OC583: ISOTOPE BIOGEOCHEMISTRY (Spring 2009)
TOPIC 5: NITROGEN CYCLE
I. Background (Fig. 1)
1. Most of Nitrogen is in atmosphere (4x1015 tons)
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-hydrosphere = 10 tons
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-soils+sediments = 10 tons
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-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 possibly 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)
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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 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., although 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)
-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
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-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
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
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-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
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)
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-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
-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)
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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.
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)
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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
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
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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
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- 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
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)
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- 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%
winter FN2 = (1.0048-1.0065)/(1.0004-1.0065) = 28%
annual 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%).
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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)
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