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: Rysgaard@natur.gl

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

mo

xin

Arc

tic

sea

ice

ID#

Po

siti

on

Ice

thic

kn

ess

(m)

Tem

per

atu

re

(�C

)

Bu

lk

sali

nit

y

(psu

)

Bu

lkN

O3-

con

cen

trat

ion

(lm

ol

l-1

sea

ice)

Bu

lkO

2

con

cen

trat

ion

(lm

ol

l-1

sea

ice)

Den

itri

fica

tio

n

acti

vit

y

(lm

ol

m-

2d

ay-

1)

An

amm

ox

acti

vit

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

erro

ro

fth

elo

wer

par

t(0

.5m

)o

fth

ese

aic

e.n

nu

mb

ero

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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