Microb Ecol (2008) 55:311–320 DOI 10.1007/s00248-007-9277-3

ORIGINAL ARTICLE

Anaerobic Oxidation () in Chesapeake Bay 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

þ Abstract Anaerobic ammonium oxidation (anammox) has correlate with NH4 accumulation rate in anoxic sediment recently been recognized as a pathway for the removal of incubations, but ra% was related to water column NO3 fixed N from aquatic . However, the quantitative concentrations and salinity. Anammox bacterial communi- significance of anammox in estuarine sediments is variable, ties were also examined by amplifying DNA extracted from and measurements have been limited to a few . We the upper Chesapeake Bay sediment with polymerase chain measured anammox and conventional activ- reaction (PCR) primers that are specific for 16S rRNA ities in sediments along salinity gradients in the Chesapeake genes of anammox organisms. A total of 35 anammox-like Bay and two of its sub-estuaries, the Choptank River and sequences were detected, and phylogenetic analysis Patuxent River. Homogenized sediments were incubated grouped the sequences in two distinct clusters belonging 14/15 þ “ ” with N amendments of NH4 ,NO3 , and NO2 to to the genus. 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 the highest ra% in the freshwater portion of the main stem of Introduction upper Chesapeake Bay, where water column NO3 concen- trations are consistently high. Intermediate levels of relative Human activities have approximately doubled terrestrial anammox (10%) were detected at locations corresponding inputs on a global scale through production of to tidal freshwater and mesohaline locations in the synthetic fertilizers and emission of NOx from fossil fuel Choptank River, whereas anammox was not detected in combustion [12]. In the United States, 25% of anthropo- the tidal freshwater location in the Patuxent River. genic N inputs are exported to coastal areas [14]. Coupling Anammox activity was also not detected in the seaward of and denitrification has long been assumed to end of Chesapeake Bay, where water column NO3 be the primary mechanism of fixed N removal from concentrations are consistently low. The ra% did not ecosystems. It has become clear over the last decade, however, that an entirely novel process, known as anaero- * : J. J. Rich ( ) B. B. Ward bic ammonium oxidation (anammox), may be an important Department of Geosciences, Princeton University, Princeton, New Jersey, USA loss term in some systems [6, 23]. e-mail: [email protected] Anammox was first reported in wastewater systems [29, : 52], and theÀÁ process is definedÀÁ as the oxidation of O. R. Dale B. Song ammonium NHþ with NO , in the absence of Department of and , 4 2 University of North Carolina at Wilmington, O2, in the following reaction: Wilmington, North Carolina, USA þ þ ! þ NO2 NH4 N2 2H2O Present address: Similar reactions were proposed earlier, based on J. J. Rich Center for Environmental Studies, Brown University, thermodynamic calculations [2] and nutrient profiles in Providence, Rhode Island, USA anoxic marine basins [32] or sediments [1], but the process 312 J.J. Rich et al. was first documented in wastewater bioreactors [29, 52]. In activity, including the Black Sea [22], off the coast of þ addition, the oxidation of NH4 to N2 by MnO2 reduction in Africa [23], and estuarine sediments [33]. Kuypers et al. marine sediments has been proposed [15, 27], but evidence [23] found a strong correlation between abundance of in support of a biological basis for this mechanism is “Scalindua” cells and anammox activity in the African lacking [47]. coast system. Penton et al. [31] detected 16S rRNA Anammox activity was first reported in natural systems sequences related to “Scalindua” in a variety of soils and in sediments of the Baltic–North Sea transition [48], where sediments, including deep ocean, freshwater wetlands, and anammox accounted for up to 67% of anaerobic N2 permafrost. production. Anammox activity has since been detected in In this study, our objective was to examine anammox other continental margin sediments [8, 36], estuarine sedi- activities in sediments from different locations in the largest ments [28, 33, 45, 50], anoxic marine waters [5, 22, 23, in the United States, the Chesapeake Bay, and two 49], and anoxic tropical freshwater [40]. Incubations of of its sub-estuaries, the Choptank River and Patuxent River. homogenized sediment, amended with 15N, are typically We also used polymerase chain reaction (PCR) to amplify used to determine relative rates of anammox and denitrifi- 16S rRNA genes that are specific to known anammox cation. Although this approach provides potential rates, from the sediments. Tal et al. [45] found evidence measurements made with homogenized sediments appear to for the anammox process in sediments of Baltimore Harbor, approximate or underestimate the percent of N2 production a branch of the Chesapeake Bay system. However, the due to anammox compared to measurements made with potential quantitative significance of anammox in the intact cores [51]. Based on slurry experiments, the percent Chesapeake Bay has not been reported, and this of N2 flux due to anammox is estimated at 0–26% in information is relevant to understanding benthic N cycling estuarine sediments [28, 33, 50]. Anammox thus appears to processes. Our work also contributes to the growing body be ecologically significant in some estuarine sediments, but of literature on the potential regulating factors affecting its contribution is variable, and reported measurements anammox. have been limited to a few estuaries. Most of what is known about the microbiology of anammox bacteria comes from studies of highly enriched Methods cultures from wastewater treatment systems [16, 42]. The organisms grow extremely slowly (minimum generation Sample Locations and Collection time of 11 days), and no pure culture has been isolated. Analysis of 16S rRNA genes has revealed that anammox The five sample locations spanned salinity and NO3 gradients organisms form a deeply branching monophyletic group of in the Chesapeake Bay and two adjacent sub-estuaries, the at least four candidate genera, including, “Brocadia”, Choptank River and Patuxent River. Hydrographic and “Kuenenia”, “Scalindua”,and“Anammoxoglobus” [19, nutrient conditions on the various sampling dates are given 39, 42]. Cells related to the anammox genus of “Scalindua” in Table 1. Temperature, salinity, and oxygen concentrations þ have been found in environmental samples with anammox were measured by CTD, and NH4 and NO3 were measured

Table 1 Site characteristics

Estuary Descriptiona Site Depth Date Bottom water (m) sampled þðÞμ ðÞμ Temperature Salinity NH4 M NO3 M O2 (mg/L) (°C)

Chesapeake Upper Bay, tidal CB1 10 Jul-2004 25.3 2.5 9.8 74.4 8.0 Bay fresh May-2005 15.0 0.4 ND 93.6 7.9 Lower Bay, CB3 11 Jul-2004 24.1 19.7 2.7 0.4 3.8 polyhaline Oct-2004 22.9 18.6 1.7 0.5 5.6 Choptank Upper, tidal fresh CT1 5 Jun-2004 26.9 1.0 1.0 70.7 7.2 River Mar-2005 10.2 0.1 ND 169.7 8.4 Lower, mesohaline CT2 7 Jun-2004 24.6 10.7 4.4 11.6 7.6 Mar-2005 7.8 11.0 ND 28.0 6.5 Patuxent Upper, tidal fresh PR 10 Nov-2004 7.5 0.1 4.5 38.1 8.3 River a Details of site locations are found in Francis et al. [10], except the Patuxent River site (38°41.03 N and 76°41.46 W) ND Not determined Anammox in Chesapeake Bay 313 with an autoanalyzer or manually, using standard colorimetric bag, the sediment was homogenized and aliquotted (1.5 ml) techniques. The sites have been thoroughly characterized by into sealed glass vials (Exetainer, 5.9 ml, 3 mm butyl Cowan and Boynton [3] and the Chesapeake Bay monitoring septa). The vials were purged with He and preincubated for program (http://www.chesapeakebay.net/wquality.htm). Bot- about an hour before additions of He-flushed stock 14 15 μ tom water is characterized by high NO3 concentrations solutions of Nor N [30 l additions; natural abundance 15 15 15 (∼30–100 μM) and low salinity (∼0–10 ppt) at the upper or NH4Cl (99.2 atm%), Na NO3 (99.5 atm%), Na NO2 Chesapeake Bay station (CB1), and higher salinity (19 ppt) (99.1 atm%), Sigma-Aldrich, Co., St. Louis, Missouri]. The ∼ μ 15/14 and low NO3 ( 0.2-6 M) in lower Chesapeake Bay (CB3). following six combinations of N and H2O only were 15 þ 15 þþ Tidal freshwater locations in the Choptank River and included in most experiments: (1) NH4 , (2) NH4 14 15 þ þ 14 14 þ þ 15 Patuxent River (CT1 and PR) experience high NO3 in NO2 ,(3) NH4 NO3 ,(4) NH4 NO2 ,(5) ∼ – μ 14 þþ 15 winter and spring ( 50 300 M) and lower NO3 in early NH4 NO3 and (6) de-ionized H2Oonly(100nmol ∼ – μ 14 − 15 þ 3 fall ( 1 50 M), with incursions of saline water in summer N or N NH4 ,NO2 ,NO3 cm sediment, final and fall in some years (<7 ppt, 2001–2005). Intermediate concentration). Time-course experiments (30 min) were done ∼ – ∼ – μ salinity ( 10 15 ppt) and NO3 concentrations ( 5 50 M) in May 2005 to determine specific anammox and denitrifica- are found in the lower Choptank River. tion activities at one of the sites, whereas end-point measure- Sediments were obtained using box-cores on the dates ments (24 h) were made to determine relative levels of specified in Table 1. The sampling locations were chosen to anammox and denitrification activity on all sampling dates. represent different hydrographic provinces in the Chesa- Biological activity was stopped by addition of ZnCl2 (100 μl, 15 peake Bay ecosystem. In the Choptank and Patuxent River, 7M) to incubation vials to be used for N–N2. Ammonium þ ðÞ14 þ15 a Plexiglas box core (PBS100 type, Ocean Instruments, San and NO3 NO2 N N concentrations were measured Diego, California) was used to obtain two to four box cores in 2 N KCl (3.3 ml) extracts from vials with and without N from each site, whereas in the Chesapeake Bay, a stainless additions. KCl extracts were shaken horizontally on a steel box core (BX640 type, Ocean Instruments, San Diego, reciprocal shaker (30 min, 300 rpm), centrifuged (2,000×g, California) was used to obtain one box core at each 5 min), and supernatants collected and frozen. Differences in þ location. These cores were subsequently sub-cored with the concentration of NH4 at the beginning and end of 24 h þ acrylic tubes (length 30 cm, 7 cm inner diameter), filling incubations were used to estimate net NH4 accumulation via the bottom half of each tube with sediment and the top half mineralization. with bottom water, to obtain 6 to 12 sub-cores at each site. The sub-cores were submerged in bottom water from the 15/14N Analyses and Calculations site and aerated to maintain oxygen gradients in the 15 15 sediment, at near in situ temperature, until the N experi- The amount of N–N2 produced during the incubations ments were conducted (within 5 days of sample collection). was measured directly in the incubation vials with a continuous flow isotope ratio mass spectrometer (Europa 15N Sediment Experiments Scientific 20-20), in-line with an automated gas preparation unit (Europa Scientific, ANCA-G Plus). The amount of 29 30 Experiments for anammox and denitrification activity N2 and N2 produced in the vials in excess of natural followed the procedures of Thamdrup and Dalsgaard [48] abundance was determined using the method of Thamdrup þ and Risgaard-Petersen et al. [34], with some modifications. and Dalsgaard [47]. NO2 was measured using – The top 0.5 1 cm of sediment from two or three sub-cores spongy-Cd [17]andNO2 was measured without Cd 15 ðÞ was pooled and homogenized. This was repeated with reduction [41]. The N-atom% of NO3 or NO2 Fn in additional sub-cores to obtain two or three homogenized sediment incubations was determined by difference, with samples from each site. Each homogenized sample was and without NO3 or NO2 addition, and corrected for the further subdivided into duplicate laboratory replicates to 15N atom% of stock solutions. The phenol/hypochlorite þ obtain an average value for each homogenized sample. The method was used to measure NH4 [21]. 29 30 coefficient of variation was consistently lower for labora- The values for N2 and N2 production and Fn of NO3 tory replicates than for homogenized samples from any or NO2 were plugged into the equations described by given sample location. Therefore, the level of replication Thamdrup and Dalsgaard [48] to calculate anammox and reported here is for the homogenized samples obtained at denitrification. By convention, the percent of N2 production each site (n=2 or 3). accounted for anammox is abbreviated as ra% [i.e., ra%= During sample processing, sediments were kept at the 100{anammox/(anammox+denitrification)}]. Due to uncer- þ experimental temperature of 12°C for sediments obtained in tainties in recovery of NH4 with our KCl extraction method þ 14 þ November, March, and May or 23°C for sediments to measure exchangeable NH4 [24], and NH4 production obtained in June, July, and October. In an N2-flushed glove during incubations through mineralization, we do not report 314 J.J. Rich et al.

15 þ values of N-atom% of NH4 , based on differences before of the PCR product was determined by using gel elec- 15 þ and after NH4 addition, and consequently, we do not trophoresis with a 1% (wt/vol) agarose gel and 1× Sigma 15 þ þ14 report actual levels of anammox in the NH4 NO3 TAE buffer (Sigma-Aldrich Co., St. Louis, Missouri). treatment. Cloning, Sequencing, and Phylogenetic Analysis Statistical Analyses of Activity Data PCR-amplified DNA fragments of the correct size (approx- imately 320 bp) were excised from the gel using the Using Excel software (Microsoft Corp., Redmond, Wash- Eppendorf Perfect Gel Clean-Up Kit, following the manu- ington), two-sample t tests were used to test for differences ’ in 29N production between 15NHþ and 15NHþ þ14 NO facturer s instructions (Eppendorf, Brinkmann Instruments 2 4 4 3 Inc., USA). Purified DNA fragments were introduced into a additions, on any given sampling date and location. Paired t pCR2A vector and transformed into Escherichia coli by tests were used to test for differences in ra% between 15NO and 15NO additions. Using Jmp (SAS Institute, using a TOPO TA cloning kit, as instructed by the 3 2 manufacturer (Invitrogen, Carlsbad, California). Cloned Inc., Cary, North Carolina), linear regression was used to inserts were verified by PCR amplification and sequencing test for correlations between ra% and the bottom water þ with the ABI 3100 automated sequencer (Applied Biosys- variables and NH4 accumulation in sediment incubations. tems, Foster City, California). DNA sequences were exam- The NO data were ln-transformed because these data 3 ined and edited using DNASTAR Lasergene SeqMan spanned two orders of magnitude, whereas the other variables were ln-transformed for comparison. Program (DNASTAR, Inc., Madison, Wisconsin). NCBI BLAST (http://www.ncbi.nih.gov) was used to find the most closely related 16S rRNA gene sequences in the public Extraction of DNA and PCR Amplification databases. The partial 16S rRNA gene sequences were aligned using ClustalW (http://www.ebi.ac.uk/clustalw/). 16S rRNA genes were examined to identify which Neighbor-joining phylogenetic trees were produced by using anammox organisms were present in Chesapeake Bay the Kimura-2 parameter method in PAUP * 4.0b10 software sediments. The CB1 site was selected for this purpose program [44]. Bootstrapping (100 replicates) was used to because anammox activity was most reliably detected at estimate the reproducibility of the trees. The 16S rRNA this location. DNA was extracted from sediments collected sequences from Chesapeake Bay have been deposited in from the CB1 site using the MoBio Power Soil DNA Kit, GenBank with accession numbers EF653646-EF653680. following the manufacturer’s instructions (MoBio Labora- tories, Inc., Carlsbad, California). A nested PCR approach was used to detect anammox bacterial 16S rRNA gene Results sequences in these sediments. First, amplification of Planctomycetales-specific 16S rRNA genes was done by 15N Experiments using the Pla46 primer (5′-GGATTAGGCATGCAAGTC- 3′)[30] and the 1392r universal bacterial primer (5′- Nine endpoint experiments (24 h) were performed ′ 15 29 GACGGGCGGTGTGTACAA-3 )[9]. Next, an anammox (Table 2). With the addition of NO3 ,productionof N2 30 specific 16S PCR was performed by using the Pla46 and N2 was detected in sediments from all locations, and ′ 30 29 29 forward primer and the Amx368r primer (5 - more N2 was produced than N2 (Table 2). Very little N2 ′ μ 15 þ CCTTTCGGGCATTGCGAA-3 )[39] with 1 l of PCR was detected in the presence of NH4 without added NO3 . 15 þ þ14 29 product from the previous reaction as template. Each PCR With addition of NH4 NO3 ,significant N2 pro- (25 μl) contained 2.5 μl 10× PCR buffer, 2.5 μl MgCl2 duction was detected in sediments from CB1 and the (25 mM), 0.2 μl Taq (Promega, Madison, Wisconsin), Choptank River. At CB3 and the Patuxent River site, there μ 29 15 þ 14 0.2 l deoxynucleoside triphosphates (0.8 mM of each was little N2 production from NH4 ,regardlessof NO3 nucleotide), and 1 μl of each primer (400 pmol), and 1 μl addition, indicating no anammox activity at these locations. 29 30 of DNA as template (10 to 100 ng). The reaction cycle Patterns of N2 or N2 accumulation in 30 min parameters of the first PCR included an initial denaturation incubations of CB1 sediment (Fig. 1) were similar to those 15 step of 4 min at 94°C, followed by 40 cycles of ampli- observed in endpoint experiments. Production of N–N2 15 15 þ þ14 fication; each cycle consisted of denaturation at 94°C for from added NO3 or NH4 NO3 was linear during 15 45 s, primer annealing at 59°C for 50 s, and primer the incubations, and production of N–N2 from added 15 þ 14 extension at 72°C for 3 min. The reaction cycle parameters NH4 was dependent on the addition of NO3 (Fig. 1). of the second PCR were the same, except the second The percent of anammox plus denitrification (total N2 cycle’s primer extension step was 72°C for 1 min. The size production) due to anammox (ra%) ranged from 0 to 22% Anammox in Chesapeake Bay 315

29 30 29 30 −3 15 þ14 þ Table 2 Production of N2 or N2 [mean±SD, nmol N2 or N2 cm sediment] in 24 h incubations, in the presence of NO3 NH4 , 15 þ 15 þ þ 14 NH4 ,or NH4 NO3 and the percent of N2 production as anammox (ra%) þ þ þ a b 15 þ14 15 15 þ14 d Site Date Number (n) NO3 NH4 NH4 NH4 NO3 Fn% ra%

29 30 29 30 29 30 N2 N2 N2 N2 N2 N2

CB1 Jul-2004 3 16±4 35±4 0.07±0.03 0.08±0.10 3.8±1.9c 0.09±0.12 93±3 22±6 May-2005 3 9±1 34±2 0.08±0.04 0.03±0.02 2.6±0.1c 0.04±0.01 96±1 15±4 CB3 Jul-2004 2 2.5±0.2 34±3 0.01±0.01 0.07±0.02 0.03±0.002 0.07±0.01 95±4 −3±8 Oct-2004 3 5±1 40±5 0.06±0.02 0.05±0.01 0.08±0.02 0.05±0.03 94±2 −1±2 CT1 Jun-2004 2 5.8±0.1 40±1 0.01±0.004 −0.02±0.02 0.39±0.01c 0.002±0.01 ND ND Mar-2005 2 4.7±0.5 37±4 −0.001±0.01 −0.04±0.03 0.05±0.01c −0.04±0.03 99±0.1 10±0.1 CT2 Jun-2004 2 7.2±0.7 39±1 −0.002±0.01 0.04±0.04 1.1±0.3c 0.06±0.04 ND ND Mar-2005 2 10±3 42±1 0.10±0.13 0.04±0.06 0.8±0.3c 0.01±0.003 97±1 10±7 PR Nov-2004 3 0.8±0.03 27±1 −0.02±0.02 −0.05±0.04 −0.02±0.02 −0.05±0.04 ND ND a CB1 Chesapeake Bay upper, CB3 Chesapeake Bay lower, CT1 Choptank River upper, CT2 Choptank River lower, PR Patuxent River b n The number of homogenized sediment samples analyzed from each location c 29 15 þ 15 þ þ14 Statistical differences in N2 production between NH4 and NH4 NO3 (P value <0.05, two-sample t test) d þ 15 Fn% is the percent of NO3 NO2 as NO3 .

15 “ ” “ (Table 2). There was no difference in ra% whether NO3 with Candidatus Scalindua brodae and Scalindua 15 ” or NO2 was added (P value=0.49, paired t test). The sorokinii (Fig. 2). The average sequence similarity highest mean ra of 22% was found at CB1, in July 2004. between the Chesapeake sequences and the closest 29 30 Based on linear regression of N2 and N2 production in matching Scalindua sequence was 95%, with the excep- CB1 samples (Fig. 1) and Fn, individual rates were 8 nmol tion of one additional sequence (CB1_AMX46-2) that Ncm−3 h−1 for anammox and 31 nmol N cm−3 h−1 for grouped outside the two main clusters but shared an denitrification; these rates yielded a slightly higher mean ra average of 92% similarity with Candidatus “S. brodae” of 21% than in 24 h incubations (15±4%, Table 2), but this and “S. Sorokinii”. difference was not significant (P value=0.11, two-sample t test). The ra% was negatively correlated with salinity and Discussion positively correlated with the ln of NO3 concentration in the bottom water (Table 3). Salinity and NO3 also were Potential Anammox and Denitrification Activities negatively correlated (r=−0.97, P value=0.002; salinity in Chesapeake Bay Sediments correlated with ln NO3 ). The ra% did not correlate with net þ NH4 accumulation in the 24 h sediment incubations or the The discovery of anammox in natural environments has other tested variables (Table 3). prompted a re-evaluation of N2 production in estuarine sediments. Incubations of homogenized sediments, Molecular Detection of Anammox Bacteria in Chesapeake amended with inorganic 15N, have been typically used to

Bay quantify the potential contribution of anammox to total N2 production (i.e., ra%). Our work extends measurements of Nested PCR of 16S rRNA genes, specific for anammox ra% in estuarine sediments to North America and contrib- bacteria, yielded the expected 320 bp fragment from utes to an understanding of some of the factors that may DNA extracted from the upper Chesapeake Bay (CB1) regulate anammox. 15 sediment. Sequencing of 320 bp fragments showed that Our results show similar patterns in N–N2 production 35 out of 40 clones were identified as anammox-like, to previous studies of anammox activity in sediments. In 15 þ þ14 29 based on BLAST searches and phylogenetic analysis. All slurries amended with NH4 NO3 , N2 production 30 35 of the sequences grouped within the Candidatus and lack of N2 production indicated 1:1 pairing of N “Scalindua” genus (Fig. 2). GenBank accession numbers atoms (Table 2), and that nitrate must have been reduced to and identification of the reference sequences in Fig. 2 are NO2 before conversion to N2, which is diagnostic of the presentedinTable4. Within the major Chesapeake Bay anammox reaction and typical for sediment experiments 15 group, two distinct clusters of sequences were found: [33, 50]. Immediate and linear production of N–N2 in Twenty-one clones formed a cluster with Candidatus 30 min time-course experiments indicated that active “”, while 13 clones formed a cluster anammox organisms and denitrifiers were present in the 316 J.J. Rich et al.

8 et al. [43] identified a putative homolog of the respiratory NO reductase gene, narG inthegenomeoftheanammox +15NO - 14NH + 3 3 4 bacterium Kuenenia stuttgartiensis. 6 Slurry incubations, as employed in this study, can be 29N 2 useful for quantifying potential mechanisms of N2 produc- 30N

sediment 2 tion, but this approach perturbs the natural chemical -3 4 gradients and spatial arrangement of organisms carrying cm

2 out N cycling processes in situ. Whether slurry measure- ments yield artificially high or low ra% values is thus a 2 concern. Trimmer et al. [51] recently compared rates of nmol N anammox and denitrification in slurries and intact cores. In sediments with low anammox activity (ra<1%), slurries and 0 intact cores yielded similar results, but when anammox 15 + – + NH4 was more significant (ra>5%), ra% was about 10 15% 0.4 higher in intact cores than in slurries [51]. Consequently, our slurry measurements probably accurately assessed the 0.3

sediment presence or absence of anammox activity, but the actual ra -3 % in sediments with detectable anammox may have been

cm 0.2 underestimated. 2 In studies that have examined anammox activity along 0.1 salinity gradients in estuaries, ra% generally decreases nmol N with increasing salinity [28, 50]. Similarly, we found low ra% in the seaward end of Chesapeake Bay. Anammox 0.0 activity occurred most reliably at high concentrations of 15 + 14 - + NH4 NO3 NO3 in the tidal freshwater part of Chesapeake Bay (CB1) 0.4 and was absent in the saline part of Chesapeake Bay (CB3), where NO3 concentrations are consistently low. 0.3 The pattern that we observed in ra% could be partly sediment -3 explained by variation in bottom water NO3 concentra-

cm 0.2 tions, salinity, or both (Table 3). As salinity and NO

2 3 usually covary in this and other estuarine ecosystems, it is

0.1 difficult to assess their independent effects. Increased

nmol N salinity is known to directly inhibit nitrification and denitrification [35], but salinity effects on anammox in 0.0 estuarine sediments have not been reported. There is 0 102030 evidence, however, that an abundant and stable supply of Time (min) NO3 in the anoxic zone is necessary to maintain active 29 30 anammox populations, and it has been hypothesized that Figure 1 Production of N2 and N2 in incubations of homogenized 15=14 15=14 þ variationinNO availability plays a role in regulating sediment amended with N NO3 or N NH4 , from the 3 upper Chesapeake Bay site (CB1), May 2005. Each symbol represents anammox activity [28, 34, 36]. We similarly hypothesize the mean±1 SE (n=2) that variation in NO3 availability is a key factor regulating ra% in sediments in the Chesapeake Bay Chesapeake Bay sediment (Fig. 1). Intriguingly, rates of ecosystem. This hypothesis needs more rigorous testing, NO3 reduction to NO2 do not limit anammox activity in however, as our dataset is limited and the effects of this and other estuarine and marine sediments, as salinity or other factors have not been ruled out. demonstrated by the fact that NO3 and NO2 additions result in the same ra% (this study and [4, 33, 50]). Anammox-Specific 16S rRNA Sequences in Chesapeake Assuming that NO2 is a freely diffusible intermediate and Bay Sediments NO3 reduction rates are high in estuarine sediments, it is not necessary to postulate that anammox organisms also On the basis of 16S rRNA sequence analysis, the upper perform the NO3 reduction step. However, some anam- Chesapeake Bay sediment community contains at least ‘ ’ mox organisms are capable of coupling NO3 reduction to three distinct anammox bacterial species in the candi- the oxidation of certain organic acids [13, 19]), and Strous date genus “Scalindua” and these are distinct from the Anammox in Chesapeake Bay 317

Figure 2 Phylogenetic tree of 16S rRNA gene sequences obtained from the upper Chesapeake Bay site (CB1 prefix)

currently known “Scalindua” species. Within the two sary when using our PCR approach to analyze the clusters of Chesapeake Bay anammox bacteria, several distribution and diversity of anammox organisms. subclusters were observed, which might represent multiple Although all known anammox bacteria share the same ecotypes of Scalindua-like bacteria in the sediment fundamental morphological and physiological traits central to community. the anammox , species of anammox bacteria may The primers we used match sequences present in all differ in ecologically meaningful ways, such as differential known anammox bacteria, indicating that other anammox utilization of certain organic acids [19]. Most of the other genera were either absent or below the detection limit of ecological aspects of anammox bacteria are not known, the PCR assay. Our PCR assay, however, was not however. Intriguingly, only Scalindua-like sequences have completely specific for anammox bacteria, as 12.5% of been detected in non-wastewater environments and the other our sequenced clones did not match known anammox anammox genera have gone undetected, despite deep sequences. Sequencing PCR products is therefore neces- phylogenetic branching in the anammox lineage. 318 J.J. Rich et al.

Table 3 Correlations between mean ra% and some of the variables NO3 concentrations in bottom waters (this study and [28, 34, measured in this study 50]]) but effects of salinity and other factors have not been Variablea RPvalue ruled. The variability in ra% over time and space as demonstrated even in our relatively small dataset is consistent − Temperature 0.17 0.75 with the dynamic nature of the Chesapeake system and is a − ln(temperature) 0.17 0.75 compelling reason for further studies of anammox and Salinityb −0.84 0.04 ln(salinity) −0.52 0.29 denitrification in this and other estuaries. The presence of NOÀÁ3 0.55 0.26 anammox bacterial communities in estuarine sediments b ln NO3 0.86 0.03 indicates a niche for these organisms that was otherwise þ NHÀÁ4 accumulation −0.51 0.31 assumedtobeoccupiedbyconventional denitrifiers, thereby þ ln NH accumulation −0.33 0.52 4 providing insights into NOx consumption processes, in a þ general, and factors that regulate anammox activity. The variables are for the bottom water at each site, except for NH4 accumulation in sediment incubations. b Significant correlations (P value<0.05) are in bold.

Conclusions Acknowledgements This work was supported by the NSF Micro- bial Biology Fellowship program (DBI-0301308 to JJR) and the NSF Biocomplexity program (OCE 99-81482 to BBW). We thank the Denitrification activity was present throughout the Chesapeake Biocomplexity team for shiptime, supplying some of the nutrient Bay ecosystem, whereas anammox activity was not nearly so data, and assistance, particularly J. Alexander, J. Cornwell, and M. ubiquitous. The presence of Scalindua-like sequences in Owens. We are also indebted to D. A. Bronk, R. Mason, and T. Chesapeake Bay sediment further implicates this group as Jordan for shiptime; T. Jordan provided nutrient data for the Patuxent River, as well. We thank T. Dalsgaard, N. Risgaard-Petersen, L. having a global role in the anammox process. Anammox Nielsen, B. Thamdrup, M. Jensen, J. Nicholls, C. Davies, and M. activity in estuarine sediments appears linked to variation in Trimmer for methodological advice and helpful discussions.

Table 4 GenBank accession numbers and origin for the reference sequences

Label Accession number Origin of 16S rRNA gene sequences Reference

Candidatus Scalindua spp. S. wagneri AY254882 Candidatus Scalindua wagneri [39] S. sorokinii AY257181 Uncultured bacterium, Black Sea [22] S. brodae AY254883 Candidatus Scalindua brodae [39] Clone 15.6 JK854 DQ368148 Uncultured bacterium, Black Sea [20] Clone 15.8 JK636 DQ368248 Uncultured bacterium, Black Sea [20] Clone BD3-11 AB015552 Uncultured bacterium, deep sea sediments [25] Candidatus Brocadia spp. B. anammoxidans AF375994 Candidatus Brocadia anammoxidans [38] B. fulgida DQ459989 Candidatus Brocadia fulgida [18] Clone 14 DQ304531 Uncultured bacterium, anammox reactor Unpublished Clone KU1 AB054006 Uncultured bacterium, sludge treatment [11] Clone Rexco 102/8 AJ871747 Uncultured bacterium, groundwater, UK Unpublished Clone Rexco 101/4 AJ871735 Uncultured bacterium, groundwater, UK Unpublished Candidatus Kuenenia spp. K. stuttgartiensis AF375995 Candidatus Kuenenia stuttgartiensis [38] Clone Pla2-19 AF202661 Uncultured bacterium, trickling filter [37] Clone Pla1-14 AF202659 Uncultured bacterium, trickling filter biofilm [37] Clone Pla1-1 AF202660 Uncultured bacterium, trickling filter biofilm [37] Clone KOLL2a AJ250882 Anammox enrichment culture, wastewater [7] Clone KU2 AB054007 Uncultured bacterium, sludge treatment [11] spp. Clone 3-8b6 AY769988 Uncultured bacterium, aquaculture biofilter [46] Clone A62 AY360085 Uncultured bacterium, Baltimore Harbor sediment [45] clone C6 AY360082 Uncultured bacterium, Baltimore Harbor sediment [45] Clone A62 AY266449 Uncultured bacterium, Baltimore Harbor sediment [45] Clone B4 AY266450 Uncultured bacterium, Baltimore Harbor sediment [45] G. obscuriglobus X56305 Gemmata obscuriglobus [26] Anammox in Chesapeake Bay 319

References 18. Kartal, B, van Niftrik, L, Sliekers, O, Schmid, MC, Schmidt, I, van de Pas-Schoonen, K, Cirpus, I, van der Star, W, van Loosdrecht, M, Abma, W, Kuenen, GJ, Mulder, J-W, Jetten, 1.Bender,M,Jahnke,R,Weiss,R,Martin,W,Heggie,DT, MSM, Strous, M. , van de Vossenberg, J (2004) Application, eco- Orchardo, J, Sowers, T (1989) Organic-carbon oxidation and physiology and biodiversity of anaerobic ammonium-oxidizing benthic nitrogen and silica dynamics in San-Clemente Basin, a bacteria. Rev Environ Sci Biotechnol 3: 255–264 continental borderland site. Geochim Cosmochim Acta 53: 19. Kartal, B, Rattray, J, van Niftrik, LA, van de Vossenberg, J, 685–697 Schmid, MC, Webb, RI, Schouten, S, Fuerst, JA, Damste, JS, 2. Broda, E (1977) Two kinds of missing in nature. Jetten, MSM, Strous, M (2007) Candidatus “Anammoxoglobus – Zeitschrift fur Allgemeine Mikrobiologie 17: 491 493 propionicus” a new propionate oxidizing species of anaerobic 3. Cowan, JLW, Boynton, WR (1996) Sediment-water oxygen and ammonium oxidizing bacteria. Syst Appl Microbiol 30: 39–49 nutrient exchanges along the longitudinal axis of Chesapeake 20. Kirkpatrick, J, Oakley, B, Fuchsman, C, Srinivasan, S, Staley, JT, Bay: seasonal patterns, controlling factors and ecological Murray, JW (2006) Diversity and distribution of Planctomycetes – significance. Estuaries 19: 562 580 and related Bacteria in the suboxic zone of the Black Sea. Appl 4. Dalsgaard, T, Thamdrup, B (2002) Factors controlling anaerobic Environ Microbiol 72: 3079–3083 ammonium oxidation with nitrite in marine sediments. Appl 21. Koroleff, F (1983) Determination of nutrients. In: Grasshoff K Environ Microbiol 68: 3802–3808 (Ed.) Methods of Seawater Analysis. Verlag Chemie 5. Dalsgaard, T, Canfield, DE, Petersen, J, Thamdrup, B, Acuna- 22. Kuypers, MMM, Sliekers, AO, Lavik, G, Schmid, M, Jorgensen, Gonzalez, J (2003) N2 production by the anammox reaction in BB, Kuenen, JG, Damste, JSS, Strous, M, Jetten, MSM (2003) the anoxic water column of Golfo Dulce, Costa Rica. Nature Anaerobic ammonium oxidation by anammox bacteria in the 422: 606–608 Black Sea. Nature 422: 608–611 6. Dalsgaard, T, Thamdrup, B, Canfield, DE (2005) Anaerobic 23. Kuypers, MMM, Lavik, G, Woebken, D, Schmid, M, Fuchs, BM, ammonium oxidation (anammox) in the marine environment. Res Amann, R, Jorgensen, BB, Jetten, MSM (2005) Massive nitrogen Microbiol 156: 457–464 loss from the Benguela upwelling system through anaerobic 7. Egli, K, Fanger, U, Alvarez, PJJ, Siegrist, H, van der Meer, JR, ammonium oxidation. Proc Natl Acad Sci U S A 102: 6478–6483 15 þ Zehnder, AJB (2001) Enrichment and characterization of an 24. Laima, MCJ (1994) Is KCl a reliable extractant of NH4 added anammox bacterium from a rotating biological contactor treat- to coastal marine sediments? Biogeochemistry 27: 83–95 ing ammonium-rich leachate. Arch Microbiol 175: 198–207 25. Li, L, Kato, C, Horikoshi, K (1999) Bacterial diversity in deep-sea 8. Engström, P, Dalsgaard, T, Hulth, S, Aller, RC (2005) Anaerobic sediments from different depths. Biodivers Conserv 8: 659–677 ammonium oxidation by nitrite (anammox): implications for N2 26. Liesack, W, Stackebrandt, E (1992) Occurrence of novel groups of production in coastal marine sediments. Geochim Cosmochim the domain Bacteria as revealed by analysis of genetic material Acta 69: 2057–2065 isolated from an Australian terrestrial environment. J Bacteriol – 9. Ferris, M, Muyzer, G, Ward, D (1996) Denaturing gradient gel 174: 5072 5078 electrophoresis profiles of 16S rRNA-defined populations inhab- 27. Luther, GW, Sundby, B, Lewis, BL, Brendel, PJ, Silverberg, N iting a hot spring microbial mat community. Appl Environ (1997) Interactions of manganese with the : Microbiol 62: 340–346 alternative pathways to dinitrogen. Geochim Cosmochim Acta – 10. Francis, CA, O’Mullan, GD, Ward, BB (2003) Diversity of 61: 4043 4052 ammonia monooxygenase (amoA) genes across environmental 28. Meyer, RL, Risgaard-Petersen, N, Allen, DE (2005) Correlation gradients in Chesapeake Bay sediments. Geobiology 1: 129–140 between anammox activity and microscale distribution of nitrite in a subtropical mangrove sediment. Appl Environ Microbiol 71: 11. Fujii, T, Sugino, H, Rouse, JD, Furukawa, K (2002) Character- 6142–6149 ization of the microbial community in an anaerobic ammonium- 29. Mulder, A, van de Graaf, AA, Robertson, LA, Kuenen, JG (1995) oxidizing biofilm cultured on a nonwoven biomass carrier. J Anaerobic ammonium oxidation discovered in a denitrifying Biosci Bioeng 94: 412–418 fluidized-bed reactor. FEMS Microbiol Ecol 16: 177–183 12. Galloway, JN, Dentener, FJ, Capone, DG, Boyer, EW, Howarth, 30. Neef, A, Amann, R, Schlesner, H, Schleifer, K (1998) Monitoring RW, Seitzinger, SP, Asner, GP, Cleveland, CC, Green, PA, a widespread bacterial group: in situ detection of planctomycetes Holland, EA, Karl, DM, Michaels, AF, Porter, JH, Townsend, with 16S rRNA-targeted probes. Microbiology 144: 3257–3266 AR, Vorosmarty, CJ (2004) Nitrogen cycles: past, present, and 31. Penton, CR, Devol, AH, Tiedje, JM (2006) Molecular evidence future. Biogeochemistry 70: 153–226 for the broad distribution of anaerobic ammonium-oxidizing 13. Güven, D, Dapena, A, Kartal, B, Schmid, MC, Maas, B, van de bacteria in freshwater and marine sediments. Appl Environ Pas-Schoonen, K, Sozen, S, Mendez, R, Op den Camp, HJM, Microbiol 72: 6829–6832 Jetten, MSM, Strous, M, Schmidt, I (2005) Propionate oxidation 32. Richards, FA (1965) Anoxic basins and fjords. In: Riley JP, by and methanol inhibition of anaerobic ammonium-oxidizing Skirrow G (Eds.) Chemical Oceanography. Academic Press, vol 1, – bacteria. Appl Environ Microbiol 71: 1066 1071 pp 611–645 14. Howarth, RW, Boyer, EW, Pabich, WJ, Galloway, JN (2002) 33. Risgaard-Petersen, N, Meyer, RL, Schmid, M, Jetten, MSM, – Nitrogen use in the United States from 1961 2000 and potential Enrich-Prast, A, Rysgaard, S, Revsbech, NP (2004) Anaerobic – future trends. Ambio 31: 88 96 ammonium oxidation in an estuarine sediment. Aquat Microb 15. Hulth, S, Aller, RC, Gilbert, F (1999) Coupled anoxic nitrifica- Ecol 36: 293–304 tion manganese reduction in marine sediments. Geochim 34. Risgaard-Petersen, N, Meyer, RL, Revsbech, NP (2005) Deni- Cosmochim Acta 63: 49–66 trification and anaerobic ammonium oxidation in sediments: 16. Jetten, M, Schmid, M, van de Pas-Schoonen, K, Sinninghe effects of microphytobenthos and NO3 . Aquat Microb Ecol 40: Damsté, J, Strous, M (2005) Anammox organisms: enrichment, 67–76 cultivation, and environmental analysis. Methods Enzymol 397: 35. Rysgaard, S, Thastum, P, Dalsgaard, T, Christensen, PB, Sloth, – þ 34 57 NP (1999) Effects of salinity on NH4 adsorption capacity, 17. Jones, MN (1984) Nitrate reduction by shaking with Cd. Water nitrification, and denitrification in Danish estuarine sediments. Res 18: 643–646 Estuaries 22: 21–30 320 J.J. Rich et al.

36. Rysgaard, S, Glud, RN, Risgaard-Petersen, N, Dalsgaard, T 44. Swofford, DL (2002) PAUP*: Phylogenetic Analysis Using (2004) Denitrification and anammox activity in Arctic marine Parsimony, Version 4. Sinauer Associates, Sunderland, MA sediments. Limnol Oceanogr 49: 1493–1502 45. Tal, Y, Watts, JEM, Schreier, HJ (2005) Anaerobic ammonia- 37. Schmid, M, Twachtmann, U, Klein, M, Strous, M, Juretschko, S, oxidizing bacteria and related activity in Baltimore inner Harbor Jetten, M, Metzger, JW, Schleifer, KH, Wagner, M (2000) sediment. Appl Environ Microbiol 71: 1816–1821 Molecular evidence for genus level diversity of bacteria capable 46. Tal, Y, Watts, JEM, Schreier, HJ (2006) Characterization and of catalyzing anaerobic ammonium oxidation. Syst Appl Micro- abundance of anaerobic ammonia oxidizing (anammox) bacteria biol 23: 93–106 in biofilters of recirculating aquaculture systems. Appl Environ 38. Schmid, M, Schmitz-Esser, S, Jetten, M, Wagner, M (2001) 16S- Microbiol 72: 2896–2904 23S rDNA intergenic spacer and 23S rDNA of anaerobic 47. Thamdrup, B, Dalsgaard, T (2000) The fate of ammonium in ammonium-oxidizing bacteria: implications for phylogeny and in anoxic manganese oxide-rich marine sediment. Geochim Cosmo- situ detection. Environ Microbiol 3: 450–459 chim Acta 64: 4157–4164 39. Schmid, M, Walsh, K, Webb, R, Rijpstra, WIC, van de Pas- 48. Thamdrup, B, Dalsgaard, T (2002) Production of N2 through Schoonen, K, Verbruggen, MJ, Hill, T, Moffett, B, Fuerst, J, anaerobic ammonium oxidation coupled to nitrate reduction in Schouten, S, Damste, JSS, Harris, J, Shaw, P, Jetten, M, Strous, M marine sediments. Appl Environ Microbiol 68: 1312–1318 (2003) Candidatus “Scalindua brodae”,spnov.,Candidatus 49. Thamdrup, B, Dalsgaard, T, Jensen, MM, Ulloa, O, Farias, L, “Scalindua wagneri”, sp nov., two new species of anaerobic Escribano, R (2006) Anaerobic ammonium oxidation in the ammonium oxidizing bacteria. Syst Appl Microbiol 26: 529–538 oxygen-deficient waters off northern Chile. Limnol Oceanogr 40. Schubert, CJ, Durisch-Kaiser, E, Wehrli, B, Thamdrup, B, Lam, P, 51: 2145–2156 Kuypers, M (2006) Anaerobic ammonium oxidation in a tropical 50. Trimmer, M, Nicholls, JC, Deflandre, B (2003) Anaerobic freshwater system (Lake Tanganyika). Environ Microbiol DOI ammonium oxidation measured in sediments along the Thames 10.1111/j.1462-2920.2006.001074.x estuary, United Kingdom. Appl Environ Microbiol 69: 6447– 41. Strickland, JD, Parsons, TR (1972) A practical handbook of 6454 seawater analysis. Fish Res Board Can 167: 1–311 51. Trimmer, M, Risgaard-Petersen, N, Nicholls, JC, Engström, P 42. Strous, M, Fuerst, JA, Kramer, EHM, Logemann, S, Muyzer, G, (2006) Direct measurement of anaerobic ammonium oxidation van de Pas-Schoonen, KT, Webb, R, Kuenen, JG, Jetten, MSM (anammox) and denitrification in intact sediment cores. Mar Ecol (1999) Missing identified as new planctomycete. Nature Prog Ser 326: 37–47 400: 446–449 52. van de Graaf, AA, Mulder, A, Debruijn, P, Jetten, MSM, 43. Strous, M (2006) Deciphering the evolution and metabolism of an Robertson, LA, Kuenen, JG (1995) Anaerobic oxidation of anammox bacterium from a community . Nature 440: ammonium is a biologically mediated process. Appl Environ 790–794 Microbiol 61: 1246–1251