1. The Ecology of Nitrogen Cycling Microorganisms in the Subtropical North Pacific Ocean

2. Thank you…
Daniela Böttjer, Brenner Wai, Donn Viviani, Sara Thomas, Christina Johnson
Dave Karl, Jon Zehr, Ed DeLong
NSF

3. A Dedicated HOT Team
NSF
NSF

4. N cycle stories from the open sea
How do seasonal to episodic changes in the upper ocean habitat influence distributions, abundances, and activities of N cycling microorganisms?
N2 fixing cyanobacteria
Ammonia oxidizing Crenarchaea
Focus on epipelagic N- cycle dynamics:
Concentrations of inorganic N low
Rapid N turnover
Intense vertical gradients in microbial habitat structure (light, nutrients, temperature, etc.)
Sensitive to physical perturbations (seasonal to episodic)

5. Time, water, and change
The 24 years of Hawaii Ocean Time-series (HOT) measurements provide a rich time-history for assessing change to a persistently oligotrophic ocean ecosystem.
The resulting data provides insight into the mean ecosystem state, and variability around the mean state.
ALOHA
Photo: Paul Lethaby

6. The upper ocean habitat
14C-PP
PAR
Chl a
N+N

7. These processes are time variable
Physical and biological controls on nitrogen availability to the upper ocean
N2 fixation
Physical:
Mixing
Upwelling, downwelling
Biological:
N2 fixation
Remineralization
(e.g. ammonification, nitrification)
Organic matter
NH4+
NO2-
NO3-
These processes are time variable
Organic matter
NO3-
NO2-
NH4+

8. Seasonal variations in upper ocean temperature, light, and nutrients

9. Seasonal climatology of primary production and particle export
Productivity and export both elevated in summer when upper ocean nutrient concentrations are lowest.
What processes supply nitrogen to support new production in this ecosystem?

10. Biological N supply to the ocean: N2 fixation
At ALOHA, N2 fixation fuels ~50% of new production (and hence carbon export)
Several groups of N2 fixing microorganisms identified
Regular occurrence of large blooms of N2 fixing microorganisms; Trichodesmium spp. and/or diatoms with endo/epi-symbionts
Changes in elemental stoichiometry of particulate and dissolved nutrient pools (decreased P availability) attributed to N2 fixation.

11. Motivating Questions
How variable in time are N2 fixing microbes and rates of N2 fixation?
What processes control variability in diazotroph population structure and rates of N2 fixation?

12. nifH → Nitrogenase (N2 fixers)
Approach
Combined rate measurements of N2 fixation together with analyses of time/space variability in distributions and activities of N cycle microbes in the sea.
nifH → Nitrogenase (N2 fixers)

13. N2 fixing cyanobacteria
at Station ALOHA based on nifH gene transcripts
Richelia-1
Richelia-2
Richelia-1
20-50 mm
Heterocystous cyanobacteria
Richelia-1
>10 mm
Richelia-2
Calothrix
2-10 mm
Group A
Unicellular cyanobacteria
Calothrix
Crocosphaera
Trichodesmium /
Katagynemene
20-50 mm
Trichodesmium spp.
Katagynemene spp.
Images courtesy of
Angel White, Rachel Foster, Grieg Steward mm mm

14. Several cultivated strains (in culture since 1985) Uncultivated
Crocosphaera
“Group A”
Several cultivated strains (in culture since 1985)
Uncultivated
Fixes N2 at night
Likely fixes N2 during day
Photosystem I & II
Photosystem I
Functional TCA cycle
No TCA cycle
Photoautotroph
Photoheterotroph
“Free” living
Symbiont?
Church et al. (2005)

15. 0-125 m
Abundances of unicellular N2 fixing cyanobacteria vary seasonally and interannually

16. Temporal variability in N2 fixation
Major fraction (~70%) of annual N2 fixation associated with microorganisms <10 mm.
Rates of N2 fixation tend to increase in the summer, driven by microorganisms > 10 mm.

17. Unicellular N2 fixers are dominate diazotroph abundances and activities most of the year
Filamentous diazotrophic cyanobacteria increase episodically through the summer and fall
0-125 m
Modified from Church et al. (2009)

18. Spatiotemporal history of a
January 2005
Spatiotemporal history of a
downwelling eddy
April 2005
Fong et al. 2008
July 2005
July 2005
“typical profile”

19. Time series measurements of near-surface ocean N2 fixation at Station ALOHA
Episodic increases in N2 fixation by diazotrophs >10 mm appear associated with mesoscale physical forcing
Modified from Church et al. (2009, GBC)
nmol N L-1 d-1

20. OPEREX: July 2009 CMORE cruise in the subtropical North Pacific Ocean
Meso- and submesoscale physical processes may drive enhanced carbon export in the open sea
OPEREX: July 2009 CMORE cruise in the subtropical North Pacific Ocean
Chlorophyll a
N2 fixing microorganism biomass accumulation
SSHa
2 white transects
Cruise track across eddies
Guidi et al. 2012
Large particle concentration

21. What is the time history associated with these processes?
Property
(0-200 m)
SSHa - +
Primary production 
Carbon export
NO3-  
NO3- : PO43-
Chl a
Prochlorococcus
Diatoms
Pelagophytes
Haptophytes
N2 fixation
What is the time history associated with these processes?
How does such mesoscale variability influence plankton ecology?

22. Variability in diazotrophs and N2 fixation
Unicellular N2 fixing microorganisms are important contributors to the upper ocean N cycle
Different groups of N2 fixing microorganisms have very different physiologies and population dynamics
Larger, filamentous N2 fixing microorganisms (Trichodesmium, heterocystous N-fixers) increase seasonally, but episodically
Mesoscale physical processes appear important drivers of episodic N2 fixation supported plankton blooms

23. Nutrient recycling: Dynamics of ammonia oxidizing Thaumarchaea
N2 fixation
amoA gene as a molecular marker to examine the time/space variability in Thaumarchaeal distributions and transcriptional activities
Organic matter
NH4+
NO2-
NO3-
amoA
Organic matter
NO3-
NO2-
NH4+

24. Nitrification and ammonia oxidation
Nitrification is the two-step process that converts ammonia to nitrate.
Ammonia oxidation: NH3 + O2 → NO2− + 3H+ + 2e-
Nitrite oxidation: NO2− + H2O → NO3− + 2H+ + 2e-
Different steps are mediated by different groups of prokaryotic microorganisms
Ammonia oxidation historically thought to be catalyzed by ammonia oxidizing bacteria (b and g-Proteobacteria)
Now thought to be predominately driven by members of the Thaumarchaeota (previously classified as mesophilic Crenarchaeota).

25. Karner et al. (2001) Könneke et al. (2005)
Archaea first identified in seawater in 1992
Found to dominate plankton biomass in the meso- and bathypelagic waters at Station ALOHA
Sequencing efforts in Sargasso Sea discovered gene encoding ammonia monooxygenase from planktonic Thaumarchaea (Venter et al. 2004)
Isolation of marine Thaumarchaea (Nitrosopumilus maritimus) from Seattle Aquarium found to grow using ammonia as sole source of energy, CO2 as carbon source
Könneke et al. (2005)

26. Only cultivated representative of marine Thaumarchaea
“Upper ocean” Thaumarchaea
Only cultivated representative of marine Thaumarchaea
“Deep ocean” Thaumarchaea
Comparison of Thaumarchaea amoA genes indicates at least two vertically partitioned groups (upper and deep ocean) –
Mincer et al. (2007), Beman et al. (2008).
At Station ALOHA there appear to be “sub-clades” of these vertically partitioned major groups.

27. Brenner Wai, Christina Johnson
Vertical distributions of these major groups are distinct, with the transition region between the euphotic zone and the upper mesopelagic comprising a dynamic transition point.
1% PAR
0.1% PAR
Brenner Wai, Christina Johnson

28. Distributions of Thaumarchaea amoA genes appear sensitive to upper ocean mixing
108
107 50
106
100
amoA genes L-1
105
Depth (m)
104
150
103
200
102
2007
2008
2009
2010
Year
What processes control the vertical distributions of Thaumarchaea?
Physics (mixing, upwelling, downwelling)?
Light?
Competition for substrates?
Top down (predators, viruses)?
Brenner Wai et al.

29. Thaumarchaea amoA expression and rates of ammonia oxidation at Stn
Thaumarchaea amoA expression and rates of ammonia oxidation at Stn. ALOHA
Transcription of the amoA gene often peaks in the transition zone between the euphotic zone and the upper mesopelagic
Ammonia oxidation rates courtesy of Mike Beman

30. Gene abundances increase by ~1000-fold between the near-surface ocean and the lower euphotic zone (200 m).
Gene transcripts increase ~10- fold toward the base of the euphotic zone.
Why are the upper ocean populations apparently so active, yet not abundant?

31. Conclusions
Time-varying changes in ocean habitat structure influence the population dynamics of N cycling microorganisms.
Ecology matters: Changes in population structure of N cycling microbes are directly influence ocean biogeochemistry.
Interactions between biogeochemistry and ecology occur across a range of scales, from decadal to seasonal to episodic.