Professional Documents
Culture Documents
DOI 10.1007/s00248-007-9277-3
ORIGINAL ARTICLE
Received: 5 December 2006 / Accepted: 21 May 2007 / Published online: 7 July 2007
# Springer Science + Business Media, LLC 2007
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
312
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
Descriptiona
Site
Depth
(m)
Date
sampled
Bottom water
Temperature
(C)
Chesapeake
Bay
Choptank
River
Patuxent
River
a
CB1
10
CB3
11
CT1
Lower, mesohaline
CT2
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
N Sediment Experiments
313
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
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.
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
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
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
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
15
+ NO3
6
- 14
NH4
29
N2
30
N2
+15NH4+
0.4
0.3
0.2
0.1
0.0
+15NH4+ 14NO3-
0.4
0.3
0.2
0.1
0.0
0
10
20
30
Time (min)
29
317
318
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
Reference
AY254882
AY257181
AY254883
DQ368148
DQ368248
AB015552
[39]
[22]
[39]
[20]
[20]
[25]
AF375994
DQ459989
DQ304531
AB054006
AJ871747
AJ871735
[38]
[18]
Unpublished
[11]
Unpublished
Unpublished
AF375995
AF202661
AF202659
AF202660
AJ250882
AB054007
[38]
[37]
[37]
[37]
[7]
[11]
AY769988
AY360085
AY360082
AY266449
AY266450
X56305
[46]
[45]
[45]
[45]
[45]
[26]
References
1. Bender, M, Jahnke, R, Weiss, R, Martin, W, Heggie, DT,
Orchardo, J, Sowers, T (1989) Organic-carbon oxidation and
benthic nitrogen and silica dynamics in San-Clemente Basin, a
continental borderland site. Geochim Cosmochim Acta 53:
685697
2. Broda, E (1977) Two kinds of lithotrophs missing in nature.
Zeitschrift fur Allgemeine Mikrobiologie 17: 491493
3. Cowan, JLW, Boynton, WR (1996) Sediment-water oxygen and
nutrient exchanges along the longitudinal axis of Chesapeake
Bay: seasonal patterns, controlling factors and ecological
significance. Estuaries 19: 562580
4. Dalsgaard, T, Thamdrup, B (2002) Factors controlling anaerobic
ammonium oxidation with nitrite in marine sediments. Appl
Environ Microbiol 68: 38023808
5. Dalsgaard, T, Canfield, DE, Petersen, J, Thamdrup, B, AcunaGonzalez, J (2003) N2 production by the anammox reaction in
the anoxic water column of Golfo Dulce, Costa Rica. Nature
422: 606608
6. Dalsgaard, T, Thamdrup, B, Canfield, DE (2005) Anaerobic
ammonium oxidation (anammox) in the marine environment. Res
Microbiol 156: 457464
7. Egli, K, Fanger, U, Alvarez, PJJ, Siegrist, H, van der Meer, JR,
Zehnder, AJB (2001) Enrichment and characterization of an
anammox bacterium from a rotating biological contactor treating ammonium-rich leachate. Arch Microbiol 175: 198207
8. Engstrm, P, Dalsgaard, T, Hulth, S, Aller, RC (2005) Anaerobic
ammonium oxidation by nitrite (anammox): implications for N2
production in coastal marine sediments. Geochim Cosmochim
Acta 69: 20572065
9. Ferris, M, Muyzer, G, Ward, D (1996) Denaturing gradient gel
electrophoresis profiles of 16S rRNA-defined populations inhabiting a hot spring microbial mat community. Appl Environ
Microbiol 62: 340346
10. Francis, CA, OMullan, GD, Ward, BB (2003) Diversity of
ammonia monooxygenase (amoA) genes across environmental
gradients in Chesapeake Bay sediments. Geobiology 1: 129140
11. Fujii, T, Sugino, H, Rouse, JD, Furukawa, K (2002) Characterization of the microbial community in an anaerobic ammoniumoxidizing biofilm cultured on a nonwoven biomass carrier. J
Biosci Bioeng 94: 412418
12. Galloway, JN, Dentener, FJ, Capone, DG, Boyer, EW, Howarth,
RW, Seitzinger, SP, Asner, GP, Cleveland, CC, Green, PA,
Holland, EA, Karl, DM, Michaels, AF, Porter, JH, Townsend,
AR, Vorosmarty, CJ (2004) Nitrogen cycles: past, present, and
future. Biogeochemistry 70: 153226
13. Gven, D, Dapena, A, Kartal, B, Schmid, MC, Maas, B, van de
Pas-Schoonen, K, Sozen, S, Mendez, R, Op den Camp, HJM,
Jetten, MSM, Strous, M, Schmidt, I (2005) Propionate oxidation
by and methanol inhibition of anaerobic ammonium-oxidizing
bacteria. Appl Environ Microbiol 71: 10661071
14. Howarth, RW, Boyer, EW, Pabich, WJ, Galloway, JN (2002)
Nitrogen use in the United States from 19612000 and potential
future trends. Ambio 31: 8896
15. Hulth, S, Aller, RC, Gilbert, F (1999) Coupled anoxic nitrification manganese reduction in marine sediments. Geochim
Cosmochim Acta 63: 4966
16. Jetten, M, Schmid, M, van de Pas-Schoonen, K, Sinninghe
Damst, J, Strous, M (2005) Anammox organisms: enrichment,
cultivation, and environmental analysis. Methods Enzymol 397:
3457
17. Jones, MN (1984) Nitrate reduction by shaking with Cd. Water
Res 18: 643646
319
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,
MSM, Strous, M. , van de Vossenberg, J (2004) Application, ecophysiology and biodiversity of anaerobic ammonium-oxidizing
bacteria. Rev Environ Sci Biotechnol 3: 255264
19. Kartal, B, Rattray, J, van Niftrik, LA, van de Vossenberg, J,
Schmid, MC, Webb, RI, Schouten, S, Fuerst, JA, Damste, JS,
Jetten, MSM, Strous, M (2007) Candidatus Anammoxoglobus
propionicus a new propionate oxidizing species of anaerobic
ammonium oxidizing bacteria. Syst Appl Microbiol 30: 3949
20. Kirkpatrick, J, Oakley, B, Fuchsman, C, Srinivasan, S, Staley, JT,
Murray, JW (2006) Diversity and distribution of Planctomycetes
and related Bacteria in the suboxic zone of the Black Sea. Appl
Environ Microbiol 72: 30793083
21. Koroleff, F (1983) Determination of nutrients. In: Grasshoff K
(Ed.) Methods of Seawater Analysis. Verlag Chemie
22. Kuypers, MMM, Sliekers, AO, Lavik, G, Schmid, M, Jorgensen,
BB, Kuenen, JG, Damste, JSS, Strous, M, Jetten, MSM (2003)
Anaerobic ammonium oxidation by anammox bacteria in the
Black Sea. Nature 422: 608611
23. Kuypers, MMM, Lavik, G, Woebken, D, Schmid, M, Fuchs, BM,
Amann, R, Jorgensen, BB, Jetten, MSM (2005) Massive nitrogen
loss from the Benguela upwelling system through anaerobic
ammonium oxidation. Proc Natl Acad Sci U S A 102: 64786483
24. Laima, MCJ (1994) Is KCl a reliable extractant of 15 NH
4 added
to coastal marine sediments? Biogeochemistry 27: 8395
25. Li, L, Kato, C, Horikoshi, K (1999) Bacterial diversity in deep-sea
sediments from different depths. Biodivers Conserv 8: 659677
26. Liesack, W, Stackebrandt, E (1992) Occurrence of novel groups of
the domain Bacteria as revealed by analysis of genetic material
isolated from an Australian terrestrial environment. J Bacteriol
174: 50725078
27. Luther, GW, Sundby, B, Lewis, BL, Brendel, PJ, Silverberg, N
(1997) Interactions of manganese with the nitrogen cycle:
alternative pathways to dinitrogen. Geochim Cosmochim Acta
61: 40434052
28. Meyer, RL, Risgaard-Petersen, N, Allen, DE (2005) Correlation
between anammox activity and microscale distribution of nitrite in
a subtropical mangrove sediment. Appl Environ Microbiol 71:
61426149
29. Mulder, A, van de Graaf, AA, Robertson, LA, Kuenen, JG (1995)
Anaerobic ammonium oxidation discovered in a denitrifying
fluidized-bed reactor. FEMS Microbiol Ecol 16: 177183
30. Neef, A, Amann, R, Schlesner, H, Schleifer, K (1998) Monitoring
a widespread bacterial group: in situ detection of planctomycetes
with 16S rRNA-targeted probes. Microbiology 144: 32573266
31. Penton, CR, Devol, AH, Tiedje, JM (2006) Molecular evidence
for the broad distribution of anaerobic ammonium-oxidizing
bacteria in freshwater and marine sediments. Appl Environ
Microbiol 72: 68296832
32. Richards, FA (1965) Anoxic basins and fjords. In: Riley JP,
Skirrow G (Eds.) Chemical Oceanography. Academic Press, vol 1,
pp 611645
33. Risgaard-Petersen, N, Meyer, RL, Schmid, M, Jetten, MSM,
Enrich-Prast, A, Rysgaard, S, Revsbech, NP (2004) Anaerobic
ammonium oxidation in an estuarine sediment. Aquat Microb
Ecol 36: 293304
34. Risgaard-Petersen, N, Meyer, RL, Revsbech, NP (2005) Denitrification and anaerobic ammonium oxidation in sediments:
effects of microphytobenthos and NO
3 . Aquat Microb Ecol 40:
6776
35. Rysgaard, S, Thastum, P, Dalsgaard, T, Christensen, PB, Sloth,
NP (1999) Effects of salinity on NH
4 adsorption capacity,
nitrification, and denitrification in Danish estuarine sediments.
Estuaries 22: 2130
320
36. Rysgaard, S, Glud, RN, Risgaard-Petersen, N, Dalsgaard, T
(2004) Denitrification and anammox activity in Arctic marine
sediments. Limnol Oceanogr 49: 14931502
37. Schmid, M, Twachtmann, U, Klein, M, Strous, M, Juretschko, S,
Jetten, M, Metzger, JW, Schleifer, KH, Wagner, M (2000)
Molecular evidence for genus level diversity of bacteria capable
of catalyzing anaerobic ammonium oxidation. Syst Appl Microbiol 23: 93106
38. Schmid, M, Schmitz-Esser, S, Jetten, M, Wagner, M (2001) 16S23S rDNA intergenic spacer and 23S rDNA of anaerobic
ammonium-oxidizing bacteria: implications for phylogeny and in
situ detection. Environ Microbiol 3: 450459
39. Schmid, M, Walsh, K, Webb, R, Rijpstra, WIC, van de PasSchoonen, K, Verbruggen, MJ, Hill, T, Moffett, B, Fuerst, J,
Schouten, S, Damste, JSS, Harris, J, Shaw, P, Jetten, M, Strous, M
(2003) Candidatus Scalindua brodae, sp nov., Candidatus
Scalindua wagneri, sp nov., two new species of anaerobic
ammonium oxidizing bacteria. Syst Appl Microbiol 26: 529538
40. Schubert, CJ, Durisch-Kaiser, E, Wehrli, B, Thamdrup, B, Lam, P,
Kuypers, M (2006) Anaerobic ammonium oxidation in a tropical
freshwater system (Lake Tanganyika). Environ Microbiol DOI
10.1111/j.1462-2920.2006.001074.x
41. Strickland, JD, Parsons, TR (1972) A practical handbook of
seawater analysis. Fish Res Board Can 167: 1311
42. Strous, M, Fuerst, JA, Kramer, EHM, Logemann, S, Muyzer, G,
van de Pas-Schoonen, KT, Webb, R, Kuenen, JG, Jetten, MSM
(1999) Missing lithotroph identified as new planctomycete. Nature
400: 446449
43. Strous, M (2006) Deciphering the evolution and metabolism of an
anammox bacterium from a community genome. Nature 440:
790794