New Stable Isotopic Tools for the Marine Nitrogen Cycle Karen L. Casciotti WHOI, Department of Marine Chemistry and Geochemistry

Biogeochemical cycles of C, N, P The fact is that a lot of important nutrient cycles are driven biologically…well, there are important bio/physical/chemical coupling… We need to understand how these processes interact and affect each other. Gruber and Galloway 2008 Nature 451: 293-296

Increasing Greenhouse Gas Levels Atmospheric CO2 at Mauna Loa Observatory AGAGE Atmospheric N2O records N2O has 300x global warming potential per mole than CO2

Oxygen Deficient Zones ETNP Oxygen (mL/L) at 250 m Arabian Sea ETSP Suboxic regions have long been recognized as important regions of the world’s oceans, although our understanding of the N cycle in these regions has undergone substantial change in the past 5-10 years WOA05 data plotted in ODV Highly productive Sources of N2O Response to anthropogenic activity--expansion/intensification Quantitative importance in marine nitrogen budget

Suboxic Nitrogen Cycle N2, N2O Particulate NO3- Surface ocean N DON, NH3 Deep ocean N2 upwelling Anammox Remineralization NH3 NO2- NO3- Nitrification NO Major questions… N2 fixation in overlying waters? Anammox vs. denitrification? who is oxidizing ammonium? Denitrification N2O Burial N2 Major questions…

What role can a microbiologist play in understanding global biogeochemistry?

Stable Isotopes of NO3-, NO2-, and N2O in Air N2 d15N (‰) = [(15N/14Nsample / 15N/14NAIR) -1 ] *1000 16O: 99.76206% 17O: 0.03790% 18O: 0.20004% in Standard Mean Ocean Water d18O (‰) = [(18O/16Osample / 18O/16OSMOW) -1 ] *1000

The Denitrifier Method: Simultaneous N and O Isotopic Analyses Pseudomonas aureofaciens or Pseudomonas chlororaphis Inject NO3- or NO2- sample Denitrification takes place NO3- NO2- NO N2O N2 N2O (g) Analyze N2O isotopes Sigman et al., 2001. Analytical Chemistry: 15N-NO3- Casciotti et al., 2002. Analytical Chemistry: 18O-NO3-

Peru Upwelling: d15NO3- 19 18 17 10 16 11 12 15 10 11 12 13 14 16 17 19 13 14

Rayleigh Model for Isotope Fractionation in a Closed System d15NH4+ (substrate) d15NO2- (instantaneous product) d15Nsubstrate ≈ d15Ninitial substrate - e * ln(f) e d15NO2- (accumulated product) e BEGINNING d15Naccumulated product ≈ d15Ninitial substrate + e * f ln(f)/(1-f) END

Linearized Rayleigh Model Substrate Product Substrate f*ln(f)/(1-f) d15N Product e d15N e -ln(f) f = the fraction of initial substrate remaining

Casciotti and Voytek, unpublished Phylogeny of June Ammonia monooxygenase Clones from Sugar Creek, Indiana Stream nitrifier community 67 = 14.2 ± 0.5‰ Casciotti and Voytek, unpublished Nitrosospira tenuis 98 79 = 24.6 ± 1.4‰ 90 60 91 81 Nitrosomonas marina = 14.2 ± 3.6‰ Nitrosomonas sp. C-113a 74 = 19.1 ± 1.2‰ 72 Nitrosomonas eutropha 96 = 32.8 ± 1.7‰ 100 Nitrosomonas europaea = 38.2 ± 1.6‰ 98

Important to know who is there and who is active Potential for variability in isotope effects within a ‘functional group’ Important to know who is there and who is active

Stable Isotope Fractionation at the Cell Level Nitrifier cell periplasm Transport? NH4+ NH4+ expt eeq eeq cytoplasm ediff eamo ehao NH3 NH3 NH2OH NO2- NO2- ediff Diffusion eoverall is dominated by ediff Without transport With transport eoverall is dominated by expt

Potential for variability within a given species Important to know physiological factors that may play a role

d15N variations in NO3- 20 Water column denitrification e  20-30 ‰ 15 d15NO3- (‰ vs. AIR) Nitrate assimilation e  5-10 ‰ 10 Nitrogen fixation d15N  0 ‰ 5 Sedimentary denitrification e  0 ‰ 0.5 1 1.5 2 [NO3-]/[NO3-]initial Sigman and Casciotti, 2001 EOS

d15N variations in NO3- d15NNO3 d15NNO3 N2 Fixation Low d15N Depth (m) High d15NO3- Depth (m) Denitrification Arabian Sea ETNP d15NN2 d15NN2 Brandes et al. (1998) L&O Gross rates of nitrogen fixation and denitrification are difficult to diagnose.

Coupled d15N and d18O analyses of nitrate N isotopes O isotopes Nitrogen fixation N2 Denitrification N2 Denitrification Norg Norg Assimilation Assimilation NO3- NO3- Nitrification Not going to have time to talk about nitrification/oxygen isotope work, but it goes here… Nitrification NH4+ NH4+ O2, H2O

Coupled N and O isotopes of NO3- 20 15 (‰ vs. VSMOW) d18O-NO3- 10 Denitrification 15e/ 18e  1 (Granger et al., 2006) Nitrate assimilation 15e/ 18e  1 5 (Granger et al., 2004) Deep Ocean 5 10 15 20 d15N-NO3- (‰ vs. AIR)

Coupled N and O isotopes of NO3- 20 15 (‰ vs. VSMOW) d18O-NO3- 10 5 Remineralization/Nitrification 5 10 15 20 d15N-NO3- (‰ vs. AIR)

Coupled N and O isotopes of NO3- 20 15 (‰ vs. VSMOW) d18O-NO3- 10 N2 fixation/ Nitrification D(15,18) 5 Remineralization/Nitrification 5 10 15 20 d15N-NO3- (‰ vs. AIR) D(15,18) = (d15N - d15Ndeep) - 15e/18e * (d18O - d18Odeep) Sigman et al., 2005

Coupled N and O isotopes of NO3- 20 15 (‰ vs. VSMOW) d18O-NO3- 10 N2 fixation/ Nitrification D(15,18) 5 Remineralization/Nitrification 5 10 15 20 d15N-NO3- (‰ vs. AIR) D(15,18) = (d15N - d15Ndeep) - 15e/18e * (d18O - d18Odeep) Sigman et al., 2005

Nitrate d15N and d18O deviations 20 N2 Fixation Low d15N Remineralized PN from Nitrogen fixation 15 Nitrite reoxidation High d15NO3- d18O-NO3- (‰ vs. VSMOW) 10 Nitrite reoxidation Denitrification Denitrification 15e/ 18e  1 5 5 10 15 20 NO3- NO2- N2 d15N-NO3- (‰ vs. AIR) D(15,18) = (d15N - d15Ndeep) - 15e/18e * (d18O - d18Odeep) Sigman et al., 2005

ETNP NO3- isotope studies Map from Cline and Kaplan, 1975 Sigman et al. 2005 --> 65% of Denitrified N is replaced by nitrification of newly fixed N. Brandes et al. 1998 Cline and Kaplan 1975 This study Voss et al. 2001 Sutka et al., 2004 Sigman et al. 2003, 2005 Altabet et al., 1999

The Denitrifier Method: Simultaneous N and O Isotopic Analyses Pseudomonas aureofaciens or Pseudomonas chlororaphis Inject NO3- or NO2- sample Denitrification takes place NO3- NO2- NO N2O N2 N2O (g) Analyze N2O isotopes Sigman et al., 2001. Analytical Chemistry: 15N-NO3- Casciotti et al., 2002. Analytical Chemistry: 18O-NO3-

The Denitrifier Method: Simultaneous N and O Isotopic Analyses Pseudomonas aureofaciens or Pseudomonas chlororaphis Inject NO3- or NO2- sample Denitrification takes place NO3- NO2- NO N2O N2 Granger et al., 2006 N2O (g) Analyze N2O isotopes Sigman et al., 2001. Analytical Chemistry: 15N-NO3- Casciotti et al., 2002. Analytical Chemistry: 18O-NO3-

Nitrite isotope subtraction Pretreatment Ascorbate NO2- Isotopic analysis Denitrifier Azide Denitrifier d15N, d18O (NO3- + NO2-) d15N, d18O (NO2-) d15N, d18O (NO3-) 2 Subtract d15N, d18O (NO3-) 1 Compare

ETNP Nitrate Isotope Data d15N d18O Casciotti and McIlvin, 2007 Marine Chemistry

ETNP Nitrate and Nitrite Isotopes d15N (‰), D(15,18) (‰), and [NO2-] (mM) d15NNO3 d15NNO2+NO3 d15NNO2 D(15,18) D(15,18) = (d15N - d15Ndeep) - 15e/18e * (d18O - d18Odeep) Casciotti and McIlvin, 2007 Marine Chemistry

ETNP Nitrate and Nitrite Isotopes d15N (‰), D(15,18) (‰), and [NO2-] (mM) d15NNO3 d15NNO2+NO3 d15NNO2 D(15,18) Casciotti and McIlvin, 2007 Marine Chemistry

Model Interpretation of D(15,18) 20 Mixing with shallower water Remineralized PN from Nitrogen fixation 15 of diazotroph biomass Remineralization d18O-NO3- (‰ vs. VSMOW) Nitrate reduction Denitrification (or Anammox) 10 Nitrite reoxidation NO3- NO2- Denitrification 15e/ 18e  1 Nitrite “reoxidation” 5 Mixing with deeper water 5 10 15 20 d15N-NO3- (‰ vs. AIR) Based on Sigman et al. 2005 model

Model Results NO3- NO2- N2 Casciotti and McIlvin, 2007 Marine Chemistry

ETNP Nitrate and Nitrite Isotopes d15N (‰), D(15,18) (‰), and [NO2-] (mM) d15NNO3 d15NNO2+NO3 d15NNO2 Nitrite reoxidation at top of omz could be…0.6 umol m-2 d-1 D(15,18) Nitrite reoxidation going on…additional information needed

Nitrite Oxidation in the ETSP Nitrite oxidation rate nmol/L/day [NO2-] mmol/L Lipschultz et al., 1990: Showed nitrite oxidation below SNM and associated w/PNM but little at the top of the OMZ in the ETSP Nitrate reduction rate nmol/L/day [O2] mmol/L

ETNP Samples d15N (‰), D(15,18) (‰), and [NO2-] (mM) d15NNO3 d15NNO2+NO3 d15NNO2 D(15,18)

Isotope effects for nitrite reactions d15N Nitrite oxidation 15eNO 15eNR NO3- NO2- d15N Nitrate reduction Initial condition NO3- 15eNR NO3- NO2- NO2- N2 15eNIR Initial condition Nitrate reduction Nitrite reduction

Nitrite reduction by (de)nitrifying bacteria 14NO2- 14NO 15NO2- 15NO Casciotti, Sigman, and Ward, unpublished

Isotope effects for nitrite reactions d15N Nitrite oxidation 15eNO 15eNR NO3- NO2- d15N Nitrate reduction Initial condition NO3- 15eNR NO3- NO2- NO2- 15eNIR N2 Initial condition Nitrate reduction Nitrite reduction

Nitrite oxidation by Nitrococcus mobilis NO2- NO3- “Inverse isotope effect” 14NO2- 14NO3- d15NNO3 15NO2- 15NO3- d15NNO2

Isotope effects for nitrite reactions d15N d15N NO3- NO3- NO3- 15eNR 15eNR NO3- NO3- NO2- NO3- NO2- 15eNIR NO2- 15eNO NO2- N2 Initial condition Nitrate reduction Nitrite reduction Initial condition Nitrate reduction Nitrite oxidation

ETNP Samples d15N (‰), D(15,18) (‰), and [NO2-] (mM)

Implications for interpretation of D(15,18) Now required nitrite oxidation rate less than 0.9 mM/yr and oxidation/reduction ratio ~0.7 Can’t rule out a contribution from N2 fixation, but… Many indicators consistent with nitrite oxidation: Location of D(15,18) minimum Low nitrite d15N Large nitrate/nitrite d15N offset Inverse isotope effect for nitrite oxidation Strength of D(15,18) minimum

Implications for interpretation of D(15,18) Now required nitrite oxidation rate less than 0.9 mM/yr and oxidation/reduction ratio ~0.7 Can’t rule out a contribution from N2 fixation, but… Many indicators consistent with nitrite oxidation: Location of D(15,18) minimum Low nitrite d15N Large nitrate/nitrite d15N offset Inverse isotope effect for nitrite oxidation Strength of D(15,18) minimum

Suboxic Nitrogen Cycle N2, N2O Particulate NO3- Surface ocean N DON, NH3 Deep ocean N2 upwelling Anammox Remineralization NH3 NO2- NO3- Nitrification NO Major questions… N2 fixation in overlying waters? Anammox vs. denitrification? who is oxidizing ammonium? Denitrification N2O Burial N2

Ongoing and Future Work Oxygen isotopic systematics of ammonia oxidation Nitrogen and oxygen isotopic systematics of nitrite oxidation Interpret coupled N and O isotopic signatures from nitrate and nitrite using more sophisticated models Extend analysis to additional ODZs (Arabian Sea; Benguela; GEOTRACES)

Acknowledgements Matthew McIlvin Danny Sigman ETNP work Carrie Tuit Penzance Assistant Scientist Endowed Support NSF Chemical Oceanography Nitrite Oxidizer work Carly Buchwald