Microb Ecol (2008) 55:311320

DOI 10.1007/s00248-007-9277-3

ORIGINAL ARTICLE

Anaerobic Ammonium Oxidation (Anammox) 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

Abstract Anaerobic ammonium oxidation (anammox) has

recently been recognized as a pathway for the removal of
fixed N from aquatic ecosystems. However, the quantitative
significance of anammox in estuarine sediments is variable,
and measurements have been limited to a few estuaries. We
measured anammox and conventional denitrification activities in sediments along salinity gradients in the Chesapeake
Bay and two of its sub-estuaries, the Choptank River and
Patuxent River. Homogenized sediments were incubated


with 14/15N amendments of NH
4 , NO3 , and NO2 to
determine relative activities of anammox and denitrification. 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
upper Chesapeake Bay, where water column NO
3 concentrations are consistently high. Intermediate levels of relative
anammox (10%) were detected at locations corresponding
to tidal freshwater and mesohaline locations in the
Choptank River, whereas anammox was not detected in
the tidal freshwater location in the Patuxent River.
Anammox activity was also not detected in the seaward
end of Chesapeake Bay, where water column NO
3
concentrations are consistently low. The ra% did not
J. J. Rich (*) : B. B. Ward
Department of Geosciences, Princeton University,
Princeton, New Jersey, USA
e-mail: jjrich@princeton.edu
O. R. Dale : B. Song
Department of Biology and Marine Biology,
University of North Carolina at Wilmington,
Wilmington, North Carolina, USA
Present address:
J. J. Rich
Center for Environmental Studies, Brown University,
Providence, Rhode Island, USA

correlate with NH
4 accumulation rate in anoxic sediment
incubations, but ra% was related to water column NO
3
concentrations and salinity. Anammox bacterial communities were also examined by amplifying DNA extracted from
the upper Chesapeake Bay sediment with polymerase chain
reaction (PCR) primers that are specific for 16S rRNA
genes of anammox organisms. A total of 35 anammox-like
sequences were detected, and phylogenetic analysis
grouped the sequences in two distinct clusters belonging
to the Candidatus Scalindua genus.

Introduction
Human activities have approximately doubled terrestrial
nitrogen inputs on a global scale through production of
synthetic fertilizers and emission of NOx from fossil fuel
combustion [12]. In the United States, 25% of anthropogenic N inputs are exported to coastal areas [14]. Coupling
of nitrification and denitrification has long been assumed to
be the primary mechanism of fixed N removal from
ecosystems. It has become clear over the last decade,
however, that an entirely novel process, known as anaerobic ammonium oxidation (anammox), may be an important
loss term in some systems [6, 23].
Anammox was first reported in wastewater systems [29,
52], and the process
is defined as
the oxidation of


ammonium NH
with
nitrite NO
2 , in the absence of
4
O2, in the following reaction:

NO
2 NH4 ! N2 2H2 O

Similar reactions were proposed earlier, based on

thermodynamic calculations [2] and nutrient profiles in
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

addition, the oxidation of NH
4 to N2 by MnO2 reduction in
marine sediments has been proposed [15, 27], but evidence
in support of a biological basis for this mechanism is
lacking [47].
Anammox activity was first reported in natural systems
in sediments of the BalticNorth Sea transition [48], where
anammox accounted for up to 67% of anaerobic N2
production. Anammox activity has since been detected in
other continental margin sediments [8, 36], estuarine sediments [28, 33, 45, 50], anoxic marine waters [5, 22, 23,
49], and anoxic tropical freshwater [40]. Incubations of
homogenized sediment, amended with 15N, are typically
used to determine relative rates of anammox and denitrification. Although this approach provides potential rates,
measurements made with homogenized sediments appear to
approximate or underestimate the percent of N2 production
due to anammox compared to measurements made with
intact cores [51]. Based on slurry experiments, the percent
of N2 flux due to anammox is estimated at 026% in
estuarine sediments [28, 33, 50]. Anammox thus appears to
be ecologically significant in some estuarine sediments, but
its contribution is variable, and reported measurements
have been limited to a few estuaries.
Most of what is known about the microbiology of
anammox bacteria comes from studies of highly enriched
cultures from wastewater treatment systems [16, 42]. The
organisms grow extremely slowly (minimum generation
time of 11 days), and no pure culture has been isolated.
Analysis of 16S rRNA genes has revealed that anammox
organisms form a deeply branching monophyletic group of
at least four candidate genera, including, Brocadia,
Kuenenia, Scalindua, and Anammoxoglobus [19,
39, 42]. Cells related to the anammox genus of Scalindua
have been found in environmental samples with anammox

activity, including the Black Sea [22], off the coast of

Africa [23], and estuarine sediments [33]. Kuypers et al.
[23] found a strong correlation between abundance of
Scalindua cells and anammox activity in the African
coast system. Penton et al. [31] detected 16S rRNA
sequences related to Scalindua in a variety of soils and
sediments, including deep ocean, freshwater wetlands, and
permafrost.
In this study, our objective was to examine anammox
activities in sediments from different locations in the largest
estuary in the United States, the Chesapeake Bay, and two
of its sub-estuaries, the Choptank River and Patuxent River.
We also used polymerase chain reaction (PCR) to amplify
16S rRNA genes that are specific to known anammox
bacteria from the sediments. Tal et al. [45] found evidence
for the anammox process in sediments of Baltimore Harbor,
a branch of the Chesapeake Bay system. However, the
potential quantitative significance of anammox in the
Chesapeake Bay ecosystem has not been reported, and this
information is relevant to understanding benthic N cycling
processes. Our work also contributes to the growing body
of literature on the potential regulating factors affecting
anammox.

Methods
Sample Locations and Collection
The five sample locations spanned salinity and NO
3 gradients
in the Chesapeake Bay and two adjacent sub-estuaries, the
Choptank River and Patuxent River. Hydrographic and
nutrient conditions on the various sampling dates are given
in Table 1. Temperature, salinity, and oxygen concentrations

were measured by CTD, and NH
4 and NO3 were measured

Table 1 Site characteristics

Estuary

Descriptiona

Site

Depth
(m)

Date
sampled

Bottom water
Temperature
(C)

Chesapeake
Bay

Choptank
River

Patuxent
River
a

Upper Bay, tidal

fresh
Lower Bay,
polyhaline
Upper, tidal fresh

CB1

10

CB3

11

CT1

Lower, mesohaline

CT2

Upper, tidal fresh

PR

10

Jul-2004
May-2005
Jul-2004
Oct-2004
Jun-2004
Mar-2005
Jun-2004
Mar-2005
Nov-2004

25.3
15.0
24.1
22.9
26.9
10.2
24.6
7.8
7.5

Salinity

2.5
0.4
19.7
18.6
1.0
0.1
10.7
11.0
0.1

NH
4 M

NO
3 M

O2 (mg/L)

9.8
ND
2.7
1.7
1.0
ND
4.4
ND
4.5

74.4
93.6
0.4
0.5
70.7
169.7
11.6
28.0
38.1

8.0
7.9
3.8
5.6
7.2
8.4
7.6
6.5
8.3

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

Anammox in Chesapeake Bay

with an autoanalyzer or manually, using standard colorimetric

techniques. The sites have been thoroughly characterized by
Cowan and Boynton [3] and the Chesapeake Bay monitoring
program (http://www.chesapeakebay.net/wquality.htm). Bottom water is characterized by high NO
3 concentrations
(30100 M) and low salinity (010 ppt) at the upper
Chesapeake Bay station (CB1), and higher salinity (19 ppt)
and low NO
3 (0.2-6 M) in lower Chesapeake Bay (CB3).
Tidal freshwater locations in the Choptank River and
Patuxent River (CT1 and PR) experience high NO
3 in
winter and spring (50300 M) and lower NO
in
early
3
fall (150 M), with incursions of saline water in summer
and fall in some years (<7 ppt, 20012005). Intermediate
salinity (1015 ppt) and NO
3 concentrations (550 M)
are found in the lower Choptank River.
Sediments were obtained using box-cores on the dates
specified in Table 1. The sampling locations were chosen to
represent different hydrographic provinces in the Chesapeake Bay ecosystem. In the Choptank and Patuxent River,
a Plexiglas box core (PBS100 type, Ocean Instruments, San
Diego, California) was used to obtain two to four box cores
from each site, whereas in the Chesapeake Bay, a stainless
steel box core (BX640 type, Ocean Instruments, San Diego,
California) was used to obtain one box core at each
location. These cores were subsequently sub-cored with
acrylic tubes (length 30 cm, 7 cm inner diameter), filling
the bottom half of each tube with sediment and the top half
with bottom water, to obtain 6 to 12 sub-cores at each site.
The sub-cores were submerged in bottom water from the
site and aerated to maintain oxygen gradients in the
sediment, at near in situ temperature, until the 15N experiments were conducted (within 5 days of sample collection).
15

N Sediment Experiments

Experiments for anammox and denitrification activity

followed the procedures of Thamdrup and Dalsgaard [48]
and Risgaard-Petersen et al. [34], with some modifications.
The top 0.51 cm of sediment from two or three sub-cores
was pooled and homogenized. This was repeated with
additional sub-cores to obtain two or three homogenized
samples from each site. Each homogenized sample was
further subdivided into duplicate laboratory replicates to
obtain an average value for each homogenized sample. The
coefficient of variation was consistently lower for laboratory replicates than for homogenized samples from any
given sample location. Therefore, the level of replication
reported here is for the homogenized samples obtained at
each site (n=2 or 3).
During sample processing, sediments were kept at the
experimental temperature of 12C for sediments obtained in
November, March, and May or 23C for sediments
obtained in June, July, and October. In an N2-flushed glove

313

bag, the sediment was homogenized and aliquotted (1.5 ml)

into sealed glass vials (Exetainer, 5.9 ml, 3 mm butyl
septa). The vials were purged with He and preincubated for
about an hour before additions of He-flushed stock
solutions of 14N or 15N [30 l additions; natural abundance
or 15NH4Cl (99.2 atm%), Na15NO3 (99.5 atm%), Na15NO2
(99.1 atm%), Sigma-Aldrich, Co., St. Louis, Missouri]. The
following six combinations of 15/14N and H2O only were

15
included in most experiments: (1) 15 NH
4 , (2) NH4




14
15
14
14
15
NO2 , ( 3 ) NH4 NO3 , ( 4 ) NH4 NO2 , ( 5 )
14
15
NH
NO
3 and (6) de-ionized H2O only (100 nmol
4
14


15
3
N or N  NH
sediment, final
4 , NO2 , NO3 cm
concentration). Time-course experiments (30 min) were done
in May 2005 to determine specific anammox and denitrification activities at one of the sites, whereas end-point measurements (24 h) were made to determine relative levels of
anammox and denitrification activity on all sampling dates.
Biological activity was stopped by addition of ZnCl2 (100 l,
7M) to incubation vials to be used for 15NN2. Ammonium
 14
15
and NO
3 NO2 N N concentrations were measured
in 2 N KCl (3.3 ml) extracts from vials with and without N
additions. KCl extracts were shaken horizontally on a
reciprocal shaker (30 min, 300 rpm), centrifuged (2,000g,
5 min), and supernatants collected and frozen. Differences in
the concentration of NH
4 at the beginning and end of 24 h
incubations were used to estimate net NH
4 accumulation via
mineralization.
15/14

N Analyses and Calculations

The amount of 15NN2 produced during the incubations

was measured directly in the incubation vials with a
continuous flow isotope ratio mass spectrometer (Europa
Scientific 20-20), in-line with an automated gas preparation
unit (Europa Scientific, ANCA-G Plus). The amount of
29
N2 and 30N2 produced in the vials in excess of natural
abundance was determined using the method of Thamdrup
and Dalsgaard [47]. Nitrate NO
2 was measured using
spongy-Cd [17] and NO
was
measured without Cd
2

reduction [41]. The 15N-atom% of NO
3 or NO2 Fn in
sediment incubations was determined by difference, with

and without NO
3 or NO2 addition, and corrected for the
15
N atom% of stock solutions. The phenol/hypochlorite
method was used to measure NH
4 [21].
The values for 29N2 and 30N2 production and Fn of NO
3
or NO
2 were plugged into the equations described by
Thamdrup and Dalsgaard [48] to calculate anammox and
denitrification. By convention, the percent of N2 production
accounted for anammox is abbreviated as ra% [i.e., ra%=
100{anammox/(anammox+denitrification)}]. Due to uncertainties in recovery of NH
4 with our KCl extraction method

14
to measure exchangeable NH
4 [24], and NH4 production
during incubations through mineralization, we do not report

314

values of 15N-atom% of NH
4 , based on differences before
and after 15 NH
addition,
and
consequently, we do not
4

14
report actual levels of anammox in the 15 NH
4 NO3
treatment.

J.J. Rich et al.

of the PCR product was determined by using gel electrophoresis with a 1% (wt/vol) agarose gel and 1 Sigma
TAE buffer (Sigma-Aldrich Co., St. Louis, Missouri).
Cloning, Sequencing, and Phylogenetic Analysis

Statistical Analyses of Activity Data

Using Excel software (Microsoft Corp., Redmond, Washington), two-sample t tests were used to test for differences

15
14
in 29N2 production between 15 NH
NH
4 and
4 NO3
additions, on any given sampling date and location. Paired t
tests were used to test for differences in ra% between
15
15
NO
NO
3 and
2 additions. Using Jmp (SAS Institute,
Inc., Cary, North Carolina), linear regression was used to
test for correlations between ra% and the bottom water
variables and NH
4 accumulation in sediment incubations.
The NO
data
were
ln-transformed because these data
3
spanned two orders of magnitude, whereas the other
variables were ln-transformed for comparison.
Extraction of DNA and PCR Amplification
16S rRNA genes were examined to identify which
anammox organisms were present in Chesapeake Bay
sediments. The CB1 site was selected for this purpose
because anammox activity was most reliably detected at
this location. DNA was extracted from sediments collected
from the CB1 site using the MoBio Power Soil DNA Kit,
following the manufacturers instructions (MoBio Laboratories, Inc., Carlsbad, California). A nested PCR approach
was used to detect anammox bacterial 16S rRNA gene
sequences in these sediments. First, amplification of
Planctomycetales-specific 16S rRNA genes was done by
using the Pla46 primer (5-GGATTAGGCATGCAAGTC3) [30] and the 1392r universal bacterial primer (5GACGGGCGGTGTGTACAA-3) [9]. Next, an anammox
specific 16S PCR was performed by using the Pla46
forward primer and the A mx368r primer (5CCTTTCGGGCATTGCGAA-3) [39] with 1 l of PCR
product 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 each
nucleotide), and 1 l of each primer (400 pmol), and 1 l
of DNA as template (10 to 100 ng). The reaction cycle
parameters of the first PCR included an initial denaturation
step of 4 min at 94C, followed by 40 cycles of amplification; each cycle consisted of denaturation at 94C for
45 s, primer annealing at 59C for 50 s, and primer
extension at 72C for 3 min. The reaction cycle parameters
of the second PCR were the same, except the second
cycles primer extension step was 72C for 1 min. The size

PCR-amplified DNA fragments of the correct size (approximately 320 bp) were excised from the gel using the
Eppendorf Perfect Gel Clean-Up Kit, following the manufacturers instructions (Eppendorf, Brinkmann Instruments
Inc., USA). Purified DNA fragments were introduced into a
pCR2A vector and transformed into Escherichia coli by
using a TOPO TA cloning kit, as instructed by the
manufacturer (Invitrogen, Carlsbad, California). Cloned
inserts were verified by PCR amplification and sequencing
with the ABI 3100 automated sequencer (Applied Biosystems, Foster City, California). DNA sequences were examined and edited using DNASTAR Lasergene SeqMan
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
databases. The partial 16S rRNA gene sequences were
aligned using ClustalW (http://www.ebi.ac.uk/clustalw/).
Neighbor-joining phylogenetic trees were produced by using
the Kimura-2 parameter method in PAUP * 4.0b10 software
program [44]. Bootstrapping (100 replicates) was used to
estimate the reproducibility of the trees. The 16S rRNA
sequences from Chesapeake Bay have been deposited in
GenBank with accession numbers EF653646-EF653680.

Results
15

N Experiments

Nine endpoint experiments (24 h) were performed

29
(Table 2). With the addition of 15 NO
N2
3 , production of
30
and N2 was detected in sediments from all locations, and
more 30N2 was produced than 29N2 (Table 2). Very little 29N2

was detected in the presence of 15 NH
4 without added NO3 .
29

14
With addition of 15 NH
N2 pro4 NO3 , significant
duction was detected in sediments from CB1 and the
Choptank River. At CB3 and the Patuxent River site, there

14
was little 29N2 production from 15 NH
4 , regardless of NO3
addition, indicating no anammox activity at these locations.
Patterns of 29N2 or 30N2 accumulation in 30 min
incubations of CB1 sediment (Fig. 1) were similar to those
observed in endpoint experiments. Production of 15NN2

15
14
from added 15 NO
NH
3 or
4 NO3 was linear during
15
the incubations, and production of NN2 from added
15
14
NH
NO
4 was dependent on the addition of
3 (Fig. 1).
The percent of anammox plus denitrification (total N2
production) due to anammox (ra%) ranged from 0 to 22%

Anammox in Chesapeake Bay

315

Table 2 Production of 29N2 or 30N2 [meanSD, nmol 29N2 or 30N2 cm3 sediment] in 24 h incubations, in the presence of
15
15
14
NH
NH
NO
4 , or
4
3 and the percent of N2 production as anammox (ra%)
Sitea

Date

Number (n)b

14
NO
3 NH4

15

CB3
CT1
CT2
PR

Jul-2004
May-2005
Jul-2004
Oct-2004
Jun-2004
Mar-2005
Jun-2004
Mar-2005
Nov-2004

3
3
2
3
2
2
2
2
3


14
NH
4 NO3

15

29

30

29

30

29

30

164
91
2.50.2
51
5.80.1
4.70.5
7.20.7
103
0.80.03

354
342
343
405
401
374
391
421
271

0.070.03
0.080.04
0.010.01
0.060.02
0.010.004
0.0010.01
0.0020.01
0.100.13
0.020.02

0.080.10
0.030.02
0.070.02
0.050.01
0.020.02
0.040.03
0.040.04
0.040.06
0.050.04

3.81.9c
2.60.1c
0.030.002
0.080.02
0.390.01c
0.050.01c
1.10.3c
0.80.3c
0.020.02

0.090.12
0.040.01
0.070.01
0.050.03
0.0020.01
0.040.03
0.060.04
0.010.003
0.050.04

N2

CB1

NH
4

15

N2

N2

N2

N2

15

14
NO
3 NH4 ,

Fn%d

ra%

933
961
954
942
ND
990.1
ND
971
ND

226
154
38
12
ND
100.1
ND
107
ND

N2

CB1 Chesapeake Bay upper, CB3 Chesapeake Bay lower, CT1 Choptank River upper, CT2 Choptank River lower, PR Patuxent River
n The number of homogenized sediment samples analyzed from each location
c

15
14
Statistical differences in 29 N2 production between 15 NH
NH
4 and
4 NO3 (P value <0.05, two-sample t test)
d


15
Fn% is the percent of NO

NO
as
NO
.
3
2
3
b

(Table 2). There was no difference in ra% whether 15 NO

3
or 15 NO
2 was added (P value=0.49, paired t test). The
highest mean ra of 22% was found at CB1, in July 2004.
Based on linear regression of 29N2 and 30N2 production in
CB1 samples (Fig. 1) and Fn, individual rates were 8 nmol
N cm3 h1 for anammox and 31 nmol N cm3 h1 for
denitrification; these rates yielded a slightly higher mean ra
of 21% than in 24 h incubations (154%, Table 2), but this
difference was not significant (P value=0.11, two-sample t
test).
The ra% was negatively correlated with salinity and
positively correlated with the ln of NO
3 concentration in
the bottom water (Table 3). Salinity and NO
3 also were
negatively correlated (r=0.97, P value=0.002; salinity
correlated with ln NO
3 ). The ra% did not correlate with net
NH
4 accumulation in the 24 h sediment incubations or the
other tested variables (Table 3).
Molecular Detection of Anammox Bacteria in Chesapeake
Bay
Nested PCR of 16S rRNA genes, specific for anammox
bacteria, yielded the expected 320 bp fragment from
DNA extracted from the upper Chesapeake Bay (CB1)
sediment. Sequencing of 320 bp fragments showed that
35 out of 40 clones were identified as anammox-like,
based on BLAST searches and phylogenetic analysis. All
35 of the sequences grouped within the Candidatus
Scalindua genus (Fig. 2). GenBank accession numbers
and identification of the reference sequences in Fig. 2 are
presented in Table 4. Within the major Chesapeake Bay
group, two distinct clusters of sequences were found:
Twenty-one clones formed a cluster with Candidatus
Scalindua wagneri, while 13 clones formed a cluster

with Candidatus Scalindua brodae and Scalindua

sorokinii (Fig. 2). The average sequence similarity
between the Chesapeake sequences and the closest
matching Scalindua sequence was 95%, with the exception of one additional sequence (CB1_AMX46-2) that
grouped outside the two main clusters but shared an
average of 92% similarity with Candidatus S. brodae
and S. Sorokinii.

Discussion
Potential Anammox and Denitrification Activities
in Chesapeake Bay Sediments
The discovery of anammox in natural environments has
prompted a re-evaluation of N2 production in estuarine
sediments. Incubations of homogenized sediments,
amended with inorganic 15N, have been typically used to
quantify the potential contribution of anammox to total N2
production (i.e., ra%). Our work extends measurements of
ra% in estuarine sediments to North America and contributes to an understanding of some of the factors that may
regulate anammox.
Our results show similar patterns in 15NN2 production
to previous studies of anammox activity in sediments. In
 29
14
slurries amended with 15 NH
N2 production
4 NO3 ,
30
and lack of N2 production indicated 1:1 pairing of N
atoms (Table 2), and that nitrate must have been reduced to
NO
2 before conversion to N2, which is diagnostic of the
anammox reaction and typical for sediment experiments
[33, 50]. Immediate and linear production of 15NN2 in
30 min time-course experiments indicated that active
anammox organisms and denitrifiers were present in the

316

J.J. Rich et al.

nmol N2 cm-3 sediment

15

+ NO3
6

- 14

NH4

29

N2

30

N2

nmol N2 cm-3 sediment

+15NH4+

0.4

0.3

0.2

0.1

nmol N2 cm-3 sediment

0.0

+15NH4+ 14NO3-

0.4

0.3

0.2

0.1

0.0
0

10

20

30

Time (min)
29

Figure 1 Production of N2 and 30N2 in incubations of homogenized

15=14
sediment amended with 15=14 N  NO
N  NH
3 or
4 , from the
upper Chesapeake Bay site (CB1), May 2005. Each symbol represents
the mean1 SE (n=2)

Chesapeake Bay sediment (Fig. 1). Intriguingly, rates of


NO
3 reduction to NO2 do not limit anammox activity in
this and other estuarine and marine sediments, as

demonstrated by the fact that NO
3 and NO2 additions
result in the same ra% (this study and [4, 33, 50]).
Assuming that NO
2 is a freely diffusible intermediate and
NO
reduction
rates
are high in estuarine sediments, it is
3
not necessary to postulate that anammox organisms also
perform the NO
3 reduction step. However, some anammox organisms are capable of coupling NO
3 reduction to
the oxidation of certain organic acids [13, 19]), and Strous

et al. [43] identified a putative homolog of the respiratory

NO
3 reductase gene, narG in the genome of the anammox
bacterium Kuenenia stuttgartiensis.
Slurry incubations, as employed in this study, can be
useful for quantifying potential mechanisms of N2 production, but this approach perturbs the natural chemical
gradients and spatial arrangement of organisms carrying
out N cycling processes in situ. Whether slurry measurements yield artificially high or low ra% values is thus a
concern. Trimmer et al. [51] recently compared rates of
anammox and denitrification in slurries and intact cores. In
sediments with low anammox activity (ra<1%), slurries and
intact cores yielded similar results, but when anammox
was more significant (ra>5%), ra% was about 1015%
higher in intact cores than in slurries [51]. Consequently,
our slurry measurements probably accurately assessed the
presence or absence of anammox activity, but the actual ra
% in sediments with detectable anammox may have been
underestimated.
In studies that have examined anammox activity along
salinity gradients in estuaries, ra% generally decreases
with increasing salinity [28, 50]. Similarly, we found low
ra% in the seaward end of Chesapeake Bay. Anammox
activity occurred most reliably at high concentrations of
NO
3 in the tidal freshwater part of Chesapeake Bay (CB1)
and was absent in the saline part of Chesapeake Bay
(CB3), where NO
3 concentrations are consistently low.
The pattern that we observed in ra% could be partly
explained by variation in bottom water NO
3 concentrations, salinity, or both (Table 3). As salinity and NO
3
usually covary in this and other estuarine ecosystems, it is
difficult to assess their independent effects. Increased
salinity is known to directly inhibit nitrification and
denitrification [35], but salinity effects on anammox in
estuarine sediments have not been reported. There is
evidence, however, that an abundant and stable supply of
NO
3 in the anoxic zone is necessary to maintain active
anammox populations, and it has been hypothesized that
variation in NO
3 availability plays a role in regulating
anammox activity [28, 34, 36]. We similarly hypothesize
that variation in NO
3 availability is a key factor
regulating ra% in sediments in the Chesapeake Bay
ecosystem. This hypothesis needs more rigorous testing,
however, as our dataset is limited and the effects of
salinity or other factors have not been ruled out.
Anammox-Specific 16S rRNA Sequences in Chesapeake
Bay Sediments
On the basis of 16S rRNA sequence analysis, the upper
Chesapeake Bay sediment community contains at least
three distinct anammox bacterial species in the candidate 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

clusters of Chesapeake Bay anammox bacteria, several
subclusters were observed, which might represent multiple
ecotypes of Scalindua-like bacteria in the sediment
community.
The primers we used match sequences present in all
known anammox bacteria, indicating that other anammox
genera were either absent or below the detection limit of
the PCR assay. Our PCR assay, however, was not
completely specific for anammox bacteria, as 12.5% of
our sequenced clones did not match known anammox
sequences. Sequencing PCR products is therefore neces-

sary when using our PCR approach to analyze the

distribution and diversity of anammox organisms.
Although all known anammox bacteria share the same
fundamental morphological and physiological traits central to
the anammox metabolism, species of anammox bacteria may
differ in ecologically meaningful ways, such as differential
utilization of certain organic acids [19]. Most of the other
ecological aspects of anammox bacteria are not known,
however. Intriguingly, only Scalindua-like sequences have
been detected in non-wastewater environments and the other
anammox genera have gone undetected, despite deep
phylogenetic branching in the anammox lineage.

318

J.J. Rich et al.

Table 3 Correlations between mean ra% and some of the variables

measured in this study
Variablea

P value

Temperature
ln(temperature)
Salinityb
ln(salinity)
NO
3

b
ln NO
3
NH
4 accumulation


ln NH
4 accumulation

0.17
0.17
0.84
0.52
0.55
0.86
0.51
0.33

0.75
0.75
0.04
0.29
0.26
0.03
0.31
0.52

The variables are for the bottom water at each site, except for NH
4
accumulation in sediment incubations.
b
Significant correlations (P value<0.05) are in bold.
a

Conclusions
Denitrification activity was present throughout the Chesapeake
Bay ecosystem, whereas anammox activity was not nearly so
ubiquitous. The presence of Scalindua-like sequences in
Chesapeake Bay sediment further implicates this group as
having a global role in the anammox process. Anammox
activity in estuarine sediments appears linked to variation in

NO
3 concentrations in bottom waters (this study and [28, 34,
50]]) but effects of salinity and other factors have not been
ruled. The variability in ra% over time and space as
demonstrated even in our relatively small dataset is consistent
with the dynamic nature of the Chesapeake system and is a
compelling reason for further studies of anammox and
denitrification in this and other estuaries. The presence of
anammox bacterial communities in estuarine sediments
indicates a niche for these organisms that was otherwise
assumed to be occupied by conventional denitrifiers, thereby
providing insights into NO
x consumption processes, in
general, and factors that regulate anammox activity.

Acknowledgements This work was supported by the NSF Microbial Biology Fellowship program (DBI-0301308 to JJR) and the NSF
Biocomplexity program (OCE 99-81482 to BBW). We thank the
Biocomplexity team for shiptime, supplying some of the nutrient
data, 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 Patuxent
River, 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 4 GenBank accession numbers and origin for the reference sequences
Label
Candidatus Scalindua spp.
S. wagneri
S. sorokinii
S. brodae
Clone 15.6 JK854
Clone 15.8 JK636
Clone BD3-11
Candidatus Brocadia spp.
B. anammoxidans
B. fulgida
Clone 14
Clone KU1
Clone Rexco 102/8
Clone Rexco 101/4
Candidatus Kuenenia spp.
K. stuttgartiensis
Clone Pla2-19
Clone Pla1-14
Clone Pla1-1
Clone KOLL2a
Clone KU2
Planctomycetes spp.
Clone 3-8b6
Clone A62
clone C6
Clone A62
Clone B4
G. obscuriglobus

Accession number

Origin of 16S rRNA gene sequences

Reference

AY254882
AY257181
AY254883
DQ368148
DQ368248
AB015552

Candidatus Scalindua wagneri

Uncultured bacterium, Black Sea
Candidatus Scalindua brodae
Uncultured bacterium, Black Sea
Uncultured bacterium, Black Sea
Uncultured bacterium, deep sea sediments

[39]
[22]
[39]
[20]
[20]
[25]

AF375994
DQ459989
DQ304531
AB054006
AJ871747
AJ871735

Candidatus Brocadia anammoxidans

Candidatus Brocadia fulgida
Uncultured bacterium, anammox reactor
Uncultured bacterium, sludge treatment
Uncultured bacterium, groundwater, UK
Uncultured bacterium, groundwater, UK

[38]
[18]
Unpublished
[11]
Unpublished
Unpublished

AF375995
AF202661
AF202659
AF202660
AJ250882
AB054007

Candidatus Kuenenia stuttgartiensis

Uncultured bacterium, trickling filter biofilm
Uncultured bacterium, trickling filter biofilm
Uncultured bacterium, trickling filter biofilm
Anammox enrichment culture, wastewater
Uncultured bacterium, sludge treatment

[38]
[37]
[37]
[37]
[7]
[11]

AY769988
AY360085
AY360082
AY266449
AY266450
X56305

Uncultured bacterium, aquaculture biofilter

Uncultured bacterium, Baltimore Harbor sediment
Uncultured bacterium, Baltimore Harbor sediment
Uncultured bacterium, Baltimore Harbor sediment
Uncultured bacterium, Baltimore Harbor sediment
Gemmata obscuriglobus

[46]
[45]
[45]
[45]
[45]
[26]

Anammox in Chesapeake Bay

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