ORIGINAL ARTICLE

Anaerobic Ammonium Oxidation (Anammox) in ChesapeakeBay Sediments

Jeremy J. Rich & Olivia R. Dale & Bongkeun Song &Bess B. Ward

Received: 5 December 2006 /Accepted: 21 May 2007 / Published online: 7 July 2007# Springer Science + Business Media, LLC 2007

Abstract Anaerobic ammonium oxidation (anammox) hasrecently been recognized as a pathway for the removal offixed N from aquatic ecosystems. However, the quantitativesignificance of anammox in estuarine sediments is variable,and measurements have been limited to a few estuaries. Wemeasured anammox and conventional denitrification activ-ities in sediments along salinity gradients in the ChesapeakeBay and two of its sub-estuaries, the Choptank River andPatuxent River. Homogenized sediments were incubatedwith 14/15N amendments of NH4 , NO

3 , and NO

2 to

determine relative activities of anammox and denitrifica-tion. The percent of N2 production due to anammox (ra%)ranged from 0 to 22% in the Chesapeake system, with thehighest ra% in the freshwater portion of the main stem ofupper Chesapeake Bay, where water column NO3 concen-trations are consistently high. Intermediate levels of relativeanammox (10%) were detected at locations correspondingto tidal freshwater and mesohaline locations in theChoptank River, whereas anammox was not detected inthe tidal freshwater location in the Patuxent River.Anammox activity was also not detected in the seawardend of Chesapeake Bay, where water column NO3concentrations are consistently low. The ra% did not

correlate with NH4 accumulation rate in anoxic sedimentincubations, but ra% was related to water column NO3concentrations and salinity. Anammox bacterial communi-ties were also examined by amplifying DNA extracted fromthe upper Chesapeake Bay sediment with polymerase chainreaction (PCR) primers that are specific for 16S rRNAgenes of anammox organisms. A total of 35 anammox-likesequences were detected, and phylogenetic analysisgrouped the sequences in two distinct clusters belongingto the Candidatus Scalindua genus.

Introduction

Human activities have approximately doubled terrestrialnitrogen inputs on a global scale through production ofsynthetic fertilizers and emission of NOx from fossil fuelcombustion [12]. In the United States, 25% of anthropo-genic N inputs are exported to coastal areas [14]. Couplingof nitrification and denitrification has long been assumed tobe the primary mechanism of fixed N removal fromecosystems. It has become clear over the last decade,however, that an entirely novel process, known as anaero-bic ammonium oxidation (anammox), may be an importantloss term in some systems [6, 23].

Anammox was first reported in wastewater systems [29,52], and the process is defined as the oxidation ofammonium NH4

with nitrite NO2

, in the absence of

O2, in the following reaction:

NO2 NH4 ! N2 2H2OSimilar reactions were proposed earlier, based on

thermodynamic calculations [2] and nutrient profiles inanoxic marine basins [32] or sediments [1], but the process

Microb Ecol (2008) 55:311320DOI 10.1007/s00248-007-9277-3

J. J. Rich (*) : B. B. WardDepartment of Geosciences, Princeton University,Princeton, New Jersey, USAe-mail: jjrich@princeton.edu

O. R. Dale :B. SongDepartment of Biology and Marine Biology,University of North Carolina at Wilmington,Wilmington, North Carolina, USA

Present address:J. J. RichCenter for Environmental Studies, Brown University,Providence, Rhode Island, USA

was first documented in wastewater bioreactors [29, 52]. Inaddition, the oxidation of NH4 to N2 by MnO2 reduction inmarine sediments has been proposed [15, 27], but evidencein support of a biological basis for this mechanism islacking [47].

Anammox activity was first reported in natural systemsin sediments of the BalticNorth Sea transition [48], whereanammox accounted for up to 67% of anaerobic N2production. Anammox activity has since been detected inother continental margin sediments [8, 36], estuarine sedi-ments [28, 33, 45, 50], anoxic marine waters [5, 22, 23,49], and anoxic tropical freshwater [40]. Incubations ofhomogenized sediment, amended with 15N, are typicallyused to determine relative rates of anammox and denitrifi-cation. Although this approach provides potential rates,measurements made with homogenized sediments appear toapproximate or underestimate the percent of N2 productiondue to anammox compared to measurements made withintact cores [51]. Based on slurry experiments, the percentof N2 flux due to anammox is estimated at 026% inestuarine sediments [28, 33, 50]. Anammox thus appears tobe ecologically significant in some estuarine sediments, butits contribution is variable, and reported measurementshave been limited to a few estuaries.

Most of what is known about the microbiology ofanammox bacteria comes from studies of highly enrichedcultures from wastewater treatment systems [16, 42]. Theorganisms grow extremely slowly (minimum generationtime of 11 days), and no pure culture has been isolated.Analysis of 16S rRNA genes has revealed that anammoxorganisms form a deeply branching monophyletic group ofat least four candidate genera, including, Brocadia,Kuenenia, Scalindua, and Anammoxoglobus [19,39, 42]. Cells related to the anammox genus of Scalinduahave been found in environmental samples with anammox

activity, including the Black Sea [22], off the coast ofAfrica [23], and estuarine sediments [33]. Kuypers et al.[23] found a strong correlation between abundance ofScalindua cells and anammox activity in the Africancoast system. Penton et al. [31] detected 16S rRNAsequences related to Scalindua in a variety of soils andsediments, including deep ocean, freshwater wetlands, andpermafrost.

In this study, our objective was to examine anammoxactivities in sediments from different locations in the largestestuary in the United States, the Chesapeake Bay, and twoof its sub-estuaries, the Choptank River and Patuxent River.We also used polymerase chain reaction (PCR) to amplify16S rRNA genes that are specific to known anammoxbacteria from the sediments. Tal et al. [45] found evidencefor the anammox process in sediments of Baltimore Harbor,a branch of the Chesapeake Bay system. However, thepotential quantitative significance of anammox in theChesapeake Bay ecosystem has not been reported, and thisinformation is relevant to understanding benthic N cyclingprocesses. Our work also contributes to the growing bodyof literature on the potential regulating factors affectinganammox.

Methods

Sample Locations and Collection

The five sample locations spanned salinity and NO3 gradientsin the Chesapeake Bay and two adjacent sub-estuaries, theChoptank River and Patuxent River. Hydrographic andnutrient conditions on the various sampling dates are givenin Table 1. Temperature, salinity, and oxygen concentrationswere measured by CTD, and NH4 and NO

3 were measured

Table 1 Site characteristics

Estuary Descriptiona Site Depth(m)

Datesampled

Bottom water

Temperature(C)

Salinity NH4 M NO3 M O2 (mg/L)

ChesapeakeBay

Upper Bay, tidalfresh

CB1 10 Jul-2004 25.3 2.5 9.8 74.4 8.0May-2005 15.0 0.4 ND 93.6 7.9

Lower Bay,polyhaline

CB3 11 Jul-2004 24.1 19.7 2.7 0.4 3.8Oct-2004 22.9 18.6 1.7 0.5 5.6

ChoptankRiver

Upper, tidal fresh CT1 5 Jun-2004 26.9 1.0 1.0 70.7 7.2Mar-2005 10.2 0.1 ND 169.7 8.4

Lower, mesohaline CT2 7 Jun-2004 24.6 10.7 4.4 11.6 7.6Mar-2005 7.8 11.0 ND 28.0 6.5

PatuxentRiver

Upper, tidal fresh PR 10 Nov-2004 7.5 0.1 4.5 38.1 8.3

a Details of site locations are found in Francis et al. [10], except the Patuxent River site (3841.03 N and 7641.46 W)ND Not determined

312 J.J. Rich et al.

with an autoanalyzer or manually, using standard colorimetrictechniques. The sites have been thoroughly characterized byCowan and Boynton [3] and the Chesapeake Bay monitoringprogram (http://www.chesapeakebay.net/wquality.htm). Bot-tom water is characterized by high NO3 concentrations(30100 M) and low salinity (010 ppt) at the upperChesapeake Bay station (CB1), and higher salinity (19 ppt)and low NO3 (0.2-6 M) in lower Chesapeake Bay (CB3).Tidal freshwater locations in the Choptank River andPatuxent River (CT1 and PR) experience high NO3 inwinter and spring (50300 M) and lower NO3 in earlyfall (150 M), with incursions of saline water in summerand fall in some years (

values of 15N-atom% of NH4 , based on differences beforeand after 15NH4 addition, and consequently, we do notreport actual levels of anammox in the 15NH4 14 NO3treatment.

Statistical Analyses of Activity Data

Using Excel software (Microsoft Corp., Redmond, Wash-ington), two-sample t tests were used to test for differencesin 29N2 production between 15NH

4 and

15NH4 14 NO3additions, on any given sampling date and location. Paired ttests were used to test for differences in ra% between15NO3 and

15NO2 additions. Using Jmp (SAS Institute,Inc., Cary, North Carolina), linear regression was used totest for correlations between ra% and the bottom watervariables and NH4 accumulation in sediment incubations.The NO3 data were ln-transformed because these dataspanned two orders of magnitude, whereas the othervariables were ln-transformed for comparison.

Extraction of DNA and PCR Amplification

16S rRNA genes were examined to identify whichanammox organisms were present in Chesapeake Baysediments. The CB1 site was selected for this purposebecause anammox activity was most reliably detected atthis location. DNA was extracted from sediments collectedfrom the CB1 site using the MoBio Power Soil DNA Kit,following the manufacturers instructions (MoBio Labora-tories, Inc., Carlsbad, California). A nested PCR approachwas used to detect anammox bacterial 16S rRNA genesequences in these sediments. First, amplification ofPlanctomycetales-specific 16S rRNA genes was done byusing the Pla46 primer (5-GGATTAGGCATGCAAGTC-3) [30] and the 1392r universal bacterial primer (5-GACGGGCGGTGTGTACAA-3) [9]. Next, an anammoxspecific 16S PCR was performed by using the Pla46forward primer and the Amx368r pr imer (5 -CCTTTCGGGCATTGCGAA-3) [39] with 1 l of PCRproduct from the previous reaction as template. Each PCR(25 l) contained 2.5 l 10 PCR buffer, 2.5 l MgCl2(25 mM), 0.2 l Taq (Promega, Madison, Wisconsin),0.2 l deoxynucleoside triphosphates (0.8 mM of eachnucleotide), and 1 l of each primer (400 pmol), and 1 lof DNA as template (10 to 100 ng). The reaction cycleparameters of the first PCR included an initial denaturationstep of 4 min at 94C, followed by 40 cycles of ampli-fication; each cycle consisted of denaturation at 94C for45 s, primer annealing at 59C for 50 s, and primerextension at 72C for 3 min. The reaction cycle parametersof the second PCR were the same, except the secondcycles primer extension step was 72C for 1 min. The size

of the PCR product was determined by using gel elec-trophoresis with a 1% (wt/vol) agarose gel and 1 SigmaTAE buffer (Sigma-Aldrich Co., St. Louis, Missouri).

Cloning, Sequencing, and Phylogenetic Analysis

PCR-amplified DNA fragments of the correct size (approx-imately 320 bp) were excised from the gel using theEppendorf Perfect Gel Clean-Up Kit, following the manu-facturers instructions (Eppendorf, Brinkmann InstrumentsInc., USA). Purified DNA fragments were introduced into apCR2A vector and transformed into Escherichia coli byusing a TOPO TA cloning kit, as instructed by themanufacturer (Invitrogen, Carlsbad, California). Clonedinserts were verified by PCR amplification and sequencingwith the ABI 3100 automated sequencer (Applied Biosys-tems, Foster City, California). DNA sequences were exam-ined and edited using DNASTAR Lasergene SeqManProgram (DNASTAR, Inc., Madison, Wisconsin). NCBIBLAST (http://www.ncbi.nih.gov) was used to find the mostclosely related 16S rRNA gene sequences in the publicdatabases. The partial 16S rRNA gene sequences werealigned using ClustalW (http://www.ebi.ac.uk/clustalw/).Neighbor-joining phylogenetic trees were produced by usingthe Kimura-2 parameter method in PAUP * 4.0b10 softwareprogram [44]. Bootstrapping (100 replicates) was used toestimate the reproducibility of the trees. The 16S rRNAsequences from Chesapeake Bay have been deposited inGenBank with accession numbers EF653646-EF653680.

Results

15N Experiments

Nine endpoint experiments (24 h) were performed(Table 2). With the addition of 15NO3 , production of

29N2and 30N2 was detected in sediments from all locations, andmore 30N2 was produced than

29N2 (Table 2). Very little29N2

was detected in the presence of 15NH4 without added NO3 .

With addition of 15NH4 14 NO3 , significant 29N2 pro-duction was detected in sediments from CB1 and theChoptank River. At CB3 and the Patuxent River site, therewas little 29N2 production from 15NH

4 , regardless of

14NO3addition, indicating no anammox activity at these locations.

Patterns of 29N2 or30N2 accumulation in 30 min

incubations of CB1 sediment (Fig. 1) were similar to thoseobserved in endpoint experiments. Production of 15NN2from added 15NO3 or

15NH4 14 NO3 was linear duringthe incubations, and production of 15NN2 from added15NH4 was dependent on the addition of

14NO3 (Fig. 1).The percent of anammox plus denitrification (total N2

production) due to anammox (ra%) ranged from 0 to 22%

314 J.J. Rich et al.

(Table 2). There was no difference in ra% whether 15NO3or 15NO2 was added (P value=0.49, paired t test). Thehighest mean ra of 22% was found at CB1, in July 2004.Based on linear regression of 29N2 and

30N2 production inCB1 samples (Fig. 1) and Fn, individual rates were 8 nmolN cm3 h1 for anammox and 31 nmol N cm3 h1 fordenitrification; these rates yielded a slightly higher mean raof 21% than in 24 h incubations (154%, Table 2), but thisdifference was not significant (P value=0.11, two-sample ttest).

The ra% was negatively correlated with salinity andpositively correlated with the ln of NO3 concentration inthe bottom water (Table 3). Salinity and NO3 also werenegatively correlated (r=0.97, P value=0.002; salinitycorrelated with ln NO3 ). The ra% did not correlate with netNH4 accumulation in the 24 h sediment incubations or theother tested variables (Table 3).

Molecular Detection of Anammox Bacteria in ChesapeakeBay

Nested PCR of 16S rRNA genes, specific for anammoxbacteria, yielded the expected 320 bp fragment fromDNA extracted from the upper Chesapeake Bay (CB1)sediment. Sequencing of 320 bp fragments showed that35 out of 40 clones were identified as anammox-like,based on BLAST searches and phylogenetic analysis. All35 of the sequences grouped within the CandidatusScalindua genus (Fig. 2). GenBank accession numbersand identification of the reference sequences in Fig. 2 arepresented in Table 4. Within the major Chesapeake Baygroup, two distinct clusters of sequences were found:Twenty-one clones formed a cluster with CandidatusScalindua wagneri, while 13 clones formed a cluster

with Candidatus Scalindua brodae and Scalinduasorokinii (Fig. 2). The average sequence similaritybetween the Chesapeake sequences and the closestmatching Scalindua sequence was 95%, with the excep-tion of one additional sequence (CB1_AMX46-2) thatgrouped outside the two main clusters but shared anaverage of 92% similarity with Candidatus S. brodaeand S. Sorokinii.

Discussion

Potential Anammox and Denitrification Activitiesin Chesapeake Bay Sediments

The discovery of anammox in natural environments hasprompted a re-evaluation of N2 production in estuarinesediments. Incubations of homogenized sediments,amended with inorganic 15N, have been typically used toquantify the potential contribution of anammox to total N2production (i.e., ra%). Our work extends measurements ofra% in estuarine sediments to North America and contrib-utes to an understanding of some of the factors that mayregulate anammox.

Our results show similar patterns in 15NN2 productionto previous studies of anammox activity in sediments. Inslurries amended with 15NH4 14 NO3 , 29N2 productionand lack of 30N2 production indicated 1:1 pairing of Natoms (Table 2), and that nitrate must have been reduced toNO2 before conversion to N2, which is diagnostic of theanammox reaction and typical for sediment experiments[33, 50]. Immediate and linear production of 15NN2 in30 min time-course experiments indicated that activeanammox organisms and denitrifiers were present in the

Table 2 Production of 29N2 or30N2 [meanSD, nmol

29N2 or30N2 cm

3 sediment] in 24 h incubations, in the presence of 15NO3 14NH4 ,15NH4 , or

15NH4 14 NO3 and the percent of N2 production as anammox (ra%)Sitea Date Number (n)b

15NO3 14 NH4 15NH4 15NH4 14 NO3 Fn%d ra%29N2

30N229N2

30N229N2

30N2

CB1 Jul-2004 3 164 354 0.070.03 0.080.10 3.81.9c 0.090.12 933 226May-2005 3 91 342 0.080.04 0.030.02 2.60.1c 0.040.01 961 154

CB3 Jul-2004 2 2.50.2 343 0.010.01 0.070.02 0.030.002 0.070.01 954 38Oct-2004 3 51 405 0.060.02 0.050.01 0.080.02 0.050.03 942 12

CT1 Jun-2004 2 5.80.1 401 0.010.004 0.020.02 0.390.01c 0.0020.01 ND NDMar-2005 2 4.70.5 374 0.0010.01 0.040.03 0.050.01c 0.040.03 990.1 100.1

CT2 Jun-2004 2 7.20.7 391 0.0020.01 0.040.04 1.10.3c 0.060.04 ND NDMar-2005 2 103 421 0.100.13 0.040.06 0.80.3c 0.010.003 971 107

PR Nov-2004 3 0.80.03 271 0.020.02 0.050.04 0.020.02 0.050.04 ND ND

aCB1 Chesapeake Bay upper, CB3 Chesapeake Bay lower, CT1 Choptank River upper, CT2 Choptank River lower, PR Patuxent Riverb n The number of homogenized sediment samples analyzed from each locationc Statistical differences in 29 N2 production between 15NH

4 and

15NH4 14 NO3 (P value

Chesapeake Bay sediment (Fig. 1). Intriguingly, rates ofNO3 reduction to NO

2 do not limit anammox activity in

this and other estuarine and marine sediments, asdemonstrated by the fact that NO3 and NO

2 additions

result in the same ra% (this study and [4, 33, 50]).Assuming that NO2 is a freely diffusible intermediate andNO3 reduction rates are high in estuarine sediments, it isnot necessary to postulate that anammox organisms alsoperform the NO3 reduction step. However, some anam-mox organisms are capable of coupling NO3 reduction tothe oxidation of certain organic acids [13, 19]), and Strous

et al. [43] identified a putative homolog of the respiratoryNO3 reductase gene, narG in the genome of the anammoxbacterium Kuenenia stuttgartiensis.

Slurry incubations, as employed in this study, can beuseful for quantifying potential mechanisms of N2 produc-tion, but this approach perturbs the natural chemicalgradients and spatial arrangement of organisms carryingout N cycling processes in situ. Whether slurry measure-ments yield artificially high or low ra% values is thus aconcern. Trimmer et al. [51] recently compared rates ofanammox and denitrification in slurries and intact cores. Insediments with low anammox activity (ra5%), ra% was about 1015%higher in intact cores than in slurries [51]. Consequently,our slurry measurements probably accurately assessed thepresence or absence of anammox activity, but the actual ra% in sediments with detectable anammox may have beenunderestimated.

In studies that have examined anammox activity alongsalinity gradients in estuaries, ra% generally decreaseswith increasing salinity [28, 50]. Similarly, we found lowra% in the seaward end of Chesapeake Bay. Anammoxactivity occurred most reliably at high concentrations ofNO3 in the tidal freshwater part of Chesapeake Bay (CB1)and was absent in the saline part of Chesapeake Bay(CB3), where NO3 concentrations are consistently low.The pattern that we observed in ra% could be partlyexplained by variation in bottom water NO3 concentra-tions, salinity, or both (Table 3). As salinity and NO3usually covary in this and other estuarine ecosystems, it isdifficult to assess their independent effects. Increasedsalinity is known to directly inhibit nitrification anddenitrification [35], but salinity effects on anammox inestuarine sediments have not been reported. There isevidence, however, that an abundant and stable supply ofNO3 in the anoxic zone is necessary to maintain activeanammox populations, and it has been hypothesized thatvariation in NO3 availability plays a role in regulatinganammox activity [28, 34, 36]. We similarly hypothesizethat variation in NO3 availability is a key factorregulating ra% in sediments in the Chesapeake Bayecosystem. This hypothesis needs more rigorous testing,however, as our dataset is limited and the effects ofsalinity or other factors have not been ruled out.

Anammox-Specific 16S rRNA Sequences in ChesapeakeBay Sediments

On the basis of 16S rRNA sequence analysis, the upperChesapeake Bay sediment community contains at leastthree distinct anammox bacterial species in the candi-date genus Scalindua and these are distinct from the

Time (min)0 10 20 30

nm

ol N

2 cm

-3

sedi

men

t

0.0

0.1

0.2

0.3

0.4

nm

ol N

2 cm

-3

sedi

men

t

0.0

0.1

0.2

0.3

0.4

nm

ol N

2 cm

-3

sedi

men

t

0

2

4

6

8

29N230N2

+15NO3-

14NH4+

+15NH4+

+15NH4+

14NO3-

Figure 1 Production of 29N2 and30N2 in incubations of homogenized

sediment amended with 15=14N NO3 or 15=14N NH4 , from theupper Chesapeake Bay site (CB1), May 2005. Each symbol representsthe mean1 SE (n=2)

316 J.J. Rich et al.

currently known Scalindua species. Within the twoclusters of Chesapeake Bay anammox bacteria, severalsubclusters were observed, which might represent multipleecotypes of Scalindua-like bacteria in the sedimentcommunity.

The primers we used match sequences present in allknown anammox bacteria, indicating that other anammoxgenera were either absent or below the detection limit ofthe PCR assay. Our PCR assay, however, was notcompletely specific for anammox bacteria, as 12.5% ofour sequenced clones did not match known anammoxsequences. Sequencing PCR products is therefore neces-

sary when using our PCR approach to analyze thedistribution and diversity of anammox organisms.

Although all known anammox bacteria share the samefundamental morphological and physiological traits central tothe anammox metabolism, species of anammox bacteria maydiffer in ecologically meaningful ways, such as differentialutilization of certain organic acids [19]. Most of the otherecological aspects of anammox bacteria are not known,however. Intriguingly, only Scalindua-like sequences havebeen detected in non-wastewater environments and the otheranammox genera have gone undetected, despite deepphylogenetic branching in the anammox lineage.

Figure 2 Phylogenetic tree of16S rRNA gene sequencesobtained from the upperChesapeake Bay site (CB1prefix)

Anammox in Chesapeake Bay 317

Conclusions

Denitrification activity was present throughout the ChesapeakeBay ecosystem, whereas anammox activity was not nearly soubiquitous. The presence of Scalindua-like sequences inChesapeake Bay sediment further implicates this group ashaving a global role in the anammox process. Anammoxactivity in estuarine sediments appears linked to variation in

NO3 concentrations in bottom waters (this study and [28, 34,50]]) but effects of salinity and other factors have not beenruled. The variability in ra% over time and space asdemonstrated even in our relatively small dataset is consistentwith the dynamic nature of the Chesapeake system and is acompelling reason for further studies of anammox anddenitrification in this and other estuaries. The presence ofanammox bacterial communities in estuarine sedimentsindicates a niche for these organisms that was otherwiseassumed to be occupied by conventional denitrifiers, therebyproviding insights into NOx consumption processes, ingeneral, and factors that regulate anammox activity.

Acknowledgements This work was supported by the NSF Micro-bial Biology Fellowship program (DBI-0301308 to JJR) and the NSFBiocomplexity program (OCE 99-81482 to BBW). We thank theBiocomplexity team for shiptime, supplying some of the nutrientdata, and assistance, particularly J. Alexander, J. Cornwell, and M.Owens. We are also indebted to D. A. Bronk, R. Mason, and T.Jordan for shiptime; T. Jordan provided nutrient data for the PatuxentRiver, as well. We thank T. Dalsgaard, N. Risgaard-Petersen, L.Nielsen, B. Thamdrup, M. Jensen, J. Nicholls, C. Davies, and M.Trimmer for methodological advice and helpful discussions.

Table 3 Correlations between mean ra% and some of the variablesmeasured in this study

Variablea R P value

Temperature 0.17 0.75ln(temperature) 0.17 0.75Salinityb 0.84 0.04ln(salinity) 0.52 0.29NO3 0.55 0.26ln NO3 b

0.86 0.03NH4 accumulation 0.51 0.31ln NH4 accumulation

0.33 0.52

a The variables are for the bottom water at each site, except for NH4accumulation in sediment incubations.

b Significant correlations (P value

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320 J.J. Rich et al.

Anaerobic Ammonium Oxidation (Anammox) in Chesapeake Bay SedimentsAbstractIntroductionMethodsSample Locations and Collection15N Sediment Experiments15/14N Analyses and CalculationsStatistical Analyses of Activity DataExtraction of DNA and PCR AmplificationCloning, Sequencing, and Phylogenetic Analysis

Results15N ExperimentsMolecular Detection of Anammox Bacteria in Chesapeake Bay

DiscussionPotential Anammox and Denitrification Activities in Chesapeake Bay SedimentsAnammox-Specific 16S rRNA Sequences in Chesapeake Bay Sediments

ConclusionsReferences

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