Measuring denitrification in sediments using
the isotope pairing technique
Outline
Basic Overview
Important Assumptions
Methodology
Additional Considerations
Advantages and Disadvantages
Anne Giblin, Jane Tucker, Ketil Koop-Jakobsen
Basic Overview
The isotope Pairing technique (IPT) uses labeled
nitrate, 15NO3, to trace denitrification. Unlike
many other tracer techniques, the 15N tracer is not
added in small quantities, it is usually added in
large quantities that often exceed ambient nitrate
concentrations. To understand how to use the
IPT it is necessary to consider the differences
between the two major pathways of denitrification.
NO3
N2
NO3
oxic
anoxic
denitrification
N2
Direct
Denitrification In the
first pathway,
denitrification is
fueled by nitrate
present in the
overlying water
(direct denitrification
or Dw, D in water).
Dw is usually linear
with [nitrate] to high
concentations
Coupled nitrification/denitrification, Dn, nitrate
produced by the nitrification of ammonium within
the sediment column is subsequently denitrified.
Limited by the nitrification rate and transport to
anoxic sediments.
oxic
NH4
NO3
anoxic
ammonification
N2
nitrification
denitrification
Denitrification measured using isotope pairing
technique relies on adding 15NO3 and following
the production rate of 30N2 and 29N2
30N
2
29N
2
28N
2
15NO
3
14NO
3
15NO
3
15NO
3
14NO
3
14NH
14NO
3
4
The IPT makes use of the fact that the
distribution of 28N2, 29N2 and 30N2 produced
during denitrification is a simple
combinatorial function of the 14NO3 and 15NO3
available for denitrification. The fraction (f)
of 14NO3 and 15NO3 is related to the
distribution of 28N2, 29N2 and 30N2 by:
28N
14NO )2
,=
(f
2
3
29N
14NO * f15NO )
=
2*(f
2
3
3
30N
2
= (f15NO3)2
If f14 = 0.5 and f15= 0.5 than
25% 28N2, 50% 29N2, and 25% 30N2
IPT in sediments
The f14NO3 and f15NO3 of the overlying water is
calculated from nitrate concentrations or
directly measured,
But..the amount of 14NO3 being produced by
nitrification in the sediments isn’t known.
IPT takes advantage of the fact that that the
production rate of 29N2 and 30N2 can be used to
calculate the amount of 28N2 produced, and
hence the total denitrification from both the
overlying water nitrate and from nitrification.
First the total rate of denitrification of labeled 15N,
D15, is calculated from the production rate (p) of
29N and 30N :
2
2
D15= p(29N2 ) + 2p(30N2)
The denitrification of 14N, D14 , can than be
calculated as:
D14 = D15 * [p(29N2 )/2p(30N2)]
The total amount of denitrification (as N) then is:
Dtotal = D15+ D14
However, the N2 produced by the added 15NO3
would not normally occur in-situ and is more of a
potential measurement of possible denitrification at a
higher nitrate concentration. The ambient
denitrification rate is considered to be D14.
The total production of N2 is usually partitioned
between: 1) the denitrification of ambient 14NO3
present in the water column (Dw); 2) denitrification
of 14NO3 produced within the sediments (Dn).
Dw = D15.(14NO3 /15NO3 ) of the nitrate in the water
column
Dn= D14 - Dw
ASSUMPTIONS
There are a number of important assumptions made
when using the IPT, some can be tested easily, others
are more difficult to test.
1) A stable nitrate concentration gradient across
the sediment water interface must be
established shortly after 15NO3 addition.
If the zone of nitrification is quite deep it may take
some time for the 15NO3 in the overlying water to
mix down to the zone of denitrification. For this
reason some investigators “pre-incubate” the
samples with label but then need new “time 0”.
Testing this assumption requires that a time course
is run and demonstrating that the denitrification rate
is linear.
2) Ambient denitrification is not affected by the label
addition and denitrification of water column nitrate
increases linearly with concentration
Ambient denitrification could be affected by the label
addition if the addition saturates the denitrification rate or if
nitrate penetrates into zones where denitrification was not
taking place.
Demonstrating that assumption 2 is being met is usually
tested by running the measurements at more than one
nitrate concentration. To meet the assumptions of the
technique the calculated D15 should increase linearly with
added nitrate, which demonstrates that the system is not
saturated, and Dn and Dw should not change with added
15NO .
3
3)That the labeled 15NO3 uniformly mixes with the
14NO being produced by nitrification.
3
Non-uniform mixing will lead to areas where 28N2
and 30N2 are produced preferentially and
denitrification will not be accurately measured. This
is difficult to test.
Elegant idea isn’t it?
In practice it means lots of cores and lots of mud –
and mud/water slurries are …..well they are muddy
Water column measurements of 29N2 and 30N2 (28N2 too
if using MIMS) can be taken and are useful to determine
linearity but the sediment must be slurried at the end to
obtain porewater gases.
1.2
20
15
10
0.4
5
0
0
F
A
J
Month
S
D
Denitrification
(mmol m-2 d-1)
Salinity (psu)
0.8
And the bigger the cores, the bigger the
mess – but you can subcore if you dare\
Slurries must be killed
ADDITIONAL CONSIDERATIONS
1) Aammox
2) Sediment in-homogenity
3) Very low rates, especially when ambient
NO3 is very low
4) Photosynthesis or other processes
producing bubbles
Anammox - anammox violates the assumptions of the
isotope pairing technique. Anammox can be corrected
for using data from slurry incubations or by doing
multiple (>4 NO3 concentrations !). Paper by RisgaardPetersen et al. (2003)
Anammox:
15NH + + 14NO 4
3
15NH +
4
+ 14NO2- → 29N2 + H2O
Denitrification:
15NO 3
5CH2O + 415NO3- + 4H+ → 230N2 +
5CO2 +7H2O
Non-uniform mixing – some
possible complications
Animal burrows are less permeable to
anions than cations
Deep nitrification associated with macrophyte
roots can combine IPT with push pull
Inter-cellular nitrate in S oxidizing
bacteria such as Thioploca
IPT with Push Pull – Ketil Koop-Jakobsen 2008
Rhizosphere
rates are low
but are
integrated
over a large
amount of
sediment
Difficult to apply in
sediments where there
is a lot of porewater
drainage, must balance
water loss against
production
BUBBLES - from photosynthesis, methane, air- can you
deal with them? They matter - don’t forget there is very
little 30N2 in air.
How to fix it? Cores
could be run with small
intentional head space
and run gases directly.
This could also be used
to increase sensitivity but
you need to monitor
pressure if there is gas
production and make
sure your system comes
to equilibrium.
* Truth in advertising I’m just trying this now
Very low rates – corrections for 14NO3 in the label become
increasingly important as rates decrease and when ambient
14NO is very low. Label is usually 99% 15NO so the
3
3
production of 29N2 has to be corrected from the (29N2)
produced by the unlabeled “spike” :
p(29N2)corr = p(29N2)obs – [(2*0.99*0.01)/(0.99)2]*p (30N2)
NH4
P/C
Immobilization
(NH4 assimilation)
A/D
NH4
Nitrogen Fixation
Ammonification
Organic N
DNRA
Nitrification
Immobilization
(NO3assimilation)
H2O- NO3
Anammox
N2
Denitrification
NO3-
20
1.2
15
0.4
5
0
0
A
J
S
D
Month
DNRA showed
the opposite
pattern with
salinity at P22
over the season
16
14
12
10
8
6
4
2
0
2.5
S a lin ity
DNRA
2.0
1.5
1.0
0.5
0.0
Mar Apr May Jun Jul Aug Sep Oct Nov Dec
DNRA (mmole N m-2 d-1)
F
Denitrification
(mmol m-2 d-1)
10
Salinity (ppt)
Salinity (psu)
0.8
DNRA can be a
major sink for
nitrate
especially in
sulfidic
sediments
DNRA is a major
sink for nitrate in
both creek bottom
and salt marsh
platform sediments
ADVANTAGES and DISADVANTAGES
Can measure very low rates
Can be combined with other techniques to get both net N2 flux
and denitrification, and information on processes, DNRA
Can provide insights into controls on denitrification
Requires larger numbers of cores than many techniques
Uniform mixing may be difficult to achieve
Somewhat more expensive
The presence of anammox must be explicitly taken into
account or the data will be in error