anincomplete TCA cycle and transporters for organic substances(Walker et al. 2010). Furthermore, archaea could incorporatethe intermediate 3-hydroxypropionate through the HP/HBcycle (Berg et al. 2010), suggesting that AOA might bemixotrophic organisms. Mußmann et al. (2011) reported thatsome AOA of soil type are not obligate autotrophic.Additionally, Jia and Conrad (2009) indicated that soil AOAcould grow without incorporating 13CO2 when nitrificationwas inhibited, suggesting that someAOAmight not be strictlyautotrophic. In this study, both acetate and leaf powderpromoted the growth of AOA, indicating that at least someAOA in this research are mixotrophic or autotrophic.Sediment of mangrove wetland receives abundant organicssuch as leaf litters constantly. The occurrence of the AOAcapable of utilizing organic substances in Mai Po NatureReserve should be the result of natural selection and evolutionin this niche.

There were a variety of AOA in the initial slurries,including phylotypes affiliated to marine and soil groups.However, the community structures of AOA wereconsiderably stable during the whole duration of theincubation in all treatments (Fig. 4a), consistent with theresults of Yarwood et al. (2013), indicating that AOA in thissediment responded to organic substances similarly. In otherwords, the different types of AOA in the sediment of thismangrove wetland might have similar metabolism in terms oforganic substances. Previous studies have shown that AOAwere widely distributed in different types of environments,ranging from different pH values (Lehtovirta-Morley et al.2011; Nicol et al . 2008), different temperatures(Hatzenpichler et al. 2008; Urakawa et al. 2008; Zhang et al.

�Fig. 4 DGGE analyses of PCR-amplified AOA amoA genes of thecontrol, acetate, and leaf powder treatments during the slurry incubation(a) and phylogenetic tree based on deduced amino acid sequences of theamoA gene sequences of ammonia-oxidizing archaea (198 amino acids)(b). The phylogenetic tree was constructed with the neighbor-joiningmethod with 1,000 bootstrapping to estimate the confidence of the treetopologies. Bootstrap values (>50 %) are indicated at the branch points.The scale bar represents 0.02 sequence divergence. The solid squares inred represent the DGGE band sequences of the present study. The solidand hollow diamonds in blue represent the clone sequences within and10 m away from the vegetated area by another study of ours

Appl Microbiol Biotechnol

2008), and different salinities (Könneke et al. 2005; Tournaet al. 2011). The widespread distribution in different habitatsindicates that different AOA have different strategies to copewith adverse conditions (Cao et al. 2013). Hence, different

AOA might have different metabolisms to adapt to variousenvironments. However, as to lifestyle, AOAmight have quitesimilar metabolism, at least in the present study. Themangroves have affected the sediments for a long time by

a

bNitrosospira

L8-aobL9-aob

Clone from SCS HQ889110L10-aob

Clone from freshwater wetland HM537770L7-aob

S0-l-1 (22/45) GQ331724S1-l-2 (19/44) GQ331637

S1-s-3 (17/44) GQ331682s0-s-1 (29/47) GQ331769S1-l-22 (1/44) GQ331657

S1-l-44 (1/44) GQ331679Nitrosomonas oligotropha AF272406

S1-s-1 (27/44) GQ331680Clone from mangrove wetland sediment HM754944Clone from Pearl River estuarine water HQ330890

S0-l-6 (23/45) GQ331729s0-s-6 (18/47) GQ331774

Nitrosomonas marina Nm22 AJ388587L4-aob

Nitrosomonas ureae AF272403S1-l-4 (23/44) GQ331639

Clone from estuarine sediment HM364222Clone from Chesapeake Bay sediment AY352942Clone from Pearl River estuarine water HQ330960Y1-a-19 from Jiaozhou Bay sediment EU244508

L3-aobClone from Pearl River estuarine sediment GU988857

L2-aobY1-a-16 from Jiaozhou Bay sediment EU244505Nitrosomonas marina HM345622

Clone from waterway canal HM589773L1-aob

Clone from anoxic granular sludge EF688276Clone from biofilm bioreactor JQ413647

Nitrosomonas eutropha AY177932Nitrosomonas sp. Nm148 AY123815Clone from marine sediment AB261465

L6-aobL5-aob

Clone from Dongjiang River sediment JN787070RSA5 from paddy soil FJ755469

Clone from freshwater wetland HM537886Clone from arable soil FN691219

Nitrosococcus oceani99

9297

92

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Clu

ster

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Clu

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Clu

ster

3C

lust

er 4

Clu

ster

5

Nitr

osos

pira

-lik

eN

itros

omon

as-l

ike

Fig. 5 DGGE analyses of PCR-amplified AOB amoA genes of thecontrol, acetate, and leaf powder treatments during the slurry incubation(a) and phylogenetic tree based on deduced amino acid sequences of theamoA gene sequences of ammonia-oxidizing bacteria (150 amino acids)(b). The phylogenetic tree was constructed with the neighbor-joiningmethod with 1,000 bootstrapping to estimate the confidence of the tree

topologies. Bootstrap values (>50 %) are indicated at the branch points.The scale bar represents 0.05 sequence divergence. The solid squares inred represent DGGE band sequences of the present study. The solid andhollow diamonds in blue represent the clone sequences within and 10 maway from vegetated area by another study in our laboratory

Appl Microbiol Biotechnol

inputting leaf litters, which probably has selected the AOAthat are adapted to this environment. As a result, AOA in thismangrove wetland have similar metabolism as to usingorganic substances. In other words, AOA composition has astrong resistance to the disturbance of organic addition(Yarwood et al. 2013). When the slurries were amended withacetate and leaf litter powder, they had concerted responses,and hence, the community structures appeared to besignificantly stable.

Effects of organic substances on AOB

All cultured AOB are obligate autotrophs (Kowalchuk andStephen 2001). When culturing AOB, the media need to befree of organic compounds, and at the same time, inhibitorsare normally necessary to prevent the growth of heterotrophicorganisms. However, cultures of AOB in laboratory normallyact differently from those in natural environments in terms ofphysiology, because in the natural environment a variety ofmicroorganisms coexist and some physiochemical factorsdifferent from those in the laboratory may exist, which couldaffect the activity of AOB. Furthermore, AOB have beenshown to utilize other energy sources such as pyruvate andhydrogen under oxygen-limited condition when ammonia isprovided (Abeliovich and Vonshak 1992; Bock et al. 1995;Stüven et al. 1992). As a result, addition of organiccompounds in the slurries may benefit AOB, although purecultures of AOB are hampered by organics (Kowalchuk andStephen 2001). In this study, addition of both acetate and leaflitter powder actually enhanced the growth of AOB. The effectof leaf litter powder was more obvious than acetate. Threeexplanations are suggested here. Firstly, organic matterbenefits AOB directly because some AOB could utilizeorganics under certain conditions. Secondly, organiccompounds could promote the growth of heterotrophicorganisms in the slurries. Some heterotrophic organismsboosted by organics may further benefit AOB, and as aconsequence, AOB could benefit from the addition of acetateand leaf litter powder indirectly. Thirdly, leaf litter powdercould provide physical surfaces as habitats for AOB in theslurries. Because of that, the boosting effect of leaf litterpowder on AOB was more pronounced than acetate in thepresent experiment.

In this experiment, organic additions affected the communitystructures of AOB significantly. For the control, although therewere some changes in the middle phase of the incubation,which might be induced by the stirring at the beginning of theslurry preparation, AOB community structures at the end of theincubation restored to the initial one. Unlike the control, thecommunity structures of amendment with acetate and leaf litterslurries at the end of the incubation became far different fromthe initial slurries; furthermore, the community structures ofslurries amended with acetate and leaf litter powder were quite

similar to each other on day 60. Previous studies showed thatAOB in the β-Proteobacteria subclass were distributed only inNitrosomonas and Nitrosospira , but the two genera include alarge quantity of species of different characteristics.Furthermore, these species span a wide range of differentenvironments and have different requirements for optimumenvironmental conditions (Kowalchuk and Stephen 2001).The effect of organics on AOB community structures maylargely come from two aspects. For one, AOB might reactdifferently to the addition of organic substances. For another,organics affected the heterotrophic organisms in the slurries,and accordingly, the physiochemical parameters of the slurrieswere also changed during the incubation (Fig. 1). Once theconditions of the slurries are altered, some dominant AOBmight lose their advantages while other minor AOB becomedominant because they benefit from the new conditions. As aresult, the addition of organics into the slurries changed thecommunity structures of AOB. As organic compounds couldchange community structures of AOB in this experiment, it isproposed that leaf litters in the mangrove wetland would alsohave an influence on AOB community structures in thesediment.

In summary, slurries, made of mangrove sediments of acoastal wetland, are more similar to the natural environmentthan bioreactors. The indigenous microorganisms coexist inthe slurry and they respond to different treatments, reflectingtheir physiological and biochemical properties. By incubationof slurries, acetate and leaf powder had an obvious effect onthe community structures and abundances of different AOA,AOB, and anammox bacteria. The noticeable responses ofAOMs in the slurry to organic additions suggested that leaflitters probably also have an influence on the AOMs inmangrove wetlands.

Acknowledgments This research was supported partially by a Ph.D.studentship from the Graduate School of The University of Hong Kong(Y-FW), Environmental and Conservation Fund grant No. 15/2011 andRGC GRF grant HKU_701913 (J-DG). Additional financial support ofthis project was by the State Key Laboratory in Marine Pollution at theCity University of Hong Kong and Environmental Toxicology Educationand Research Fund of this laboratory. We would like to thank Ms. JessieLai and Ms. Kelly Lau for technical support in chemical analysis, and Dr.Meng Li for assistance in field sampling.

References

Abeliovich A,VonshakA (1992) Anaerobicmetabolism ofNitrosomonaseuropaea . Arch Microbiol 158(4):267–270. doi:10.1007/bf00245243

Agogue H, Brink M, Dinasquet J, Herndl GJ (2008) Major gradients inputatively nitrifying and non-nitrifying Archaea in the deep NorthAtlantic. Nature 456(7223):788–791

Altschul SF, GishW,MillerW,Myers EW, Lipman DJ (1990) Basic localalignment search tool. J Mol Biol 215(3):403–410

Appl Microbiol Biotechnol

Alves RJE, Wanek W, Zappe A, Richter A, Svenning MM, Schleper C,Urich T (2013) Nitrification rates in Arctic soils are associated withfunctionally distinct populations of ammonia-oxidizing archaea.ISME J. doi:10.1038/ismej.2013.35

Auguet J-C, Nomokonova N, Camarero L, Casamayor EO (2011)Seasonal changes of freshwater ammonia-oxidizing archaealassemblages and nitrogen species in oligotrophic alpine lakes.Appl Environ Microbiol 77(6):1937–1945. doi:10.1128/aem.01213-10

Berg IA, Kockelkorn D, Ramos-VeraWH, Say RF, Zarzycki J, HüglerM,Alber BE, Fuchs G (2010) Autotrophic carbon fixation in archaea.Nat Rev Micro 8(6):447–460

Bock E, Schmidt I, Stüven R, Zart D (1995) Nitrogen loss caused bydenitrifying Nitrosomonas cells using ammonium or hydrogen aselectron donors and nitrite as electron acceptor. Arch Microbiol163(1):16–20. doi:10.1007/s002030050165

Brandes JA, Devol AH, Deutsch C (2007) New developments in themarine nitrogen cycle. Chem Rev 107(2):577–589. doi:10.1021/cr050377t

Brochier-Armanet C, Boussau B, Gribaldo S, Forterre P (2008)Mesophilic crenarchaeota: proposal for a third archaeal phylum,the Thaumarchaeota. Nat Rev Micro 6(3):245–252. doi:10.1038/nrmicro1852

Cao H, Hong Y, Li M, Gu J-D (2011a) Diversity and abundance ofammonia-oxidizing prokaryotes in sediments from the coastalPearl River estuary to the South China Sea. Antonie Leeuwenhoek100(4):545–556. doi:10.1007/s10482-011-9610-1

Cao H, Li M, Dang H, Gu J-D (2011b) Responses of aerobic andanaerobic ammonia/ammonium-oxidizing microorganisms toanthropogenic pollution in coastal marine environments. MethodEnzymol 496:35–62

Cao H, Li M, Hong Y, Gu J-D (2011c) Diversity and abundance ofammonia-oxidizing archaea and bacteria in polluted mangrovesediment. Syst Appl Microbiol 34(7):513–523. doi:10.1016/j.syapm.2010.11.023

Cao H, Hong Y, Li M, Gu J-D (2012) Community shift of ammonia-oxidizing bacteria along an anthropogenic pollution gradient fromthe Pearl River Delta to the South China Sea. Appl MicrobiolBiotechnol 94(1):247–259. doi:10.1007/s00253-011-3636-1

Cao H, Auguet J-C, Gu J-D (2013) Global ecological pattern ofammonia-oxidizing archaea. PLoS ONE 8:e52853. doi:10.1371/journal.pone.0052853

Chain P, Lamerdin J, Larimer F, Regala W, Lao V, Land M, Hauser L,Hooper A, Klotz M, Norton J, Sayavedra-Soto L, Arciero D,Hommes N, Whittaker M, Arp D (2003) Complete genomesequence of the ammonia-oxidizing bacterium and obligatechemolithoautotroph Nitrosomonas europaea . J Bacteriol 185(9):2759–2773. doi:10.1128/jb.185.9.2759-2773.2003

Chen X-P, Zhu Y-G, Xia Y, Shen J-P, He J-Z (2008) Ammonia-oxidizingarchaea: important players in paddy rhizosphere soil? EnvironMicrobiol 10(8):1978–1987

Clark I, Timlin R, Bourbonnais A, Jones K, Lafleur D,Wickens K (2008)Origin and fate of industrial ammonium in anoxic groundwater—15N evidence for anaerobic oxidation (anammox). GroundWaterMonit Remediat 28(3):73–82. doi:10.1111/j.1745-6592.2008.00206.x

Dale OR, Tobias CR, Song B (2009) Biogeographical distribution ofdiverse anaerobic ammonium oxidizing (anammox) bacteria inCape Fear River Estuary. Environ Microbiol 11(5):1194–1207

Dang H, Chen R, Wang L, Guo L, Chen P, Tang Z, Tian F, Li S, KlotzMG (2010) Environmental factors shape sediment anammoxbacterial communities in hypernutrified Jiaozhou Bay, China. ApplEnviron Microbiol 76(21):7036–7047. doi:10.1128/aem.01264-10

Francis CA, Roberts KJ, Beman JM, Alyson ES, Oakley BB (2005)Ubiquity and diversity of ammonia-oxidizing archaea in water

columns and sediments of the ocean. Proc Natl Acad Sci USA102(41):14683–14688

Hamersley MR, Woebken D, Boehrer B, Schultze M, Lavik G, KuypersMMM (2009) Water column anammox and denitrification in atemperate permanently stratified lake (Lake Rassnitzer, Germany).Syst Appl Microbiol 32(8):571–582. doi:10.1016/j.syapm.2009.07.009

Han P, Gu J-D (2013) More refined diversity of anammox bacteriarecovered and distribution in different ecosystems. Appl MicrobiolBiotechnol 97:3653–3663

Han P, Huang Y-T, Lin J-G, Gu J-D (2013) A comparison of two 16SrRNA gene-based PCR primer sets in unraveling anammox bacteriafrom different environmental samples. Appl Microbiol Biotechnol.doi:10.1007/s00253-013-5305-z

Hatzenpichler R, Lebedeva EV, Spieck E, Stoecker K, Richter A, DaimsH, Wagner M (2008) A moderately thermophilic ammonia-oxidizing crenarchaeote from a hot spring. Proc Natl Acad SciUSA 105(6):2134–2139

Head IM, Hiorns WD, Embley TM, McCarthy AJ, Saunders JR (1993)The phylogeny of autotrophic ammonia-oxidizing bacteria asdetermined by analysis of 16S ribosomal RNA gene sequences. JGen Microbiol 139(6):1147–1153. doi:10.1099/00221287-139-6-1147

Heiri O, Lotter AF, Lemcke G (2001) Loss on ignition as a method forestimating organic and carbonate content in sediments:reproducibility and comparability of results. J Paleolimnol 25(1):101–110

Hong Y-G, Li M, Cao H, Gu J-D (2011) Residence of habitat-specificanammox bacteria in the deep-sea subsurface sediments of the SouthChina Sea: analyses of marker gene abundance with physicalchemical parameters. Microb Ecol 62(1):36–47. doi:10.1007/s00248-011-9849-0

Hu Z, Lotti T, de Kreuk M, Kleerebezem R, van Loosdrecht M, Kruit J,Jetten MSM, Kartal B (2013) Nitrogen removal by a nitritation-anammox bioreactor at low temperature. Appl Environ Microbiol.doi:10.1128/aem.03987-12

Jaeschke A, Camp HJMOD, Harhangi H, Klimiuk A, Hopmans EC,Jetten MSM, Schouten S, Damsté JSS (2009) 16S rRNA gene andlipid biomarker evidence for anaerobic ammonium-oxidizingbacteria (anammox) in California and Nevada hot springs. FEMSMicrobiol Ecol 67(3):343–350

Jetten MSM, Lv N, Strous M, Kartal B, Keltjens JT, Op den Camp HJM(2009) Biochemistry and molecular biology of anammox bacteria.Crit Rev Biochem Mol Biol 44(2–3):65–84. doi:10.1080/10409230902722783

Jia Z, Conrad R (2009) Bacteria rather than Archaea dominate microbialammonia oxidation in an agricultural soil. Environ Microbiol 11(7):1658–1671. doi:10.1111/j.1462-2920.2009.01891.x

Jiang QQ, Bakken LR (1999) Comparison of Nitrosospira strainsisolated from terrestrial environments. FEMS Microbiol Ecol30(2):171–186. doi:10.1111/j.1574-6941.1999.tb00646.x

KönnekeM, Bernhard AE, Torre JRDL,Walker CB,Waterbury JB, StahlDA (2005) Isolation of an autotrophic ammonia-oxidizing marinearchaeon. Nature 437(7058):543–546

Kalvelage T, Lavik G, Lam P, Contreras S, Arteaga L, Loscher CR,Oschlies A, Paulmier A, Stramma L, Kuypers MMM (2013)Nitrogen cycling driven by organic matter export in the SouthPacific oxygen minimum zone. Nature Geosci 6(3):228–234. doi:10.1038/ngeo1739

Kartal B, Kuenen JG, van Loosdrecht MCM (2010) Sewage treatmentwith anammox. Science 328(5979):702–703. doi:10.1126/science.1185941

Kartal B, Rattray J, van Niftrik LA, van de Vossenberg J, Schmid MC,Webb RI, Schouten S, Fuerst JA, Damsté Jaap S, Jetten MSM,Strous M (2007) Candidatus “Anammoxoglobus propionicus” a

Appl Microbiol Biotechnol

new propionate oxidizing species of anaerobic ammonium oxidizingbacteria. Syst Appl Microbiol 30(1):39–49

Kartal B, Van Niftrik L, Rattray J, Van De Vossenberg JLCM, SchmidMC, Sinninghe Damsté J, Jetten MSM, Strous M (2008)Candidatus ‘Brocadia fulgida’: an autofluorescent anaerobicammonium oxidizing bacterium. FEMS Microbiol Ecol 63(1):46–55. doi:10.1111/j.1574-6941.2007.00408.x

Ke X, Angel R, Lu Y, Conrad R (2013) Niche differentiation of ammoniaoxidizers and nitrite oxidizers in rice paddy soil. Environ Microbiol15(8):2275–2292. doi:10.1111/1462-2920.12098

Ke X, Lu Y (2012) Adaptation of ammonia-oxidizing microorganisms toenvironment shift of paddy field soil. FEMSMicrobiol Ecol 80:87–97

Koops H-P, Pommerening-Röser A (2001) Distribution andecophysiology of the nitrifying bacteria emphasizing culturedspecies. FEMS Microbiol Ecol 37(1):1–9

Koper TE, Stark JM, Habteselassie MY, Norton JM (2010) Nitrificationexhibits Haldane kinetics in an agricultural soil treated withammonium sulfate or dairy-waste compost. FEMS Microbiol Ecol74(2):316–322. doi:10.1111/j.1574-6941.2010.00960.x

Kowalchuk GA, Stephen JR (2001) Ammonia-oxidizing bacteria: amodel for molecular microbial ecology. Annu Rev Microbiol55(1):485–529. doi:10.1146/annurev.micro.55.1.485

KuypersMMM, Lavik G,Woebken D, SchmidM, Fuchs BM, Amann R,Jørgensen BB, JettenMSM, Hayes JM (2005)Massive nitrogen lossfrom the Benguela upwelling system through anaerobic ammoniumoxidation. Proc Natl Acad Sci USA 102(18):6478–6483

Kuypers MMM, Sliekers AO, Lavik G, Schmid M, Jorgensen BB,Kuenen JG, Sinninghe Damsté JS, Strous M, Jetten MSM (2003)Anaerobic ammonium oxidation by anammox bacteria in the BlackSea. Nature 422(6932):608–611

Lam P, Lavik G, Jensen MM, van de Vossenberg J, Schmid M, WoebkenD, Gutiérrez D, Amann R, Jetten MSM, Kuypers MMM (2009)Revising the nitrogen cycle in the Peruvian oxygen minimum zone.Proc Natl Acad Sci USA 106(12):4752–4757. doi:10.1073/pnas.0812444106

Lehtovirta-Morley LE, Stoecker K, Vilcinskas A, Prosser JI, Nicol GW(2011) Cultivation of an obligate acidophilic ammonia oxidizer froma nitrifying acid soil. Proc Natl Acad Sci USA 108(38):15892–15897. doi:10.1073/pnas.1107196108

Leininger S, Urich T, Schloter M, Schwark L, Qi J, Nicol GW, Prosser JI,Schuster SC, Schleper C (2006) Archaea predominate amongammonia-oxidizing prokaryotes in soils. Nature 442(7104):806–809

Li H, Chen S, Mu BZ, Gu J-D (2010) Molecular detection of anaerobicammonium-oxidizing (Anammox) bacteria in high-temperaturepetroleum reservoirs. Microb Ecol 60(4):771–783

Li M, Cao H-L, Hong Y-G, Gu J-D (2011a) Seasonal dynamics ofanammox bacteria in estuarial sediment of the Mai Po NatureReserve revealed by analyzing the 16S rRNA and hydrazineoxidoreductase (hzo) genes. Microbes Environ 26(1):15–22

Li M, Cao H, Hong Y, Gu J-D (2011b) Spatial distribution andabundances of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) in mangrove sediments. Appl MicrobiolBiotechnol 89(4):1243–1254. doi:10.1007/s00253-010-2929-0

Li M, Ford T, Li X, Gu J-D (2011c) Cytochrome cd1-containingnitrite reductase encoding gene nirS as a new functionalbiomarker for detection of anaerobic ammonium oxidizing(anammox) bacteria. Environ Sci Technol 45(8):3547–3553.doi:10.1021/es103826w

Li M, Hong Y-G, Cao H-L, Gu J-D (2011d) Mangrove trees affect thecommunity structure and distribution of anammox bacteria at ananthropogenic-polluted mangrove in the Pearl River Delta reflectedby 16S rRNA and hydrazine oxidoreductase (HZO) encoding geneanalyses. Ecotoxicology 20(8):1780–1790. doi:10.1007/s10646-011-0711-4

Long A, Heitman J, Tobias C, Philips R, Song B (2013) Co-occurringanammox, denitrification, and codenitrification in agricultural soils.Appl Environ Microbiol 79(1):168–176. doi:10.1128/aem.02520-12

Martens-Habbena W, Berube PM, Urakawa H, de la Torre JR, Stahl DA(2009) Ammonia oxidation kinetics determine niche separation ofnitrifyingArchaea and Bacteria. Nature 461(7266):976–979. doi:10.1038/nature08465

Molinuevo B, García MC, Karakashev D, Angelidaki I (2009) Anammoxfor ammonia removal from pig manure effluents: effect of organicmatter content on process performance. Bioresour Technol 100(7):2171–2175

Mußmann M, Brito I, Pitcher A, Sinninghe Damsté JS, Hatzenpichler R,Richter A, Nielsen JL, Nielsen PH, Müller A, Daims H, Wagner M,Head IM (2011) Thaumarchaeotes abundant in refinery nitrifyingsludges express amoA but are not obligate autotrophic ammoniaoxidizers. Proc Natl Acad Sci USA 108(40):16771–16776. doi:10.1073/pnas.1106427108

Mulder A, van de Graaf AA, Robertson LA, Kuenen JG (1995)Anaerobic ammonium oxidation discovered in a denitrifyingfluidized bed reactor. FEMS Microbiol Ecol 16(3):177–183

Muyzer G, de Waal EC, Uitterlinden AG (1993) Profiling of complexmicrobial populations by denaturing gradient gel electrophoresisanalysis of polymerase chain reaction-amplified genes coding for16S rRNA. Appl Environ Microbiol 59(3):695–700

Nicol GW, Leininger S, Schleper C, Prosser JI (2008) The influence ofsoil pH on the diversity, abundance and transcriptional activity ofammonia oxidizing archaea and bacteria. EnvironMicrobiol 10(11):2966–2978

Nicolaisen MH, Ramsing NB (2002) Denaturing gradient gelelectrophoresis (DGGE) approaches to study the diversity ofammonia-oxidizing bacteria. J Microbiol Methods 50(2):189–203.doi:10.1016/s0167-7012(02)00026-x

Nold SC, Zhou J, Devol AH, Tiedje JM (2000) Pacific Northwest marinesediments contain ammonia-oxidizing bacteria in the β subdivisionof the Proteobacteria . Appl Environ Microbiol 66(10):4532–4535.doi:10.1128/aem.66.10.4532-4535.2000

Okano Y, Hristova KR, Leutenegger CM, Jackson LE, Denison RF,Gebreyesus B, Lebauer D, Scow KM (2004) Application of real-time PCR to study effects of ammonium on population size ofammonia-oxidizing bacteria in soil. Appl Environ Microbiol 70(2):1008–1016. doi:10.1128/aem.70.2.1008-1016.2004

Park SJ, Park BJ, Rhee SK (2008) Comparative analysis of archaeal 16SrRNA and amoA genes to estimate the abundance and diversity ofammonia-oxidizing archaea in marine sediments. Extremophiles12(4):605–615

Penton CR, Devol AH, Tiedje JM (2006) Molecular evidence for thebroad distribution of anaerobic ammonium-oxidizing bacteria infreshwater and marine sediments. Appl Environ Microbiol 72(10):6829–6832. doi:10.1128/aem.01254-06

Pester M, Schleper C, Wagner M (2011) The Thaumarchaeota: anemerging view of their phylogeny and ecophysiology. CurrOpin Microbiol 14(3):300–306. doi:10.1016/j.mib.2011.04.007

Purkhold U, Pommerening-Röser A, Juretschko S, Schmid MC, KoopsH-P, Wagner M (2000) Phylogeny of all recognized species ofammonia oxidizers based on comparative 16S rRNA and amoAsequence analysis: implications for molecular diversity surveys.Appl Environ Microbiol 66(12):5368–5382. doi:10.1128/aem.66.12.5368-5382.2000

Quan Z-X, Rhee S-K, Zuo J-E, Yang Y, Bae J-W, Park JR, Lee S-T, ParkY-H (2008) Diversity of ammonium-oxidizing bacteria in a granularsludge anaerobic ammonium-oxidizing (anammox) reactor. EnvironMicrobiol 10(11):3130–3139

Rysgaard S, Glud RN (2004) Anaerobic N2 production in Arctic sea ice.Limnol Oceanogr 49(1):86–94

Appl Microbiol Biotechnol

Sabumon PC (2007) Anaerobic ammonia removal in presence of organicmatter: a novel route. J Hazard Mater 149(1):49–59

Schleper C, Jurgens G, Jonuscheit M (2005) Genomic studies ofuncultivated archaea. Nat Rev Micro 3(6):479–488

Schmid M, Twachtmann U, Klein M, Strous M, Juretschko S, Jetten M,Metzger JW, Schleifer K-H, Wagner M (2000) Molecular evidencefor genus level diversity of bacteria capable of catalyzing anaerobicammonium oxidation. Syst Appl Microbiol 23(1):93–106. doi:10.1016/s0723-2020(00)80050-8

Schmid M, Walsh K, Webb R, Rijpstra WI, van de Pas-Schoonen K,Verbruggen MJ, Hill T, Moffett B, Fuerst J, Schouten S, DamstéJSS, Harris J, Shaw P, Jetten M, Strous M (2003) Candidatus“Scalindua brodae”, sp. nov., Candidatus “Scalindua wagneri”, sp.nov., two new species of anaerobic ammonium oxidizing bacteria.Syst Appl Microbiol 26(4):529–538

Schubert CJ, Durisch-Kaiser E, Wehrli B, Thamdrup B, Lam P, KuypersMMM (2006) Anaerobic ammonium oxidation in a tropicalfreshwater system (Lake Tanganyika). Environ Microbiol 8(10):1857–1863

Singh JN, Mudgal V (2000) Assessment of mineral content of tree leaflitter of Nokrek biosphere reserve and its impact on soil properties.Trop Ecol 41(2):225–232

Smith TJ, Boto KG, Frusher SD, Giddins RL (1991) Keystone speciesand mangrove forest dynamics: the influence of burrowing by crabson soil nutrient status and forest productivity. Estuar Coast Shelf Sci33(5):419–432

Stüven R, Vollmer M, Bock E (1992) The impact of organic matter onnitric oxide formation by Nitrosomonas europaea . Arch Microbiol158(6):439–443. doi:10.1007/bf00276306

Stein LY, Arp DJ, Berube PM, Chain PSG, Hauser L, Jetten MSM, KlotzMG, Larimer FW, Norton JM, Camp HJMOD, Shin M, Wei X(2007) Whole-genome analysis of the ammonia-oxidizingbacterium, Nitrosomonas eutropha C91: implications for nicheadaptation. Environ Microbiol 9(12):2993–3007

StrousM, Fuerst JA, Kramer EHM, Logemann S, Muyzer G, van de Pas-Schoonen KT, Webb R, Kuenen JG, Jetten MSM (1999) Missinglithotroph identified as new planctomycete. Nature 400(6743):446–449

Strous M, Pelletier E, Mangenot S, Rattei T, Lehner A, Taylor MW, HornM, Daims H, Bartol-Mavel D, Wincker P, Barbe V, Fonknechten N,Vallenet D, Segurens B (2006) Deciphering the evolution andmetabolism of an anammox bacterium from a community genome.Nature 440(7085):790

Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011)MEGA5: molecular evolutionary genetics analysis using maximumlikelihood, evolutionary distance, and maximum parsimonymethods. Mol Biol Evol 28(10):2731–2739. doi:10.1093/molbev/msr121

Thamdrup B (2013) Oceanography: coastal oceanic nitrogen loss. NatureGeosci 6(3):160–161

TournaM, Stieglmeier M, Spang A, KönnekeM, Schintlmeister A, UrichT, Engel M, Schloter M, Wagner M, Richter A, Schleper C (2011)Nitrososphaera viennensis, an ammonia oxidizing archaeon fromsoil. Proc Natl Acad Sci USA 108(20):8420–8425. doi:10.1073/pnas.1013488108

Urakawa H, Tajima Y, Numata Y, Tsuneda S (2008) Low temperaturedecreases the phylogenetic diversity of ammonia-oxidizing archaeaand bacteria in aquarium biofiltration systems. Appl EnvironMicrobiol 74(3):894–900. doi:10.1128/aem.01529-07

van de Vossenberg J, Woebken D, Maalcke WJ, Wessels HJCT, DutilhBE, Kartal B, Janssen-Megens EM, Roeselers G, Yan J, Speth D,Gloerich J, GeertsW, van der Biezen E, PlukW, Francoijs K-J, Russ

L, Lam P, Malfatti SA, Tringe SG, Haaijer SCM, Op den CampHJM, Stunnenberg HG, Amann R, Kuypers MMM, Jetten MSM(2013) The metagenome of the marine anammox bacterium‘Candidatus Scalindua profunda’ illustrates the versatility of thisglobally important nitrogen cycle bacterium. Environ Microbiol15(5):1275–1289. doi:10.1111/j.1462-2920.2012.02774.x

Venter JC, Remington K, Heidelberg JF, Halpern AL, Rusch D, Eisen JA,Wu D, Paulsen I, Nelson KE, Nelson W, Fouts DE, Levy S, KnapAH, Lomas MW, Nealson K, White O, Peterson J, Hoffman J,Parsons R, Baden-Tillson H, Pfannkoch C, Rogers Y-H, SmithHO (2004) Environmental genome shotgun sequencing of theSargasso Sea. Science 304(5667):66–74. doi:10.1126/science.1093857

Verhamme DT, Prosser JI, Nicol GW (2011) Ammonia concentrationdetermines differential growth of ammonia-oxidising archaea andbacteria in soil microcosms. ISME J 5(6):1067–1071. doi:10.1038/ismej.2010.191

Walker CB, de la Torre JR, Klotz MG, Urakawa H, Pinel N, Arp DJ,Brochier-Armanet C, Chain PSG, Chan PP, Gollabgir A, Hemp J,Hügler M, Karr EA, Könneke M, Shin M, Lawton TJ, Lowe T,Martens-Habbena W, Sayavedra-Soto LA, Lang D, Sievert SM,Rosenzweig AC, Manning G, Stahl DA (2010) Nitrosopumilusmaritimus genome reveals unique mechanisms for nitrificationand autotrophy in globally distributed marine crenarchaea. ProcNatl Acad Sci USA 107(19):8818–8823. doi:10.1073/pnas.0913533107

Wang J, Gu J-D (2012) Dominance of Candidatus Scalindua species inanammox community revealed in soils with different duration ofrice paddy cultivation in Northeast China. Appl MicrobiolBiotechnol 97(4):1785–1798. doi:10.1007/s00253-012-4036-x

Wang Y-F, Feng Y-Y, Ma X, Gu J-D (2013) Seasonal dynamics ofammonia/ammonium-oxidizing prokaryotes in oxic and anoxicwetland sediments of subtropical coastal mangrove. ApplMicrobiol Biotechnol 97(17):7919–7934. doi:10.1007/s00253-012-4510-5

Wang Y-F, Gu J-D (2013) Higher diversity of ammonia/ammonium-oxidizing prokaryotes in constructed freshwater wetland thannatural coastal marine wetland. Appl Microbiol Biotechnol 97:7015–7033. doi:10.1007/s00253-012-4430-4

Whitby CB, Saunders JR, Rodriguez J, Pickup RW, McCarthy A (1999)Phylogenetic differentiation of two closely related Nitrosomonasspp. that inhabit different sediment environments in an oligotrophicfreshwater lake. Appl Environ Microbiol 65(11):4855–4862

Winogradsky S (1890) Investigations on nitrifying organisms. Ann InstPasteur 4:213–321

Yang G-F, Zhang Q-Q, Jin R-C (2013) Changes in the nitrogen removalperformance and the properties of granular sludge in an Anammoxsystem under oxytetracycline (OTC) stress. Bioresour Technol 129:65–71

Yarwood S, Brewer E, Yarwood R, Lajtha K, Myrold D (2013) Soilmicrobe active community composition and capability ofresponding to litter addition after 12 years of no inputs. ApplEnviron Microbiol 79(4):1385–1392. doi:10.1128/aem.03181-12

Zhang CL, Ye Q, Huang Z, Li W, Chen J, Song Z, Zhao W, Bagwell C,Inskeep WP, Ross C, Gao L, Wiegel J, Romanek CS, Shock EL,Hedlund BP (2008) Global occurrence of archaeal amoA genes interrestrial hot springs. Appl Environ Microbiol 74(20):6417–6426.doi:10.1128/aem.00843-08

Zhang Y, Ruan X-H, Op den Camp HJM, Toine JM S, Mike SM J,Schmid MC (2007) Diversity and abundance of aerobic andanaerobic ammonium-oxidizing bacteria in freshwater sedimentsof the Xinyi River (China). Environ Microbiol 9(9):2375–2382

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