Diapositive 1

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NITROGEN FIXATION IN THE EQUATORIAL
PACIFIC RATES, DIVERSITY, NUTRIENT LIMITATION
AND BIOGEOCHEMICAL IMPLICATIONS
EUC-Fe Kilo Moana Cruise 0625 Data Workshop,
Seattle
Sophie Bonnet WiSE Postdoctoral Fellow
University of Southern California - Los Angeles
Doug Capone Lab, Department of Biological Sciences
18-19 September 2007
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NITROGEN FIXATION WHY DO WE CARE?
N2
Endless source of nitrogen
atmosphere
ocean
Prokaryotes
Diazotrophs (prokaryotes)
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NITROGEN FIXATION WHY DO WE CARE?
New Nitrogen
Prokaryotes
Sustains new primary production
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EVOLUTION IN THE PERCEPTION OF N2 FIXATION
Large cyanobacteria (gt 10µm) - Trichodesmium
Explain only 40-59 of nitrogen fixation predited
by geochemical methods (N, stable isotopes)
(Mahaffey et al., 2005)
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N2 FIXATION IN THE EQUATORIAL PACIFIC
N2 fixation suggested in the warm pool - delta
15N settling particles (Yoshikawa et al.,
2005) - total organic nitrogen measurements
(Hansell and Feely, 2000) ? No direct
measurements
Surface nitrate gradient (S. Pal)
Surface DFe gradient (L. Ossiander)
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PROTOCOLE
Labelling technique 15N2 (Montoya et al. 1996)
15N (atom ) Europa Integra continuous flow mass
spectrometer
Incubation in presence of 15N2 gas
Gentle filtrations (2 size fractions)
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PROTOCOLE

• Equatorial stations
• Vertical profiles (50 - 10 - 1 - 0.1 surface
  irradiance)
• Each depth
• total N2 fixation (2 replicates)
• size fractionnation (2 replicates per fraction)
• gt 10µm
• lt 10µm

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TRACE METAL CLEAN CONDITIONS!!!
NITROGENASE
N2 8H 8e- ? 2 NH3 H2
NH3
Fe-Mo protein (7 Fe atoms)
Diazotrophs 10-fold higher Fe requirements than
than non diazotrophic phytopk
Station 25 TMC versus non TMC conditions
Culture Fe limits growth and N2 fixation of
Trichodesmium (Rueter 1988 Pearl et al., 1992)
Field Fe limits nitrogen fixation in the North
tropical Atlantic (Mills et al., 2004)
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TRACE METAL CLEAN CONDITIONS!!!

• Trace metal clean sampling AND incubation
• Go flo (in the bubble)
• Clean tubes (Suprapur HCl)
• Clean bottles (Suprapur HCl)

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Before..
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RESULTS (Total N2 Fixation - nmol N.l-1.h-1)
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RESULTS (Size fractionnation)
25 of Total N2 fixation
gt 10µm
Station 27 25 22 20 18 16 14
Trichomes.l-1 1849 175 20 0 0 0 0 0
Adrian Marchetti
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RESULTS (Size fractionnation)
25 of Total N2 fixation
gt 10µm
Characterisation of the assemblage by molecular
tools
75 of Total N2 fixation
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RESULTS - Molecular data
Collaboration Isabelle Biegala - IRD Marseille
(France)
3 size fractions gt10µm 3-10µm 0.2-3µm
TSA-FISH technique whole cell specific
fluorescent probes hybridization (Biegala et al.
2003, 2005).
Abundance of unicellular diazotroph in each size
fraction
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RESULTS - molecular data
Dominance of pico diazotrophs
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RESULTS - molecular data
Free picodiazotrophs (station 18)
Symbiosis with dinoflagelates (stations 10 27)
Tricho (station 27)
Pictures from Isabelle Biegala
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RESULTS - molecular data

• Summary
• 75 of N2 fixation occurs in the small size
  fraction
• molecular data small size fraction pico
  diazotrophs (free or attached)
• Cf lagon noumea (Biegala et al., in rev)

micro
nano
pico
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BIOGEOCHEMICAL IMPLICATIONS
Areal rates 32 to 268 µmol N.m-2.d-1
Contribution of N2 fixation to PP 0.8
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Different species / different ecological niches?
gt 10µm
lt 10µm
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Different species / different ecological niches?

• What controls N2 fixation in the ocean?
• P Sañudo-Wilhelmy et al. 2001 Dyhrman et al.
  2002 Krauk et al. 2006 Sohm Capone 2006
• Fe Rueter 1988 Pearl et al., 1992 Mills et
  al., 2004
• Temperature Staal et al., 2003 Breitbarth et
  al., 2006
• ? CO2 Hutchins et al., 2007

Different controlling factors for different
diazotrophs in the open ocean.
Nature of limitation of diazotrophy changes over
time and space
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Trichodesmium dominates at high DFe levels
gt 10µm
DFe
DFe data from L. Ossiander
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Different species / different ecological niches
Fe-Mo protein (7 Fe atoms)
NITROGENASE
Unicellulars diazotrophs
Trichodesmium
High cellular Fe quotas 30 µmol Fe mol
C (Berman-Franck et al., 2001)
?
Higher S/V (x4) ? outcompet Tricho at low Fe?
Fe limits N2 fixation in 75 of the global
ocean!!!
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The role of Fe on unicellular diazotrophs
(Post-doc)
Crocosphaera watsonii (WH 8501)
Bonnet, Unpublished data
Cellular quotas 0.3 µmol Fe mol
C (Berman-Franck et al., 2007)
100 times lower than Trichos
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Different species / different ecological niches
gt 10µm
gt 10µm
DFe
DFe data from L. Ossiander
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IS IT NECESSARY TO WORK UNDER TMC CONDITIONS?
Why? Fe unintended fertil? (DFe Lia)
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RESULTS FROM Fe FERTILISATIONS

• Different combinations of nutrients added
• Fe
• P
• N
• Si
• deepwater

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Grow out exp
No effect of Fe , P and FeP additions on N2
fixation
Units nmol.l-1.d-1
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WHY WORKING UNDER TMC CONDITIONS?
Why do we observe such differences in TMC versus
non TMC?
Hypothesis other metals exert a control on N2
fixation
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WHY WORKING UNDER TMC CONDITIONS?
Produce large quantities of B12
Crocosphaera watsonii (WH 8501)
2 fmol.cell-1 (Bonnet, Panzeca)
Vitamin B12
Co
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The role of Cobalt
B12 synthesis N expensive process
C63H88CoN14O14P

• Dual ecological function of diazotrophs in
  N-depleted environments?
• Source of new N (sustains new PP)
• Source of B12 for eukaryotes

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SUMMARY

• First rates measured in the eq pac N2 fixation
  well established
• E-W gradient (very high rates close to PNG)
• - Minor biogeochemical importance in HNLC waters
• - Major importance in the warm pool (up to 70 of
  PP)
• The small size fraction represents 75 of total
  N2 fixation
• Dominance picodiazotrophs - importance of
  symbiosis
• Necessity to work under trace metal clean
  conditions

Article in prep. For L0. Bonnet, S., Biegala,
I., Capone, D.G., Ossiander, L., Dutrieux, P.,
Murray, J. Nitrogen fixation in the Equatorial
Pacific Ocean.