ORIGINAL PAPER
Denitrification activity and oxygen dynamics in Arctic sea ice
Søren Rysgaard Æ Ronnie N. Glud Æ Mikael K. Sejr ÆMartin E. Blicher Æ Henrik J. Stahl
Received: 23 February 2007 / Revised: 9 October 2007 / Accepted: 9 October 2007 / Published online: 31 October 2007
� Springer-Verlag 2007
Abstract Denitrification and oxygen dynamics were
investigated in the sea ice of Franklin Bay (70�N), Canada.
These investigations were complemented with measure-
ments of denitrification rates in sea ice from different parts
of the Arctic (69�N–85�N). Potential for bacterial denitri-
fication activity (5–194 lmol N m-2 day-1) and anammox
activity (3–5 lmol N m-2 day-1) in melt water from both
first-year and multi-year sea ice was found. These values
correspond to 27 and 7%, respectively, of the benthic
denitrification and anammox activities in Arctic sediments.
Although we report only potential denitrification and
anammox rates, we present several indications that active
denitrification in sea ice may occur in Franklin Bay (and
elsewhere): (1) despite sea ice-algal primary production in
the lower sea ice layers, heterotrophic activity resulted in net
oxygen consumption in the sea ice of 1–3 lmol l-1 sea ice
per day at in situ light conditions, suggesting that O2
depletion may occur prior to the spring bloom. (2) The
ample organic carbon (DOC) and NO3- present in sea ice
may support an active denitrification population. (3) Mea-
surements of O2 conditions in melted sea ice cores showed
very low bulk concentrations, and in some cases anoxic
conditions prevailed. (4) Laboratory studies using planar
optodes for measuring the high-resolution two-dimensional
O2 distributions in sea ice confirmed the very dynamic and
heterogeneous O2 distribution in sea ice, displaying a
mosaic of microsites of high and low O2 concentrations.
Brine enclosures and channels were strongly O2 depleted in
actively melting sea ice, and anoxic conditions in parts of
the brine system would favour anaerobic processes.
Keywords Arctic � Sea ice � Bacteria � Nitrogen �Oxygen
Introduction
Studies have shown that O2 depletion occurs in association
with the ice thaw due to liberation of O2-depleted melt
water, and this may create favourable conditions for
anaerobic bacteria and processes in the sea ice matrix (Glud
et al. 2002). Bacterial strains of denitrifying species have
been isolated from Antarctic sea ice (Staley and Gosink
1999), and, recently, the potential for bacterial denitrifica-
tion activity was observed in sea ice from the Baltic Sea
(Kaartokallio 2001) and NE Greenland (Rysgaard and Glud
2004), which also exhibited the potential for anammox.
Given its average global extent of 19–29 9 106 km2, sea
ice may provide a vast low-temperature habitat for these
bacterial processes. At present, very few studies have
considered bacterial denitrification and anammox activities
in sea ice (above references) whereas an increasing number
of observations exists from sediments in the Arctic (Koike
S. Rysgaard (&) � M. E. Blicher
Greenland Institute of Natural Resources,
Kivioq 2, Box 570, 3900 Nuuk, Greenland
e-mail: [email protected]
R. N. Glud � H. J. Stahl
Marine Biological Laboratory, University of Copenhagen,
Strandpromenaden 5, 3000 Helsingør, Denmark
Present Address:R. N. Glud � H. J. Stahl
Scottish Association of Marine Sciences (SAMS),
Dunstaffnage Marine Laboratory Dunbeg,
Oban Argyll PA37 1QA, Scotland, UK
M. K. Sejr
Department of Marine Ecology,
National Environmental Research Institute,
Vejlsøvej 25, 8600 Silkeborg, Denmark
123
Polar Biol (2008) 31:527–537
DOI 10.1007/s00300-007-0384-x
and Hattori 1979; Henriksen et al. 1993; Devol et al. 1997;
Glud et al. 1998; Rysgaard et al. 2004). As bacterial
denitrification and anammox remove nitrogen from the
environment through their respiratory pathways, these
processes will influence the availability of nutrients for
primary producers and thus affect the productivity of Arctic
ecosystems. At present, more data are needed on bacterial
denitrification and anammox activity in sea ice from dif-
ferent geographic localities and at different times of the
year to determine the importance of sea ice relative to
benthic nitrogen removal. In this study, we provide mea-
surements of denitrification and anammox in annual and
multi-year sea ice in the Arctic (69�N–85�N) and compare
these with benthic rates measured by the isotope pairing
technique in Franklin Bay (present study) and previously
published values from other Arctic areas.
Materials and methods
Study sites
The study covers a detailed investigation on sea ice from
Franklin Bay (70�N), Canada, during 4–15 April 2004 of
temperature and salinity conditions, permeability, nutri-
ents, chlorophyll a levels, sea ice-algal productivity and
heterotrophic activity, including anaerobic bacterial N2
production. These measurements are supplemented with
additional measurements of potential anaerobic N2 pro-
duction in sea ice (melted sea ice) from Young Sound, a
Northeast Greenland fjord in May–June 2002, a multi-year
sea ice floe in September 2002 in the Greenland Sea, Disko
Bay, West Greenland, during April 2001, and the Arctic
Canada
Greenland
Scoresbysund
AngmagssalikGodthåb
Godhavn
Thule
YS
GS
DB
FB
AO1AO2
AO3
Fig. 1 Sea ice localities included in the present study. The intensive
study area Franklin Bay is labelled FB, Young Sound, YS, the
Greenland Sea, GS, Disko Bay, DB, and the three Arctic Ocean study
areas, AO1, AO2 and AO3 Ta
ble
1D
enit
rifi
cati
on
and
anam
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Arc
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ID#
Po
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Ice
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(m)
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)
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Den
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An
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y
(lm
ol
m-
2d
ay-
1)
n
DB
69
�17
N,
53�5
4W
a0
.5±
0.1
-2
.3±
0.3
3.2
±0
.31
.2±
0.4
10
2±
13
0±
00
±0
15
FB
70
�02
N,
12
6�1
8W
a1
.8±
0.1
-3
.2±
0.6
5.4
±0
.31
.5±
1.2
44
±1
15
±2
0±
04
YS
74
�18
N,
20�1
5W
b1
.5±
0.1
-2
.2±
0.5
4.3
±0
.25
.2±
1.2
12
8±
32
10
±2
0±
08
GS
79
�21
N,
11�0
8W
b2
.2±
0.2
-1
.3±
0.1
5.1
±1
.0–
–4
5±
11
3±
23
AO
18
2�3
0N
,6
2�1
7W
a4
.3±
0.2
-2
.1±
0.2
3.4
±0
.71
.4±
0.6
82
±2
21
94
±5
65
±1
3
AO
28
4�5
6N
,5
9�5
5W
a1
.7±
0.2
-2
.6±
0.8
4.5
±0
.41
.3±
0.7
12
9±
25
45
±3
23
±1
3
AO
38
5�2
8N
,5
9�2
8W
a1
.6±
0.2
-3
.7±
0.7
5.3
±0
.60
.9±
0.5
13
0±
14
87
±5
24
±3
2
Dat
aar
em
ean
val
ues
±st
and
ard
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ro
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par
t(0
.5m
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aic
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nu
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fco
res
(64
cm2)
aP
rese
nt
stu
dy
bR
ysg
aard
and
Glu
d(2
00
4)
528 Polar Biol (2008) 31:527–537
123
Ocean in April 2006. A map of the study sites is presented
in Fig. 1 and locations in Table 1.
Sea ice
Sea ice cores were collected with a MARK II coring sys-
tem (Kovacs Enterprises). Vertical temperature profiles of
5–10 cm resolution were recorded with a thermometer
(Testo thermometer) at the centre of the cores through 3-
mm holes drilled immediately after coring. Sea ice was
then cut into 10-cm sections, which were kept cold and
brought to the field laboratory or vessel for further pro-
cessing. In the laboratory, sea ice was melted within 6 h,
GF/F filtered and frozen (-18�C) until nutrient analysis
within 1 month. Filters were extracted for 24 h in 96%
ethanol and analyzed fluorometrically for chlorophyll a
(TD-700 Turner design fluorometer). Salinity of the melted
sections (bulk salinity) was determined from conductivity
and temperature with a probe (Orion 0136 10MD) cali-
brated to a PORTASAL salinometer. Brine and gas
volumes in sea ice were calculated according to Cox and
Weeks (1983). Concentrations of NO3- + NO2
-, NH4+, and
PO43- were measured by standard techniques (Grasshoff
et al. 1983). Dissolved organic carbon (DOC) samples were
filtered through combusted GF/F filters and analyzed with a
Shimadzu DOC-5000 Analyzer. DOC calibrations were
made with 45-ll injections of potassium hydrogen
phthalate in UV-treated Milli-Q water. All carbon data
were corrected for the instrument blank of 12 lM, and each
measurement represents the mean of 3–7 injections.
A parallel set of cores was sampled and brought back to
the laboratory and cut into sections as described above.
From each 10-cm section, the central part (100–150 g) was
cut free by removing an equal thickness of ice from all
sides of the section, and immediately placing the central
part in a gas-tight laminated NEN/PE plastic bag (Hansen
et al. 2000) fitted with a 50-cm gas-tight Tygon tube and a
valve for sampling. The weight of the bag and the sea ice
sample was recorded. Artificial seawater (25–50 ml;
Grasshoff et al. 1983) of known weight and O2 concen-
tration was then added together with 50 ll HgCl2. The
plastic bag was then closed immediately and excess air
quickly removed through the valve. The sea ice was melted
in the artificial sea water (Grasshoff et al. 1983, salinity of
35) at 0�C, and the melt water mixture transferred to a gas-
tight vial (12 ml Exetainer1, Labco High Wycombe, UK)
containing 50 ll HgCl2. (saturated solution). Gas bubbles
released from the melting sea ice were likewise transferred
to Exetainers. Standard methods of analysis were used: O2
in melt water was measured by Winkler titration and gas-
eous O2 by gas chromatography. The bulk concentration of
O2 in the sea ice (Ci) was calculated as
Ci ¼CmVm � CaVa
Vi
ð1Þ
where Cm is the O2 concentration in the melt water mixture
(gas bubble + water), Vm the weight of the melt water
mixture, Ca the O2 concentration in the artificial sea water,
Va the weight of the artificial sea water, and Vi the weight
of the sea ice.
Permeability measurements of the sea ice were per-
formed in situ according to the procedure of Freitag and
Eicken (2003). In short, holes (91 mm in diameter) of
variable depth were drilled in the sea ice and the water
level measured in the hole over time. At the end of each
measurement the total sea ice thickness and the fraction of
ice under the blind hole were measured by drilling.
Primary production measurements were made on sec-
tions of the sea ice using 14C incubations three times during
4–15 April. In short, an equal volume of GF/F-filtered
water (50 g) from the water column was added to the sea
ice (50 g), which was allowed to melt at 0�C. Apart from
this procedure no further attempts were made to salt buffer
the solution. The melt water was transferred to two light
bottles and one dark bottle (3 9 120 ml) together with
4 lCi H14CO3 and incubated around noon in the opening in
the sea ice close to the sea ice/water column interface for
2 h. During incubation, the hole was covered with a
transparent Plexiglas plate topped with snow to ensure the
correct in situ light regime (Glud et al. 2007). After incu-
bation, the bottles were kept in the dark until filtration,
which was performed within 2 h. The entire content of
each bottle was filtered onto 25-mm GF/F filters. The filters
were transferred to scintillation vials together with 200 ll
1 N HCl and frozen until counting. Excess inorganic 14C
was removed by directing a flow of air into the vials for
24 h before addition of scintillation fluid. Samples were
counted on a liquid scintillation analyzer (Packard
1990TR). The concentration of dissolved inorganic carbon
(DIC) was measured on the diluted melted sea ice samples
using a CO2 analyzer (Coulometer CM5012, UIC Inc.,
Joliet, IL, USA). The 14C fixation was corrected for
unspecific labeling measured in the dark-incubated bottles
and daily primary production rates were quantified from
measurements around noon corrected to the amount of light
in a diurnal cycle and for the dilution from GF/F filtered
sea water. In the present study period, ca. 9 h with values of
[2 lmol photons m-2 s-1 below the sea ice was observed
during a diurnal cycle.
Light saturation curves of sea ice-algal photosynthesis
were obtained in situ in Franklin Bay (Canada) using a
waterproof pulse amplitude modulated fluorometer (Div-
ing-PAM, Walz, Germany). In parallel with the 14C
primary production measurements, sea ice cores was dril-
led, and rapid light curves (RLC) of the relative Electron
Polar Biol (2008) 31:527–537 529
123
Transport Rate (ETR) (a proxy for primary production—
Rysgaard et al. 2001) was measured over 1–2 min by the
saturation pulse method. These measurements were per-
formed at the sea ice/water interface immediately after core
retrieval in a dark chamber on location. During the short
time of measurement the cores were in contact with air. In
this way, snapshots can be observed of the in situ adapta-
tion of the photosynthetic apparatus in sea ice algae under
ambient irradiance conditions. The effective quantum yield
of charge separation up in photosystem II (PSII) was
assessed during measurement of light curves by application
of defined actinic light levels via a measuring cable of the
instrument as described by Kuhl et al. (2001) and Rysgaard
et al. (2001).
Finally, three cores were recovered in early April and the
bottom 50 cm sliced into 10-cm sections to obtain infor-
mation on the O2 consumption of the sea ice. The different
sections were melted at 2�C, transferred to gas-tight Exe-
tainers and incubated at 0�C in a temperature-controlled
room with 5 lmol photons m-2 s-1, representing the very
low irradiance at the sea ice/water interface. At four dif-
ferent time intervals (0–12 days) a set of Exetainers was
collected to determine the O2 concentration. Oxygen con-
sumption was calculated from the O2 decrease with time.
Planar optodes
10 l of seawater (salinity 33) was collected below the sea ice
in the fjord Kobbefjord close to Nuuk (64�N), Greenland,
and preserved with HgCl2 (0.5 ml 5% w/v to 2 l of seawater)
to prevent any biological activity. The water was recircu-
lated through a gas-tight transparent 126-ml (1.4 9 9 9
10 cm) insulated aquarium. Sea ice formation and thawing
was regulated by adjusting the temperature of an aluminum
‘‘cold plate’’ in contact with the surface of the seawater-
filled aquarium. The temperature of the recirculating sea-
water was maintained at -1�C and air flushing of an external
reservoir ensured 100% saturation of the recirculating water.
The inner surface of the front wall of the aquarium
was equipped with a planar optode foil based on the
O2-quenchable luminophore, ruthenium (III)-Tris-4.7-
diphenyl-1.10-phenathroline (Ru(diph)3) embedded in
plasticized PVC (O-nitrophenyl octyl ether) immobilized
on a transparent polyester support foil (Mylar, Dupont,
USA). The measuring principle of oxygen planar optodes
has previously been described in detail (Glud et al. 1996;
Holst et al. 1998) and is therefore presented only briefly
below. The luminophore was excited by blue LEDs (Lux-
eon), equipped with short-pass excitation filters (C-54,
Linos). The emitted oxygen-sensitive red luminescent
signals were imaged with a 12-bit digital CCD camera
(PCO Computer Optics). The camera was equipped with a
17-mm/f1.4 lens covered by a long-pass emission filter
(OG 570, Schott) to remove any reflected light from the
excitation source. At the given optical configuration the
Peltier-cooled chip covered an area of 6.9 9 5.1 cm at a
resolution of ca. 100 lm pixels-1. To quantify the oxygen
distribution in front of the planar optode we applied a
lifetime-based sensing scheme (Holst et al. 1998). The
luminescent light was measured after the eclipse of the
excitation light in two 4.0-ls time frames separated by a
0.2-ls interval. From these two images the oxygen-sensi-
tive lifetime (s) luminescence was calculated assuming a
mono-exponential decay curve. The lifetime images were
calibrated to yield oxygen images by a two-point calibra-
tion procedure using a rearranged modified Stern-Volmer
equation (Glud et al. 1996; Holst et al. 1998):
C ¼ s0 � sKsvðs� 0:2s0Þ
ð2Þ
Where s and s0 represent the luminescent lifetime at a given
oxygen saturation (C) and at anoxia, respectively. Ksv is a
constant expressing the quenching efficiency of the immo-
bilized luminophore. The non-quenchable fraction of the
luminescence was set at the empirically derived image con-
stant of 0.2 (Glud et al. 1996). The lifetime approach made it
possible to use transparent optodes (Holst and Grunwald
2001; Frederiksen and Glud 2006), facilitating the alignment
between oxygen images and structures in the sea ice.
One challenge of applying the present O2 planar optodes
to sea ice is that the sensor signal is temperature sensitive
(Klimant et al. 1997a, b). Calibration tests in a temperature
range from -6 to +10�C showed that Ksv increased by
0.4% per �C while s0 declined by 0.3% per �C (data not
shown). This means that a temperature 1�C higher than the
calibration temperature would cause the O2 values inferred
from the planar optode to be overestimated by 2.4%. We
calibrated the planar optode at 0�C, but as the temperature
microdistribution within the sea ice is unknown, it is not
possible to calibrate the sensor accurately, and the O2
images of the sea ice are thus only indicative of qualitative
changes in the O2 distribution.
When the temperature of the ‘‘cold plate’’ was lowered
to -18�C, sea ice started to form in front of the planar
optode and grew at a rate of 4 9 10-5 cm s-1 down along
the sensor to a thickness of 5 cm. Increasing the temper-
ature in the cold plate to 0�C caused the sea ice to gradually
retreat while O2 images were recorded of the sea ice in
contact with the planar optode.
Denitrification and anammox
Sea ice cores were collected and cut into 5 to 10-cm sec-
tions. These were kept cold and brought to the field
530 Polar Biol (2008) 31:527–537
123
laboratory, where the sections were melted at 0�C and each
sample flushed with helium to remove all O2, which orig-
inated mainly from the entrapped gas bubbles and the
sampling procedure. Three treatments were applied; (a)
addition of 50 lmol l-1 15NO3- (99.6 atom %), (b) addition
of 50 lmol l-1 15NH4+ (99.6 atom %), and (c) addition of
50 lmol l-1 15NH4+ (99.6 atom %) and 50 lmol l-1 14NO3
-
(0.367 atom %). The anoxic melt water containing the three
different isotopic treatments was then transferred to gas-
tight vials (Exetainer1, Labco High Wycombe, UK) and
incubated for 7–14 days at 0�C in the dark. The incubation
was stopped by introducing a 4-ml helium headspace and
adding 200 ll of a ZnCl2 solution (50 % w/v) to preserve
samples until later analysis. The 4-ml sample withdrawn
while introducing the 4-ml helium headspace was frozen
(-18�C) until later isotope analysis of the NO3- and NH4
+
pools. The abundances and concentrations of 14N15N and15N15N were analyzed on a gas chromatograph coupled to a
triple-collector isotopic ratio mass spectrometer (ANCA-
GSL, SerCon Ltd., UK) as described by Risgaard-Petersen
and Rysgaard (1995). The isotopic distributions in the NO3-
and NH4+ pools were likewise analyzed by mass spec-
trometry after reduction to N2 according to the procedures
of Risgaard-Petersen and Rysgaard (1995). Production of
N2 through denitrification and N2 production by anaerobic
NH4+ oxidation (anammox) was calculated according to
Nielsen (1992) and Thamdrup and Dalsgaard (2002),
respectively. Denitrification and anammox rates were also
measured in the sediment below the sea ice in Franklin Bay
at a site 220 m deep. Standard measurement procedures
were used (Rysgaard et al. 2004).
Results
During our study in Franklin Bay, atmospheric tempera-
tures ranged from -10 to -26�C and sea ice thickness was
ca. 1.8 m. Sea ice exhibited a vertical mean temperature
range of -14�C (top) to -2�C (bottom) and a corre-
sponding bulk salinity range of 4–8 (Fig. 2a). Brine salinity
ranged from 50 to 230 and the calculated brine volume
ranged between 0.02 in the upper 1.3 m to a maximum of
0.12 at the ice–water interface (Fig. 2b). The gradual
increase in brine volume towards the water was reflected in
the permeability, which exhibited a gradual increase to a
maximum of 6 9 10-11 m2 at the water–ice interface
(Fig. 2c). Thus, sea ice was permeable in the lower ice
layers, but impermeable in the upper 1 m of the sea ice
during the present investigation period. Bulk concentrations
of DOC in the sea ice ranged from 50 to 120 lmol l-1 sea
ice (Fig. 2c). Nitrate (0.3–4 lmol l-1 sea ice), NH4+ (1–
5 lmol l-1 sea ice) and PO43- (0.2–1.7 lmol l-1 sea ice)
were present throughout the sea ice matrix, with increased
bulk concentrations in the bottom sea ice layers (Fig. 2d).
The Chlorophyll a profiles mirrored the nutrient distri-
bution and gradually increased to 3.5 lg l-1 sea ice in the
bottom layers of the sea ice (Fig. 3a, b). Hence, chlorophyll
a was not only restricted to the underside of the sea ice, but
also detected in the lower 80 cm of the sea ice matrix.
However, no chlorophyll a was observed in the upper 1 m
of the sea ice where permeability was very low. Primary
productivity ranged from 0.1 to 0.3 lg C l-1 sea ice day-1
with highest activity in the bottom layers of the sea ice. The
algal community was dark-adapted and the index of light
adaptation Ek vas calculated at 5 lmol photons m-2 s-1,
and pronounced photoinhibition occurred at irradiances
[12 lmol photons m-2 s-1.
Oxygen was consumed in the lower 0.5 m of the sea ice
(Fig. 4a) at rates 100 times higher than those of primary
production (Fig. 3a). Furthermore, the bulk O2 concentra-
tion was low in the sea ice and in some cores anoxic
conditions occurred in the lower parts of the sea ice.
Several O2 profiles were obtained from sea ice in various
locations (Table 1) and very low concentrations were often
a b c d
Fig. 2 Field observations from
a 1.8 m thick ice floe from
Franklin Bay, Canada April
2004. a Vertical temperatures
(open circle) and bulk salinity
(filled circle). b Brine volume
(open circle), brine salinity
(filled circle) and permeability
(bars). c Bulk concentration of
dissolved organic carbon
(DOC). d Bulk concentrations
of phosphate (open diamond),
nitrate (open circle) and
ammonium (filled circle). Errorbars are standard error of the
mean, n = 5
Polar Biol (2008) 31:527–537 531
123
observed. Potential for bacterial denitrification capacity
was observed in the lower parts of the sea ice in Franklin
Bay (Fig. 4b). This seemed to be a general pattern in the
other visited localities having sea ice thicker than 0.5 m
(Table 1). Area-integrated denitrification rates in sea ice
ranged from 5 to 194 lmol N m-2 day-1. Anammox
activity was also observed in the sea ice, but at much lower
rates (3–5 lmol N m-2 day-1).
Irradiance(µmol photones m-2s-1)
a b
Fig. 3 a Concentration of
chlorophyll a (filled circles) and
primary production (bars) in a
1.8 m thick ice floe from
Franklin Bay, Canada, April
2004. b Relative electron
transport rates (ETR) versus
irradiance measured in situ as
rapid light curves at the sea
ice/water interface immediately
after core retrieval at an
incoming irradiance of 106
(open circle) lmol photons
m-2 s-1. The index of light
adaptation Ek was calculated
according to Platt et al. (1980)
at 5 lmol photons m-2 s-1
a b
Fig. 4 a Oxygen consumption
(bars) and O2 distribution (opencircle) in sea ice of Franklin
Bay. The vertical dotted linerepresents atmospheric O2
saturation at -1.8�C and a
salinity of 33. b Denitrification
rates at the same locality. Errorbars are standard error of the
mean, n = 4
532 Polar Biol (2008) 31:527–537
123
Discussion
Potential rates of denitrification in sea ice were observed at
all locations, except for a locality in Disko Bay with sea ice
0.5 m thick, and ranged from 5 to 194 lmol N m-2 day-1
(Table 1). The highest rate was observed in the bottom 0.5-
m layer of a multi-year sea ice floe 4.3 m thick, presumably
because it represented a stable platform where the micro-
bial community had had time to develop into a more
mature stage. The anammox rate in sea ice was low (0–
5 lmol N m-2 day-1) compared with denitrification rates,
corresponding to only 0–7% of the latter process. The
lower rate of anammox as compared with denitrification
could be related to the fact that anammox bacteria have
slower growth rates and require a more stable environment
to flourish. Denitrifying bacteria may adapt to varying O2
and NO3- concentrations very quickly due to their rapid
doubling times (hours), whereas anammox bacteria display
doubling times of at least 11 days under laboratory con-
ditions (Strous et al. 1998), and thus require more stable
conditions to establish active, dominant populations. The
potential for anaerobic N2 production in sea ice from var-
ious Arctic regions (69�N–85�N) found in the present study
supports recent findings of active denitrification and
anammox in melt water of arctic sea ice (Kaartokallio
2001; Rysgaard and Glud 2004; Schmid et al. 2007) and
findings of denitrifying bacterial strains in Antarctic sea ice
(Staley and Gosink 1999). Denitrifiers are basically aerobic
bacteria having the additional capacity for reducing nitro-
gen oxides when oxygen becomes limiting (Randall and
Ingraham 1981). Hence, they do not require strict anoxic
conditions for growth; in fact, growth is more reliable and
rapid in laboratory cultures when oxic medium is inocu-
lated with a denitrifying culture, which initially consumes
all O2 before the denitrification pathway takes over (Tiedje
1988). Berg et al. (2003) reported that denitrification in
arctic sediments was inhibited at O2 concentrations above
20 lM. In contrast, the anammox process appears to occur
only under strictly anoxic conditions. As little as 1.1 lM of
O2 is sufficient to completely inhibit anammox activity in a
bioreactor (Strous et al. 1998). The inhibition is reversible,
and in experiments with intermittent aeration, the rate of
anammox recovered to previous rates (Jetten et al. 1999).
The present incubations were performed at 0�C, which is
somewhat higher than the in situ temperature of -1.3 to
-3.7�C in the lower ice layers (Table 1). Thus, our rates
are slightly overestimated when a Q10 of 2.4 for denitrifi-
cation is applied (Rysgaard et al. 2004). Furthermore, it has
been shown that the Q10 value for anammox is slightly
lower (Rysgaard et al. 2004) compared with that of deni-
trification, which indicates that anammox compared
with denitrification will be relatively higher at lower
temperatures.
Even though sea ice apparently harbours the potential
for active denitrification, it is an important question whe-
ther denitrification actually occurs in situ and to what
extent sufficient carbon and electron acceptors are present
along with anoxic niches in intact sea ice. To evaluate the
chemical microenvironment in the inaccessible, dynamic
matrix presented by sea ice is far from trivial; however,
several of our findings suggest that anoxic conditions,
which favour the reduction of NO3- to N2, may occur in sea
ice: (1) the organic carbon (DOC) and NO3- present in sea
ice (Fig. 2, Table 1) may support an active denitrifying
population, and in Franklin Bay these pools would be
sufficient to sustain the measured potential denitrification
rates for [100 days.
The lack of salt buffer in our primary productivity
measurements—might have resulted in algae lysis and
elevated DOC levels—however the phototrophic biomass
was low in early spring and matches other findings from
the Arctic (Gradinger et al. 1999; Glud et al. 2007), and our
DOC levels compared with the lower ranges measured in
Antarctic sea ice (Kennedy et al. 2002) and with Arctic sea
ice (Rysgaard and Glud 2004) showing that the DOC
values measured during the present study are not unusually
high as a result of algal lysis during ice melting. Further-
more, the primary productivity as well as light adaptation
values observed during the present study aligns well with
previously published values in early spring from other
high-Arctic areas (Cota 1985; Mock and Gradinger 1999;
Rysgaard et al. 2001).
(2) The low bulk O2 concentrations in sea ice (Fig. 4a,
Table 1) as compared with the underlying water are the net
result of brine leakage and O2 consumption in the sea ice.
Chlorophyll a levels and sea ice algal primary productivity
were low during the study period in Franklin Bay (early
April), representing a pre-bloom situation (Fig. 3). Most of
the studies in Table 1 were preformed early in the year
prior to any potential sea ice-algal bloom. Thus, the algal
communities were in the initial growth phase but well
adapted to the very low light regime below the sea ice,
which supports earlier findings that sea ice algae are
extremely shade-adapted organisms (Cota 1985; Kirst and
Wiencke 1995; Kuhl et al. 2001). Primary production as
measured with 14C was 0.3 lg C l-1 sea ice day-1, or
0.03 lmol C l-1 sea ice day-1, during our field campaign
(Fig. 3a). However, melt water incubated under 5 lmol
photons m-2 s-1 expressed a net O2 consumption of 1–
3 lmol l-1 sea ice day-1 (Fig. 4a). Given the low in situ
diurnal light conditions, the sea ice was thus net hetero-
trophic during this time of year (Fig. 4a) e.g. more oxygen
was consumed than produced. Using a net O2 consumption
of 3 lmol l-1 sea ice day-1 it would require ca. 15 days to
consume the O2 pool in the sea ice of ca. 50 lmol l-1,
provided that the brine water did not exchange with the
Polar Biol (2008) 31:527–537 533
123
ambient sea water (Figs. 3, 4). Previous studies performed
during the summer thaw have reported that microbial
activity within enclosed sea ice incubated at in situ light
conditions led to complete anoxia in the lower 0.5 m of the
sea ice within 6–7 days (Rysgaard and Glud 2004). For
logistic reasons the present study was performed in small
vials incubated at 0�C at 5 lmol photons m-2 s-1 in the
laboratory. We cannot exclude that these conditions
favored heterotrophic activity due to grazing, or unfavor-
able growth conditions for the more sensitive phototrophic
community. However, the present study compares with
earlier findings of significant heterotrophic activity in sea
ice and an available pool of dissolved organic carbon
(Thomas et al. 1995; Krembs et al. 2002; Rysgaard and
Glud 2004).
(3) The bulk O2 concentration in the melt water is the
sum of O2 in the brine and in the air bubbles of the sea ice.
Following the melt of sea ice in our experimental setup, the
air bubbles came into equilibrium with the melt water and,
therefore, we cannot distinguish between O2 in the brine
and within air bubbles based on the present data-set.
However, given the size of the air volume in the resultant
melt water (0–80 ml l-1 melt water) a large fraction of the
O2 could have been contained in bubbles. The brine system
in sea ice is a complex medium (Weissenberger et al. 1992)
where some bubbles are in contact with the brine system
and others are isolated without potential for gas exchange
with the brine. Thus, one would expect the O2 available in
the brine for heterotrophic activity to be lower than the
bulk values presented here, and anoxic conditions in parts
Fig. 5 Black-and-white images
(a, c, e) along with
simultaneously obtained 2D O2
images of the lower 3 cm of a
sea ice core during ice melt. The
photos represent a time series
after the temperature of the cold
plate was increased from -18 to
0�C (at time 0 min). The
heterogeneous distribution of O2
within the sea ice matrix is
apparent, and gradual thawing
induces O2 depletion in brine
enclosures and channels (see
arrows). As the sensor is
temperature-sensitive, and we
have no information on
temperature micro-distribution,
the O2 images cannot be fully
calibrated (see text for details).
An offset of 1�C per �C would
lead to a 2.4% overestimation of
the O2 saturation. Scale Images
across are 45 mm
534 Polar Biol (2008) 31:527–537
123
of the brine system to develop more rapidly. However, if
all O2 is contained in the brine one would expect much
higher O2 conditions in the brine system considering the
measured bulk O2 concentrations of c. 50 lmol l-1 sea ice
(Fig. 4a) and a brine volume of 0.05–0.15 (Fig. 2b). Thus,
we need further proofs for low O2 concentrations in the
brine channels as provided and discussed below.
(4) A recent study has shown that O2 is expelled along
with the high-density brine from growing sea ice, and that
O2 depletion occurs during sea ice thaw following the
release of O2-depleted melt water (Glud et al. 2002).
Temperature variation in the atmosphere may therefore
cause very dynamic O2 conditions in the brine of sea ice, as
O2 is driven from the solid sea ice matrix and transported
out of the sea ice together with high-density brine during
freezing (Gleitz et al. 1995), and O2-depleted water enters
the brine during melting. Thus, supersaturated and under-
saturated conditions may occur in sea ice. During the
present study in Franklin Bay, the atmospheric temperature
varied from -10 to -20�C, resulting in temperature vari-
ations of 6�C in the upper 1.2 m over a 2-week period and
variations around the freezing point at the sea ice–water
column interface. In some of the other localities investi-
gated (Young Sound, NE Greenland) we have observed
variable air temperatures during winter ranging from -35 to
1�C. Temporal and spatial variations in snow cover thick-
ness will also introduce great temperature variations in the
sea ice matrix even during very cold winter conditions.
Such changes in sea ice temperatures were further investi-
gated in a temperature-controlled laboratory set-up using
two-dimensional O2 planar optodes in order to evaluate the
effect of melting on the O2 distribution in sea ice.
Our data confirm earlier findings (Glud et al. 2002) that
the O2 concentration of the brine enclosures in the sea ice
decreased with increasing air temperature (Fig. 5). The first
images (Fig. 5a) reflect conditions at the onset of thawing
and the resolved O2 distribution corresponds to the O2
dissolved in the polymer matrix of the sensor. After
changing the temperature of the cold plate the underside of
the sea ice retreated linearly at a pace of 0.3 mm min-1. As
thawing progressed, low O2 saturation became evident in
the brine channels and at the sea ice/water interface, where
the low-density melt water formed a lens between sea ice
and seawater (Fig. 5d). In the later stages of thawing, the
brine channels clearly reflected extreme O2 depletion
(Fig. 5f). Within the sea ice, local areas displayed super and
undersaturated O2 conditions, resulting in a mosaic-like O2
distribution pattern. The melting sea ice must express ver-
tical and horizontal temperature gradients, and as the O2
sensor is temperature-sensitive, the sensor cannot be
Table 2 Denitrification and anammox in Arctic sediments
Station Water depth
(meters)
Temperature
(�C)
Bottom-water
salinity (psu)
NO3-
(lM)
Denitrification
activity
(lmol m-2 day-1)
Anammox
activity
(lmol m-2 day-1)
Anammox:
denitrification
(%)
67�50N, 166�50Wb – 4.0 400 – –
69�17N, 53�54Wc 50 -0.5 32.8 7.8 154 46 23
69�16N, 53�56Wc 100 0.5 33.2 15.3 251 92 27
69�16N, 53�56Wc 50 -1.5 33.2 10.4 265 60 18
69�29N, 18�07Wd 329 7.0 – 12.0 180 – –
70�02N, 126�18Wa 220 -0.1 34.5 16.2 37 75 67
70�28N, 21�57Wc 40 4 32.3 2.4 193 3 1
72�52N, 25�07Wc 40 1.7 31.8 0.3 33 1 3
74�18N, 20�15Wc 36 -1.3 32.9 2.5 257 5 2
74�18N, 20�16Wc 85 -1.7 33.0 3.2 47 25 35
75�41N, 19�31Wc 40 -1.3 32.6 1.4 38 10 21
76�45N, 18�40Wc 40 -1.7 32.3 2.0 218 3 1
76�57N, 15�00Wd 138 2.8 – 10 305 – –
76�58N, 15�34Wd 155 2.6 – 11 620 – –
77�37N, 07�38Wb 40 -1.7 0.7 3.4 69 8 10
77�33N, 19�05Wd 175 -1.7 – 12 175 – –
77�45N, 15�03Wd 115 0.2 – 9 295 – –
a Present studyb Henriksen et al. (1993)c Rysgaard et al. (2004)d Glud et al. (1998)
Polar Biol (2008) 31:527–537 535
123
calibrated accurately. Horizontal temperature gradient from
the laboratory to the interior of the aquarium could have
induced a stimulated melting rate along the planar optode
fixed to the aquarium wall. This, however, would not
compromise the conclusion that melt water is O2 depleted
and would presumably only reduce the heterogeneity of
intact sea ice. As outlined in ‘‘Materials and methods’’ the
O2 offset of the sensor in the present configuration corre-
sponded to a 2.4-% overestimation of air saturation per �C
above 0�C. Thus, we cannot conclude that complete anoxia
occurs in the brine, but the temperature of the brine chan-
nels must be close to the freezing point. Therefore, the O2
concentration in the underside of the ice and within the
brine channels is presumably well below 20% air saturation
in Fig. 5f. The data show that O2 conditions exhibit a very
heterogeneous distribution in sea ice and that one may find
microsites with high O2 concentrations close to low-oxygen
areas, i.e. conditions that favour denitrification.
The sea ice-associated denitrification rates ranging from
0 to 194 lmol N m-2 day-1 (mean rate = 56 lmol N m-2
day-1) found at all sites investigated in the present study
(Table 1) are considerable compared with the activity in
sediment from various Arctic areas with rates ranging from
33 to 620 lmol N m-2 day-1 (mean rate = 208 lmol
N m-2 day-1). Thus, denitrification occurring in sea ice
may on average correspond to 27% of the activity in sed-
iment in ice-covered arctic areas, whereas anammox
activity in sea ice corresponds to only 7% of the activity in
sediment (Tables 1, 2). This comparison assumes that
oxygen concentrations in the sea ice are sufficiently low to
allow active denitrification and anammox activity in the sea
ice. More studies of denitrification in sea ice are, however,
needed to allow spatial and temporal extrapolation of the
present findings. Furthermore, techniques for resolving
microenvironmental conditions, while maintaining the
microstructure of the sea ice, are required to study the
microbial activity in natural intact sea ice. Planar optodes
(Glud et al. 1996, 2001) and PAM-imaging (Grunwald and
Kuhl 2004) represent such new technologies that may help
us investigate activities under natural conditions.
Acknowledgments We thank A. Haxen for analytical work. S. R., R.
N. G. and M. K. S. were supported by the Danish Natural Science
Research Council and by DANCEA, the Danish Cooperation for
Environment in the Arctic, Danish Ministry of the Environment. This
work is a contribution to the Canadian Arctic Shelf Exchange Study
(CASES) and to the Zackenberg Basic and Nuuk Basic Programmes in
Greenland.
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