OC583: ISOTOPE BIOGEOCHEMISTRY (Spring 2009)
TOPIC 5: NITROGEN CYCLE
I. Background (Fig. 1)
1. Most of Nitrogen is in atmosphere (4x1015 tons)
10
-hydrosphere = 10 tons
12
-soils+sediments = 10 tons
15
-lithosphere = 2x10 tons
2. Isotopes: 14N = 99.6% and 15N = 0.4%
-AIR is the 15N standard (15N = 0 ‰)
3. Range in 15N is ~ -20 to +20 ‰
4. Processes affecting nitrogen cycling and 15N of organic matter in plants and soils in
forest ecosystems (Fig. 2)
a. Assimilation Processes:
-ammonium and nitrate uptake: NH4 or NO3  Organic N
-nitrogen fixation: N2  Organic N
-assimilation can occur via photosynthetic plants and microbes
b. Dissimilation Processes
-mineralization: organic N  NH4
-nitrification: NH4  NO3
-denitrification: NO3  N2O  N2
-annamox (in marine systems): NO2 + NH4  N2
c. Multiple pathways of N transformation affect the abundance and isotopic
composition of N in natural systems
- this complicates our ability to use 15N as a tracer to unravel these N
transformation pathways in both land and aquatic systems
II. NITROGEN ASSIMILATION: isotope fractionation effects
A. Land Plants
Reference: Nitrogen Isotope Studies in Forest Ecosystems, Chapter 2 in Lajtha and
Michener (eds) Stable Isotopes in Ecology and Environmental Science, Blackwell Publ.
1. The 15N of the bulk of terrestrial plants typically ranges from -6 to +6 ‰ (Fig. 3)
2
2. Since in many situations the supply of nitrogen limit plant growth, generally, one
might expect little isotopic fractionation during nitrogen uptake
-i.e., the 15N of the plant would approximately equal the 15N of the inorganic
nitrogen source (e.g., nitrate, ammonia) under truly limiting conditions
-such limiting conditions may not be met if the cells can leak N back into the
environment
3. Culturing experiments show a large range of KIE during plant utilization of nitrate
(Fig. 4)
-
range is from –3 to –24 ‰ (Mariotti, 1980, Kohl and Shearer, 1980)
KIE depends on growth conditions (nitrate concentrations, light, growth rate)
4. In contrast, Mariotti (1982) found that actively growing cultivated plants showed no
net fractionation during NO3 assimilation, i.e. the 15N of the plant was 0.3±0.7‰ lower
than the NO3 (n=38) (Fig. 4)
-these experiments were apparently done at realistic NO3 concentrations for fertile
(cultivated?) soils, i.e. 2.1 mM NO3 (or 4.4 mg NO3-N per kg soil)
5. In short, the range of culture derived KIE for nitrate (and likely ammonium)
assimilation is large and dependent on culturing conditions
-whether the values represent natural systems is definitely questionable.
6. For plants with the microbial assemblage necessary to use or fix atmospheric nitrogen,
these plants (e.g. alder) have an alternative N source for synthesizing amino acids
- during N2 fixation, N2 is reduced to NH4 (N2 + H + energy  NH4 )
- the enzyme used for N2 fixation (nitrogenase) is very susceptible to oxidation
by O2
7. N2 fixing plants have 15N = ~0±3 ‰ which is very close to the 15N of atmospheric
N2 at 0 ‰ (the 15N standard) (Fig. 5)
- this observation suggests that there is little fractionation during N2 fixation
-not clear why this is the case
-i.e., if the molecular diffusion of N2 in air pockets in soils is important,
the KIE during diffusion would be -8.7 ‰ (analogous to –4.4 ‰ fractionation during CO2
diffusion in air)
2
3
-however, if molecular diffusion of N2 in water (in soil) is limiting, then
KIE is only -1.3‰
-whether there is a significant fractionation effect during the breaking of the N-N
tripled bond and conversion of N2 to the N-H bond in NH3 hasn’t been determined
-one explanation is that there is a rate limiting step before the reduction of N2 to
N-H step that doesn’t fractionate (e.g., aqueous diffusion, enzyme catalysis, )
7. Note: commercially made fertilizers are depleted in 15N/14N because they are produced
from atmospheric N2 using the Haber process (N2 + 3H2  2 NH3 ) with little
fractionation because of high temperatures (300-500 ºC) and almost complete conversion
-15N of commercially produced NH4 is -1±3 ‰ and for NO3 is 2±2 ‰.
-this 15N difference could provide an isotopic tracer of Nitrogen from
commercial versus natural NO3 or NH4 sources, i.e., cultivated versus native plants
B. Marine Plankton
KIE during NO3 and NH4 uptake by plankton using culture studies
1. KIE during NH4 and NO3 uptake by plankton (Fig. 6)
-measured a fractionation effect of -3 to -10 ‰ for NH4 assimilation
-measured a fractionation effect of 0 to -15 ‰ for NO3 assimilation
-mean KIE about –7 ‰
-NO3 and NH4 concentration were very high (millimolar) vs ocean (micromolar)
(Wada and Hattori, Geomicrobiology J. 1: 85-101, 1978)
2. Wada and Hattori (1980) measured in cultures a fractionation effect during NH4 and
NO3 uptake that depended inversely on growth rate and decreased from -12 to -6 ‰ (for
NH4) and –1 ‰ (for NO3) with higher light intensity and growth rate (Fig. 7)
- cultures grown at millimolar NO3 and NH4 concentrations
3. Culturing a blue-green algae (Anabena sp.), which is a N2 fixer, on three different N
sources (N2, NO3, and NH4) (Fig. 8) (Macko et al 1987 Chem Geol. 65:79-92)
-found that the overall 15N discrimination was -2 ‰ for N2, -12+-2 ‰ for NO3
and -14±0.2 ‰ for NH4
-these lab experiments were done in 20-60 mM NO3 and NH4 concentrations
where growth was not limited by nitrogen substrate supply
3
4
4. Culture-derived KIE for NO3 assimilation at almost ambient NO3 concentrations (Fig.
9)
-culturing diatoms at NO3 concentrations of <100 uM, rather than mM levels
-find KIEs that vary from = -5 to –25 ‰ depending on species and culturing
conditions
-also found KIE for oxygen isotopes on NO3 was of same magnitude as for
nitrogen isotopes on NO3
- (Granger et al, Limnol Oceanog., 2004)
KIE during NO3 and NH4 uptake by plankton using field studies
1.Wada finds the 15N of organic N in plankton in the Pacific fall into three categories
described by the ambient NO3 concentration (Fig. 10). Wada explained these categories
as follows:
a. At high NO3 concentrations, plankton can express the full KIE during NO3
assimilation yielding low 15N for PON (box I in Fig 10)
b. At near zero NO3 concentrations, when plankton use essentially all the NO3
available this yields no net fractionation effect and results in high 15N values for PON
(+7 to 10 ‰) which at the limit equal the 15N of the NO3 (box II in Fig 10)
c. At near zero NO3 concentrations, when plankton that can fix N2 dominate, this
yields near zero 15N for PON (box III in Fig 10)
3. In support of Wada’s categorization, Altabet and Francois find a clear inverse
relationship between the 15N of PON and NO3 concentration (Fig. 11)
-they conclude that the fractionation effect during the uptake of NO3 by plankton
depends on the NO3 concentration, that is, the higher the NO3 concentration the larger the
fractionation effect expressed
4. This inverse dependence of the 15N of PON on NO3 concentration is similar to the
situation for CO2, i.e., the 13C of plankton decreases as CO2aq concentration increases
-in contrast, if NO3 concentrations are low (limiting) then little or no overall
fractionation effect is expressed during uptake
4
5
5. At steady-state, a simple mass and isotope balance for NO3 in the mixed layer would
imply that the 15N of the PON formed should equal to the 15N of the net NO3 flux
(vertical) into the euphotic zone
-in the situation at Sta PAPA described by Altabet (Fig. 12) a calculation of the
15
 N of the net upward NO3 flux yields a value of about +3 ‰, i.e., (NO3z*RzNO3o*Ro)/(NO3z-NO3o)
-this suggests the 15N of the plankton organic N should be +3 ‰, which is about
9‰ depleted versus the surface 15N for NO3 of ~12 ‰
-this result agrees with Altabet’s calculated KIE of - 9 ‰ during NO3 uptake
based on a Rayleigh distillation closed system interpretation (why apply Rayleigh to a
near or at steady-state situation?)
6. Similarly, Sigman’s measurements of the concentration and 15N of a vertical profile of
NO3 in the Southern Ocean yield a 15N of the net upward NO3 flux of –3 ‰ which when
compared to a 15N of NO3 at + 6 ‰ implies a KIE of –9 ‰ in good agreement with
Altabet’s measurements (Fig. 12)
-In both these regions (Southern Ocean and N. Pacific), there is abundant NO3 in
surface waters so KIE is likely not NO3 supply limited
7. There are very few 15N values for open ocean NH4 because concentrations are so low,
thus our estimates of in-situ 15N fractionation effects during NH4 utilization are limited
-method needs >2 uM NH4 concentrations for 15N analysis which is difficult to
find in open ocean conditions
8. In estuaries, where there are higher NH4 concentration due to anthropogenic sources of
NH4 and nutrients, there have been measurements of the 15N of NH4+
-e.g, 15N-NH4 varies from +10 to +40 ‰ in Delaware estuary (Velinsky et al.
Mar. Chem. 26:, p.351 1989) where NH4 concentrations are up to 40uM (Fig. 13)
-The highly enriched 15N-NH4 values can be derived from two processes:
-First, sewage plant effluent which is enriched in 15N because the human
waste is high on the food chain and there is a 15N trophic level derived
enrichment of NH4 waste
-this situation likely is seen in fall conditions with effluent from waste
treatment plants near Philadelphia
-Second, Plankton utilization of NH4 during photosynthesis should also
enrich 15N in NH4 assuming there is a fractionation during its uptake
5
6
-some evidence of this situation in spring when 15N increases downriver at the
same time NH4 concentration decreases
-the 15 ‰ enrichment between 100km and 50 km associated with a ~50%
decrease in NH4 concentration yields a closed system (Rayleigh) derived KIE of ~
-21 ‰ for NH4 uptake.
-one previous estimate of the KIE for NH4 uptake in an estuary is –10 ‰ (Fig. 14)
9. A summary of the KIE measurements during NO3, NH4 and N2 assimilation indicates a
fairly consistent difference between lab and field results (Fig. 14)
- KIEs during NO3 uptake of 0 to -24 ‰ at high (mM) NO3 concentrations in
culture experiments exceeds the 0 to -9 ‰ KIE measured in the field at
ambient (uM) NO3 concentrations
-The lower the NO3 concentration the lower the KIE with little or no KIE when
NO3 concentration approaches zero and a KIE of –9 ‰ when NO3 is abundant >5
uM (based on Sigman and Altabets field measurements)
- KIEs during NH4 uptake of 0 to -15 ‰ at high (mM) concentrations in culture
experiments is within the range of the –10 to -27 ‰ range estimated from field
measurements at ambient (uM) NH4 concentrations (however there are very
few KIE measurements for NH4 uptake in the field because the NH4
-
concentrations are usually very low
During N2 fixation there is little KIE 0±3 ‰, for both culture and field
measurements which possibly implies a N2 supply limitation
- How useful are culture-derived KIEs?
C. Mechanism of 15N fractionation effect during nitrate assimilation
(Granger et al Limnol. Oceanogr., 49(5), 2004, 1763–1773.)
1. Steps during NO3 assimilation. (Fig. 15)
a. Diffusion of nitrate to cell surface.
b. Active transport of NO3 across cell membrane.
c. Reduction of NO3 to nitrite (NO2) (using nitrate reductase)
d. Reduction of nitrite to NH4 in chloroplasts (using nitrite reductase)
e. Synthesis of amino acids from NH4.
6
7
2. The fractionation effect during the diffusion of external NO3 (in water) to the cell
surface is likely small (~ 1 ‰ or so, measured?). During the active transport of
NO3 across the cell membrane there is likely a KIE (but of unknown magnitude)
3. During one culturing experiment, the measured 15N (and 18O) of internal nitrate
pool was ~ 15 ‰ enriched relative to external NO3 pool in one diatom species (T.
Weissflogii) (from Granger et al) (Fig. 15)
- implies a significant fractionation effect during the reduction of NO3 to NO2.
- experimental measurements in lab of this KIE range from –15 to –30 ‰
- remember the 15N of the internal nitrate pool depends on both the 15N of the
NO3 supplied (fractionation during active transport?) and the 15N of the NO3
lost (via NO3 reduction)
4. There is some experimental data to suggest a significant correlation between the
concentration of nitrate reductase and N assimilation rates, which suggests that the
nitrate reduction step may be limiting in N assimilation.
5. If nitrate reduction is limiting and all the nitrite produced within the cell is
ultimately converted to ammonium then no isotope fractionation is imparted
during the nitrite to NH4 reduction step, i.e., the 15N of the ammonium produced
equals the 15N of the internal nitrite pool
6. Ammonia is converted to glutamate via the enzyme glutamate dehydrogenase
which is a reversible reaction or to glutamine via glutamine synthetase which is a
non-reversible reaction
-glutamine synthesis (GS) has been shown to be the dominant pathway in
diatoms and likely is the pathway for most marine phytoplankton at micromolar or
less NH4+ concentrations
-
- the KIE for the glutamine synthetase pathway was measured for one marine
bacteria (Vibrio harveyi) at -8 ‰ (pH=7) to -12 ‰ (pH=8.6), i.e. 15Nglutamine –
15NNH4 (Hoch et al., Limnol Oceanogr, 1992)
7. If all the NH4 is eventually converted to glutamine then there is no net KIE.
- however, if some of the NH4 can leak out of the cell, then the KIE during the
synthesis of glutamine will be expressed and affect the 15N of the internal
ammonium pool and, in turn, the 15N of the glutamine formed from NH4
7
8
8. One can view the steps in N assimilation somewhat analogous to O’Leary’s two
step model for photosynthesis
a. Step 1: Diffusion and active transport supplies NO3
b. Step 2: Reduction of nitrate to nitrite and then NH4 and synthesis of amino
acids from NH4
9. The 15N of plankton likely depends on the supply rate of NO3 relative to amino
acid synthesis rate and the leakiness of the internal pools of nitrate, nitrite and
ammonium in the cell
-
-
-
- this situation is somewhat analogous to the 13C of plant depending on
the relative rates of CO2 supply versus carboxylation (k3/k2), as presented
in the 2-step model of photosynthesis
however, there are more steps and more compounds that can leak out of cell
thus variations in the 15N of plankton likely depend on the magnitude of
nitrate, nitrite and NH4 leakage, which in turn likely depends on how limiting
is the supply of NO3 or NH4 to the cell, and the KIE during the steps
- if the ecosystem is NO3 supply limited (which is often the case in the
ocean) and all the N that is transported across the cell is eventually
synthesized to amino acids (no leakage) then there is no net fractionation
during N uptake beyond the KIE associated with NO3 transport to the cell
membrane surface (small KIE likely <1 ‰)
-under these conditions, the 15N of the plankton would approach
the 15N of the NO3 dissolved in seawater
in contrast, if the ecosystem is not NO3 limited and leakage of nitrate, nitrite
or ammonium out of the cell occurs, then the KIE during the NO3 reduction
steps and NH4 assimilation will be expressed
under these conditions, the 15N of the plankton will be less than the 15N of
the NO3 in seawater and depend on the KIE during the nitrate reduction steps,
amino acid synthesis steps and leakage rates for NO3, NO2 and NH4.
10. Thus observed variations in the 15N of plankton should be related to ambient
NO3 concentrations, as hypothesized by Wada (1980) (see Fig. 10), because the
likelihood of N limitation increases as NO3 concentrations decrease
D. Using 15N as a tracer of N fixation
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9
Paper: The role of N2 fixation at Station ALOHA in the subtropical N. Pacific
(Karl et al, Nature 388: 533-538, 1997)
1. The role of N2 fixation as a nitrogen source in the ocean is an important issue for
oceanographers.
- N2 fixation is a way to circumvent traditional nitrate limitation for marine
photosynthesis (ocean productivity)
- However only certain plankton have the capacity to fix N2
- N2 fixation most likely occurs where NO3 supply is limiting
- N2 fixation depends on other variables like iron supply and water column
stratification intensity
3. N2 fixation is potentially important in the subtropical gyres of the ocean where
NO3 concentrations are very low (nanomolar) and NO3 limitation is significant.
4. At Sta ALOHA (near Hawaii) in the oligotrophic subtropical gyre, Karl et al
measured the 15N of the particulate organic matter (OM) collected in sediment
traps to represent the POM sinking out of the photic layer and found 1.5 ‰ during
the summer and 4.8 ‰ during the winter (Fig. 16)
5. They measured the 15N of Trichodesmium, a N2 fixing plankton, to be 0.4 ‰
(close to the 0 ‰ expected)
6. They assumed the 15N of non-N2 fixing plankton (utilizing nitrate) was 6.5 ‰
based on previous measurements of the 15N of subsurface NO3 equal to 5-6 ‰
-this assumes no fractionation during NO3 uptake—good assumption?
7. They used a simple isotope and mass balance to determine that 50% of the nitrogen
exported by POM was N2 fixation derived
FN2 + FNO3 = 1
FN2*(15N/14N)N2 + FNO3*(15N/14N)NO3 = (15N/14N)trap
FN2 = (15N/14N)trap – (15N/14N)NO3
(15N/14N)N2 – (15N/14N)NO3
-assume the 15N of the N2–fixation based OM is 0.4 ‰ based on Trichodesmium
and 15N of NO3-based OM is 6.5 ‰ based on the 15N of
summer FN2 = (1.0015-1.0065)/(1.0004-1.0065) = 80%
9
10
winter
annual
FN2 = (1.0048-1.0065)/(1.0004-1.0065) = 28%
FN2 = 48%
7. This estimate agreed reasonably well with the contribution of N2 fixation based on
nitrate and phosphorous mass balances (34±14%) and bottle measurements of N2 fixation
rates (31±18%).
7. In contrast at BATS (near Bermuda), using similar 15N measurements of
sediment trap material, DON and NO3 imply that N2 fixation is a negligible source
of nitrogen at this oligotrophic subtropical site in the N. Atlantic. (Fig. 16)
a. I.e., the 15N of sinking particles actually exceeds the 15N of the nitrate,
which they hypothesize is a result of plankton excreting 15N depleted
dissolved organic nitrogen (DON)
III. NITROGEN DISSIMILATION (Fig. 17)
A. Remineralization of Soil Organic Nitrogen under oxic conditions
1. The mean 15N of soil organic nitrogen measured during 8 separate studies varied from
0 to 11 ‰ with an overall mean of +6±3 ‰ (n=300), which was slightly greater than
mean plant 15N of +3 ‰ growing in the soil at these study sites
2. 15N of soil often increases with depth (Fig. 18)
- generally, soil organic matter (OM) at the surface has 15N close to plant 15N
- 15N of soil OM increases and organic nitrogen (%N) decreases with depth
- one explanation could be that there is a KIE during the mineralization process
that enriches the remaining OM in soils in 15N
- mineralization sequence is Org N  NH4  NO3 (last step via nitrification)
- in these soils there is a decrease in C/N with depth. What does this imply?
- what about bacterial biomass in soils (with a different 15N)? is it significant?
3. There is some evidence (one experiment) that the NO3 produced during organic matter
decomposition (remineralization) is depleted in 15N, whereas the ammonia initially
produced is enriched in 15N (relative to the 15N of the organic matter) (Fig. 19)
- At face value implies that there is a reverse fractionation during ON  NH4
mineralization step nitrification (NH4NO3)
10
11
-
-
-
-
However, it is complicated to compare 15N of NO3 and NH4 with OM in soils
because four processes, with individual KIEs, are potentially occurring
simultaneously, i.e., mineralization, nitrification, denitrification and nitrate
and ammonium utilization
One explanation could be the 15N-NH4 enrichment is a result of KIE effect
during nitrification which yields depleted 15N-NO3, which in turn becomes
more enriched over time as a result of KIE during denitrification
Difficult to separate and quantify the effects of each process without rate
measurements (e.g., using 15N labeled substrates) and individual KIEs for each
process (culture experiments, with their own issues?)
Also in some soil environments one might have to account for equilibrium
fractionation effects during sorption of NH4 onto clays or other soils surfaces
B. Remineralization of Marine Organic Nitrogen
1. Altabet finds the 15N of sinking particulate organic nitrogen (PON) collected in
sediment traps at 1000-3700m varies from 0 to 5 ‰ in the N. Atlantic (Fig. 20)
-finds that the 15N of sinking PON decreases with increasing depth
-not clear whether this 15N decrease represents fractionation during
remineralization or different integration times for deep versus shallow sediment traps
-could it reflect adsorption of 15N depleted dissolved organic nitrogen onto
particle surfaces?
2. Altabet and Francois finds 15N of sinking PON at 150m is enriched by ~ 5 ‰ versus
suspended PON in Equatorial Pacific (Fig. 20)
-does this represent a difference in plankton species (and their 15N) sinking out of
photic layer compared to living in photic layer or a KIE during mineralization of OM?
- what would be one explanation of the latitudinal trend in the 15N of the PON
(either suspended or settling out)?
-from Altabet and Francois paper (1994) in book “Carbon Cycling in the Glacial
Ocean, (ed,. Zahn) “
3. Altabet and Francois find that the 15N of PON in surface sediments is similar to and
has the same latitudinal trend as the 15N of the PON collected at 150m in Equatorial
Pacific (Fig 20)
- implies no KIE during OM mineralization in sediments
11
12
4. Given the few 15N data on fresh OM, settling OM and sedimentary OM there isn’t any
consistent evidence to support significant KIE during OM mineralization
- but comparison complicated due to potentially different plankton species and spatial
and temporal variations in 15N of OM
C. Denitrification
1. In low O2 environments, denitrification is a major pathway of N dissimilation that can
impart significant isotopic fractionations to the nitrate pool (Fig. 17)
2. Denitrification is the principle process where fixed N in the ocean is lost as N2 to the
atmosphere (Fig. 21)
- denitrification converts nitrate to N2
- denitrification occurs where dissolved O2 concentration is very low
- low O2 levels occur where organic matter remineralization (decomposition) rates
are high and/or O2 supply is low (e.g., bottom of lakes, sediments, soils)
- in certain regions of the ocean (Oxygen Minimum Zones (OMZs), etc.) low O2
concentrations and denitrification occurs where there is very slow ventilation
rate of subsurface (thermocline) waters (e.g. eastern tropical Pacific and
Atlantic and Arabian Sea )
3. Sequence of Denitrification (Fig. 22)
NO3 reduction: NO3-  NO2- (nitrite)  N2O (nitrous oxide)  N2
k1
k2
k3
- time rate of change of the concentration and isotopic composition of any
intermediate species depends on the reaction rates and isotopic fractionation during the
production and consumption steps, e.g., for nitrite
d(NO2)/dt = K1*NO3 - K2*NO2
d(15NO2)/dt = K’1*(15NO3) - K’2*(15NO2), where K’s are reaction rate
constants and K’ represents the isotopically substituted molecule
at steady-state:
K1*NO3 = K2*NO2
(15N/14N )NO2 = 1/2 *(15N/14N )NO3
-where 1 = K’1/K1 and 2 = K’2/K2
12
13
4. If denitrification is a one way (non-reversible) reaction sequence, without alternate
addition or loss of any of the intermediate compounds, then the 15N/14N of the NO3 lost
during the first step has to equal the 15N/14N of the N2 produced during the last step at
steady-state (Fig. 22)
a. RNO3*NO3-NO2 = RNO2*NO2-N2O = RN2O*N2O-N2 = RN2
b. however, since there may be different fractionation effects during each step in
the NO3 reduction sequence, the 15N of each of the intermediate pool is likely to
be very different
5. The isotopic pathway for the denitrification sequence is complicated in many situations
by the nitrification, i.e., the “reverse process”, occurring simultaneously
-for example, NO2 and NO3- can be simultaneously produced by nitrification
NH4 + O2  NO2 + O2  NO3
- the recent identification of anammox (NO2 + NH4  N2 + 2H2O ) as a possibly
important process to produce N2 would affect the concentration and isotopic
composition of the nitrite (NO2) pool and, thus, the 15N of the nitrous oxide
(N2O) and final N2 products during denitrification.
-in presence of multiple reaction pathways, the concentrations and 15N/14N of the
NO3, NO2, N2O and N2 are affected by the reaction rate constants and ’s for all
processes
6. Note: Because the product of the rate-limiting step is likely to have low concentrations,
one often has difficulty measuring the isotopic composition of that species
-as a result, overall fractionation effect (net) is measured without knowing the
individual ’s or, even, where the limiting step occurs in the reaction sequence
C. Isotopic Fractionations during Denitrification
1. Laboratory experiments
a. In most experiments, the 15N of nitrate is measured as the nitrate pool is
reduced and the KIE is determined using a Rayleigh approach (Fig. 23)
b. Mariotti et al (Plant and Soil 62: 413-430, 1981) found a kinetic fractionation
factor (k15/k14) of 0.973 (-27 ‰) for denitrification at 20-30°C.
-they did soil incubations with supplemented NO3 additions
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14
-since this was a mixed culture of naturally occurring bacteria and the change in
the 15N of the NO3 was used to estimate the KIE, the measured net could possibly
include KIE during nitrification
b) Miyake and Wada 1971 cultured marine bacteria
-found net = 0.983± 0.003 (-17 ‰) (based on 7 time measurements)
c) Brandes measured effects of denitrification in bottle incubations of subsurface
water from denitrifying zones (Eastern subtropical Pacific) and found 0.980±?
(-20 ‰)
d) Granger et al. (Limno&Oceanogr. 53: 2533-, 2008) measured KIE that varied
from -5 to -25 ‰ for lab cultures of five strains of aquatic bacteria (Fig. 23)
2. Summary of culture and field estimates of KIE during denitrification from Granger et
al., 2008 (Fig. 24)
- lab (and field) derived KIEs in –20 to –30 ‰ range
3. Case Study: The impact of denitrification on the 15N of NO3 and N2 in Oxygen
Minimum Zones in the ocean (Brandes et al. Limnol&Oceanogr. 43: 1680-, 1998)
a. In the Eastern Tropical N. Pacific (ETNP) and Arabian Sea (AS), where the dissolved
O2 concentrations are low, water column denitrification is an important process that
results in a significant NO3 loss in the water column (Fig 25)
b. The magnitude of the NO3 loss due to denitrification is determined using measured
phosphate concentrations and assuming a Redfield ratio between NO3 and PO4 (~16) to
calculate the expected NO3 and then comparing to measured NO3
- where denitrification has removed NO3, there will be a nitrate deficit
c.There is a close correlation between the depth of maximum NO3 deficit and maximum
15N of NO3 and 15N of dissolved N2 gas (Fig. 25)
-circumstantial evidence for significant KIE during denitrification
d. A closed system Rayleigh distillation calculation would yield an ε of ~-25 ‰. (Fig. 26)
-ignores any NO3 production or consumption via other pathways (i.e., oxic
organic matter degradation, nitrification) and assumes a closed system (i.e., no mixing
with outside environment)
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e. If one opens up the system to mixing, then the calculated  accounts for the input of
"non-fractionated" NO3 into the region by mixing
-this situation is described mathematically as follows:
d(NO3)/dt = -D + Kmix*(NO3src - NO3),
where D= denitrification, src = the NO3 of water being mixed into the region and
Kmix represents the mixing rate with water parcels outside the region of denitrification
d(15NO3)/dt = -D*(15N/14N)*d + Kmix*(NO3src*(15N/14N)src-NO3*(15N/14N))
e. An “along isopycnal mixing” mixing model at steady-state would imply that the 15N
of the net NO3 flux into the region equaled the 15N of the nitrate lost to denitrification.
i.e. (15N/14N)NO3*d =
NO3 src *(15 N/14 N)src - NO3 (15 N/14 N)
(NO3src - NO3 )
- In the ETNP, the mixing model yields an ε of –30±3 ‰ and in the AS one
calculates an ε of –25±5 or –28±4 ‰ depending on the choice of the 15N and NO3
concentration for the outside source region (Fig. 26)
-in both cases, treating the system as open (vs closed) increased the magnitude of
the required KIE to explain the 15N vs NO3 trends
f. The 15N of dissolved N2 pool is also affected by the N2 produced during denitrification
-first, there is an excess of dissolved N2 over background levels in denitrifying
regions which is the results of N2 produced during denitrification
-second, there is a 15N minimum for dissolve N2 gas that corresponds to the 15N
maximum for NO3 (Fig. 25)
-assuming all the NO3 deficit (~10 umol/kg) yields N2 gas, then ~5 uM of N2 is
produced which would be ~ 1% of the background dissolved N2 concentration
- to produce a 0.3 ‰ depletion in the 15N of N2 as observed in ETNP and AS
(Fig. 25) for a 1% increment in N2 concentration, then the 15N of the N2 added must be
must be ~-30 ‰ depleted relative to ~ 0.5 ‰ for 15N of N2
-this rough calculation implies a net overall KIE of ~ -36 ‰ for denitrification
(NO3  N2), assuming all NO3 undergoing denitrification winds up as N2 without any
addition or loss of intermediates (NO2, N2O). This assumption could be questioned.
D. Isotopic fractionations during nitrification
1. Nitrification is the oxidation of ammonium to nitrite and subsequently to nitrate
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NH4+ + 1.5O2  NO2- + H2O +2H+
NO2- + 0.5O2  NO3- certain bacteria and archaea have the capacity for this process
- the nitrification process requires oxygen although it can occur in low O2
environments
-often a key process, when coupled with denitrification, in sewage treatment
removal of NH4
2. The KIE during nitrification (Fig. 27)
a. Lab cultures
- Mariotti et al (1981 Plant Soil 62: 413) used cultures of Nitrosomonas europaea
(mostly mM levels of NH4) yield range of -18 to -40 ‰ with the 14NH4 being consumed
faster than the 15NH4
-Recently, Casciotti et al (in Geobiomicrobiology, 2003) cultured different species
of Nitrosonomas and measured KIE values that ranged from -14 to -38 ‰ which seems to
be species dependent.
-Casciotti et al speculate that the apparently species dependent KIE may be
a result of different enzymes used for the nitrification in different species
b.Field Study: Horrigan et al (East Coast Shelf Science, 1990) present estimates of KIE
of –13 to –16 ‰ for nitrification in Chesapeake Bay although it’s not clear wheterh other
processes affect the 15N of NO3 and NH4
c.Under ambient conditions of low NH4 concentrations often encountered in the surface
ocean, there would be a tendency towards lower overall KIEs (net) (compared to
cultures) since the internal pool of NH4 could get very enriched under low NH4 supply
rates.
-if NH4 transport to cell limited nitrification (i.e., no NH4 leakage from cell), then
the KIE would approach the KIE associated with the NH4 transport (diffusion, active
transport across membrane), i.e., likely small
E. Nitrogen Cycling in Sediments (Fig. 28)
1. The nitrogen cycle in sediments is complex because of the multiply processes
concurrently occurring
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-e.g., several processes affect the concentration and 15N of NO3 in sediment
(or soil) pore waters (e.g., diffusion from overlying water, nitrification,
denitrification)
2. There are likely substantial KIEs during denitrification and nitrification
a. However, if all the NH4 or NO3 is consumed in certain pockets or
microzones of the sediments then little or no KIE may be expressed
b. Such a situation for denitrification (KIE  0 ‰) has been observed by
Brandes and Devol (Geochim Cosmochim Acta 1997) in coastal sediments.
3. By measuring the 15N and 18O of multiple species (NO3, NH4, NO2, N2O, N2)
combined with rate measured for nitrification and denitrification it may be
possible to unravel the causes of the 15N signatures associated with N cycling in
sediments
-see for example, papers by Brandes and Devol (GCA 1997, above) and
Lehmann (in Marine Chemistry 2004)
D. Using 15N to constrain the global ocean N budget
(based on paper by Brandes and Devol in Glob Biogeo Cycles 2002 )
1. The fixed or combined nitrogen (i.e., NO3 + NO2 + NH4) budget for the ocean has a
fair amount of uncertainty associated with it. (Fig. 29)
Sources: riverine input, atmospheric precipitation, N2 fixation
Sinks: denitrification, burial of organic N
2. Is the ocean’s combined N budget balanced?
a. riverine and atmospheric inputs are relatively small as is organic N burial
b. over the last decade the estimates of N2 fixation have increased from ~10
to ~100 Tg/yr but this rate is poorly known
c. likewise estimates of denitrification have increased from 30 to >100 Tg/yr
d. there is substantial uncertainty in estimates of both the N2 fixation rate and
the denitrification rate to prevent answering the question of whether the
ocean’s combined N budget is balanced.
3. Use 15N measurements to help constrain ocean combined N budget. (Fig. 29)
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a. 15N of N Sources: riverine = +4±4 ‰, atmospheric = -4±5 ‰ N2 fixation
= -1±1 ‰
b. 15N of N Sinks: organic N burial = 6±4 ‰, denitrification (water column)
= -20±3 ‰, denitrification (sediments) = 4±2 ‰
c. the key assumption is that if denitrification occurs in the water column a
significant KIE is expressed (~ -25 ‰), whereas if denitrification occurs in
the sediments then the KIE is negligible (~ 0 ‰)
- this is based on the measurements of KIE in water column and
sediments by Brandes and Devol
2. Brandes and Devol used N2 and 15N2 budgets to constrain the global ocean rate of N2
fixation rate and denitrification rates which are difficult to directly measure on a global
scale (Fig. 30)
-the mass and isotope budgets for combined nitrogen (N)
ΔN/Δt = River + Atm + Fix – Dentr – Burial
Δ15N/Δt = River*(15N/14N)riv + Atm*(15N/14N)atm + Fix*(15N/14N)fix –
Dentr(15N/14N)dentr – Burial*(15N/14N)orgN
-
use previous estimates of riverine and atmospheric inputs and organic N burial
-
loss
estimate the 15N/14N of the riverine and atmospheric N sources, organic N
burial, fixed N2 and N lost to denitrification
3. There is a significant difference between the 15N of NO3 lost via water column versus
sedimentary denitrification (Fig. 31)
- so the estimate of N2 fixation and denitrification rates will depend on the
assumed proportion of sedimentary vs water column denitrification
- assume the water column denitrification rate estimate at 75 Tg/yr is
reasonable and then solve for the amount of sediment denitrification and N2
fixation needed to yield balanced N and 15N budgets
4. Results of N and 15N budget constraints (Fig. 32)
a. A sedimentary denitrification rate of 240±40 Tg /yr is needed to offset
both the N2 fixation rate and a 15N of ~-1 ‰ for N2 fixation.
b. This implies a large N2 fixation rate of 200±100 Tg/yr
c. There are substantial uncertainties in the results
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5. The high rate of ocean N2 fixation implied by the N and 15N budget is much
higher than previous estimates based on ocean N2 fixation rate measurements
which yield a global rate of << 100 Tg/yr.
a. However extrapolating the few ocean rate measurements to a global scale
is very uncertain.
b. Thus either the N2 fixation rate in the ocean is much higher than we
thought OR the ocean’s N budget is not balanced
c. (If the N2 fixation rate is so high, why don’t we see 15N depleted organic
N buried in sediments? The 15N of sedimentary organic N is similar to
15N of NO3)
d. Is the ocean losing NO3 if currently the denitrification rate exceeds the N2
fixation rate?
6. Some evidence that the 15N of seawater NO3 has changed over the last 100,000’s
years which could imply that the ocean’s N budget has changed over time
-the evidence is indirect, i.e., the measured 15N of organic matter in sediments
-the approach assumes that if denitrification rates (water column) increased, then
the 15N of the nitrate pool would increase and the nitrate concentration would decrease
- the 15N of the organic nitrogen reflects the 15N of the NO3, which in turn
depends on the amount of denitrification
7. Altabet (Nature 2002) finds that the 15N of organic nitrogen in sediments from the
Arabian Sea (presently an area of intense denitrification) have varied substantially on
millennial time scales over the last 60,000 years (Fig. 33)
-finds 15N record correlates well with 18O ice core record which is an air
temperature proxy
-i.e., during periods of warmer air temps, more denitrification occurred
-he speculates that increases in denitrification rates in this region resulted from
higher productivity rates, which in turn resulted from higher rates of upwelling, i.e.,
stronger monsoons, during warm events.
-higher rates of denitrification enriched the 15N of the NO3 and, in turn, plankton
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8. Altabet finds that changes in the marine 15N organic matter record, when smoothed
over the 3000 yr residence time of NO3, look very similar to the changes in atmospheric
pCO2 record reconstructed from ice cores on millennial time scales (Fig. 34)
-he speculates that during extended periods of high 15N, ocean-wide nitrate
concentrations decreased due to increased (water column) denitrification and rates of
photosynthesis in the ocean decreased which, in turn, caused high concentrations of
atmospheric CO2
V. Questions
1. Why does the 15N of phytoplankton likely depends on nitrate concentration.
2. How does one calculate the 15N of the net nitrate flux?
3. Why is the 15N of NO3 and NH4 in soils and sediments difficult to interpret?
4. Why do the fractionation effects calculated for an open system differ from those
calculated for a closed system?
5. What key conditions should be met to make laboratory-derived values of fractionation
factors useful?
6. What are the problems associated with the applying a 2-step model (supply and
demand) to nitrogen assimilation?
7.Using the Rayleigh relationship, show why a plot of 15N vs ln[NO3] yields a slope
equal to the fractionation effect during assimilation in Fig. 12.
8. Use steady-state approach, rather than Rayleigh, to determine the KIE during NO3
uptake using the data presented in Fig. 12. Why might this approach be more appropriate
than a Rayleigh approach?
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