1. 1 Nitrogen Cycle Most of Nitrogen is in the atmosphere. 14 N = 99.6% 15 N = 0.4% Air is standard for  15 N Range is –20 to +20 ‰

2. 2 Assimilation and Dissimilation Nitrogen Cycling Pathways Assimilation NO 3 and NH 4 uptake (NO 3 or NH 4  org N) N 2 Fixation (N 2  org N) Dissimilation -Mineralization (org N  NH 4 ) -Nitrification (NH 4  NO 3 ) -Denitrification (NO 3  N 2 ) -Anammox (NO 2 + NH 4  N 2 ) (in marine systems)

3. 3  15 N Range in Terrestrial Plants Non N-fixers = -6 to +6 ‰ N-Fixers = 0±3 ‰

4. 4 KIE during Nitrate Assimilation by Terrestrial Plants Large range of KIE values measured in culture experiments Range: - 3 to –24 ‰ (Mariotti, 1980) -3 to –7 ‰ (Kohl and Shearer, 1980) -0.3±0.6 ‰ (38 species) (Mariotti, 1982) Measured KIE depends significantly on culture conditions (growth rate, NO 3 concentration) Typically, KIE increased with increased nitrate concentration.

5. 5 N 2 Fixation in Plants Plants have  15 N = 0±3 ‰ similar to  15 N of N 2 in air. - molecular diffusion of N 2 is air (soil pockets) yields KIE = -9 ‰. N 2 diffusion in water has KIE = -1.3 ‰ -also you would expect a significant KIE during the breaking of a N-N bond to convert to NH 4 -this seems to imply that aqueous N 2 diffusion limits N 2 fixation. What other explanation? Haber Process (N 2 + 3H 2  2NH 3 ) yields commercial fertilizers with  15 N of ~ 0±3 ‰ (high temp (>300ºC) and complete conversion  little KIE)

6. 6 KIE during NO 3 and NH 4 Uptake in Plankton Cultures KIE ranged from 0 to –15 ‰ with a mean of ~ –7 ‰

7. 7 Effect of growth rate of KIE in cultures As growth rate increased, KIE decreased. High concentrations (mmolar) of NO 3 and NH 4. NO3 Substrate + NH4 Substrate A A

8. 8 KIE during N 2, NO3 and NH4 uptake (same species Anabaena) KIEs N 2 = -2 ‰ NO 3 = -11 to –13 ‰ NH 4 = -14 ‰

9. 9 KIE during NO 3 assimilation under closer to ambient concentrations (uM vs mM) How useful are culture derived KIEs?

10. 10  15 N variations in marine plankton Three Ocean Regimes NO 3 replete (I) NO 3 limited (II) N 2 fixation (III)

11. 11  15 N of Plankton (PON) in Mixed Layer depends on NO 3 Concentration

12. 12 Effect of KIE on  15 N of NO 3 KIE during nitrate assimilation enriches  15 N of remaining NO 3 pool Both regions yields a KIE = -9.1 ‰. What is advantage of this approach? Southern Ocean Sta Papa

13. 13  15 N of NH 4 in Delaware Estuary KIE for NH4 uptake = -20±? ‰ (Rayleigh) -NH 4 uptake -nitrification -sewage NH 4 input

14. 14 Summary of KIE during N Assimilation How useful are culture KIE determinations?

15. 15 Steps in nitrate assimilation ~15 ‰ offset between internal and external NO 3 pools

16. 16  15 N based N Budget at ALOHA and BATS At ALOHA, N 2 fixation contributes 50% of new nitrogen. At BATS, N 2 fixation is a negligible N source. ALOHA near Hawaii BATS near Bermuda

17. 17 Assimilation and Dissimilation Nitrogen Cycling Pathways Assimilation NO 3 and NH 4 uptake (NO 3 or NH 4  org N) N 2 Fixation (N 2  org N) Dissimilation -Mineralization (org N  NH 4 ) -Nitrification (NH 4  NO 3 ) -Denitrification (NO 3  N 2 ) -Anammox (NO 2 + NH 4  N 2 ) (in marine systems)

18. 18  15 N of OM trends in soils  15 N vs %N  15 N vs C/N Are these trends a result of KIE during remineralization?

19. 19  15 N of NO 3 and NH 4 released during a Soil Incubation Experiment NO 3 NH 4  15 N The NO 3 produced during OM degradation is 15 N depleted, whereas the NH 4 is enriched (initially) relative to OM. Complicated interpretation because there is OM mineralization, nitrification and denitrification occurring simultaneously, all of which likely have different KIEs.

20. 20  15 N of Organic N in Sediment Traps N. Atlantic Bloom ConditionsEquatorial Pacific

21. 21 Denitrification Converts nitrate (NO 3 )  Nitrite (NO 2 )  Nitrous Oxide (N 2 O)  Nitrogen gas (N 2 ) Major NO 3 loss pathway in the ocean. Anammox is another pathway to convert combined N to Nitrogen gas (NH 4 + NO 2  N 2 + 2H 2 O) Denitrification occurs under low Oxygen concentrations (e.g., sediments, soils, certain ocean regions (OMZs), etc.)

22. 22 Denitrification If none of the NO 2 or N 2 O produced via denitrification is removed, diluted with other sources or affected by reactions other than denitrification (e.g., anammox), then the 15 N/ 14 N of the N 2 produced during denitrification would equal the 15 N/ 14 N of nitrate times the KIE for NO 3 reduction to NO 2. ( 15 N/ 14 N ) NO3 *  NO3-NO2 = ( 15 N/ 14 N ) NO2 *  NO2-N2O ( 15 N/ 14 N ) NO2 *  NO2-N2O = ( 15 N/ 14 N ) N2O *  N2O-N2 ( 15 N/ 14 N ) N2O *  N2O-N2 = ( 15 N/ 14 N ) N2

23. 23 Culture Measurements of KIE for Denitrification Typically, the KIE for denitrification is measured by the measuring the 15 N/ 14 N trend of nitrate pool

24. 24 Summary of KIE for Denitrification in Cultures, Soils and Ocean (Granger, Limnol&Oceanogr, 2008)

25. 25 Depth Profiles of NO 3 deficit,  15 N of NO 3 and N 2 in ETNP and Arabian Sea Similar depth for: - Nitrate deficit max. -  15 N NO 3 max. -  15 N N 2 min.

26. 26 Estimate of KIE for Denitrification in ETNP and AS: field data Closed System: KIE = -25 ‰ (ETNP) and –22 ‰ (AS) Open System: KIE = -30±3 ‰ (ETNP) and –25±5 to –28±4 ‰ (AS) = ETNP  = AS

27. 27 KIE during Nitrification in Cultures Previous lab cultures = -18 to –40 ‰ ( Mariotti 1981 ) Field Study (Chesapeake Bay) = -13 to –16 ‰ ( Horrigan et al., 1990)

28. 28 Nitrogen Cycling in Sediments Ocean Water Sediments

29. 29 Ocean’s Combined Nitrogen Budget Large uncertainties in the rates of N 2 fixation and denitrification.

30. 30 Combined N and 15 N Budgets ΔN/Δt = River + Atm + Fix – Denitr – Burial Δ 15 N/Δt = River*( 15 N/ 14 N) riv + Atm*( 15 N/ 14 N) atm + Fix*( 15 N/ 14 N) fix – Denitr( 15 N/ 14 N) dentr – Burial*( 15 N/ 14 N) orgN Estimate three of the rates: river, atmos and burial Estimate the 15 N/ 14 N of all the terms Solve for N 2 fixation and denitrification rates.

31. 31 KIE for denitrification One significant complication is that the KIE assigned for denitrification depends significantly on the proportion of denitrification that occurs in the water column (KIE~ -20 ‰) versus in the sediments (KIE ~ -1 ‰). Thus the solution to the N and 15 N budget depends on the proportion of sedimentary vs water column denitrification. sediments Water column

32. 32 Combined N and 15 N Budgets Results: The only way to get an isotopic balance is to have very high rates of denitrification and N 2 fixation. Sedimentary Denitrification Rate = 200 - 280 Tg/yr N 2 Fixation Rate = 100 - 330 Tg/yr (direct meas. << 100Tg/yr) -substantial uncertainty in budget approach.

33. 33  15 N of Organic N in Ocean Sediments Correlation between changes in  15 N of organic N in sediments in the Arabian Sea with ice core  18 O over last few glacial cycles.

34. 34 Correlation between marine  15 N and atmospheric pCO 2 changes Is there a link between denitrification controlling NO 3 concentrations in the ocean and biological drawdown of atmospheric CO 2 ?