1. Insight into the fate of newly-fixed nitrogen
in a high-CO2 ocean
Thank you for the introduction. Today I will talk to you about a central experiment for my PhD project which looks at the fate of newly-fixed nitrogen in a high-CO2 ocean.
Allanah Paul
GEOMAR
Helmholtz Centre for Ocean Research Kiel (Germany)
C-MORE Summer Course
Hawaii 2013

2. Present: Nitrogen fixation in the ocean
Surface water nitrate concentration (µM)
Source: Gledhill and Buck (2012), Frontiers in Microbiological Chemistry 3: 69
Nitrogen availability limits primary productivity in large areas of the ocean.
Nitrogen fixation adds new nitrogen to the marine environment and may stimulate productivity where nitrate is limiting.
Poor global coverage of direct measurements of nitrogen fixation rates.
Individual mechanisms of nitrogen transfer in natural systems investigated in some regions.
N2 fixation rates (μmol N m-2 d-1)
Currently, primary productivity in much of the world’s ocean is limited by the availability of nitrate. This figure is a map of the surface nitrate concentrations in the ocean. The purple colour which dominates the picture indicates low nitrate concentrations.
Nitrogen fixation adds new nitrogen and can relieve nitrate limitation, particularly in the oligotrophic ocean, and stimulate phytoplankton productivity. However, there is a paucity of data on direct nitrogen fixation rate measurements globally. Some globally important nitrogen fixers have only just been discovered within the last decade or so. Experiments looking into mechanisms of nitrogen transfer between trophic levels indicated it is a 50:50 split between zooplankton grazing and dissolved organic nitrogen release but again this knowledge is lacking for different plankton communities. It seems like we still have a lot to learn.
Source: Luo, Y.-W., et al. (2012), Earth System Science Data 4(1):47–73
A. Paul
C-MORE Hawaii 2013

3. Global change: Ocean acidification
Year 2100
Atmospheric pCO2 (ppm)
Year 2000
Ocean pH (units)
Year 2000
To add to this, we are currently also in a period of change. Anthropogenic release of carbon dioxide has created a rapid phase of change in atmospheric CO2 concentrations. Well recognised is the effect of this CO2 on global temperature, termed global warming. However the ocean and atmosphere have a constant exchange of gases and other material. The atmospheric CO2 dissolves in the ocean and as a consequence, the ocean pH decrease mirrors the increase in the atmosphere due to changes in the marine carbon chemistry. This has been the subject of great interest in the past few years. Research has shown that the physiology of many organisms will be affected and will likely result in shifts in the community composition of plankton in the future. But what will this mean for nitrogen fixers and their role in adding new nitrogen to the ocean?
Source: Riebesell et al. (Eds.), Guide to best practices for ocean acidification research and data reporting, 260 p. Luxembourg: Publications Office of the European Union
Year 2100
A. Paul
C-MORE Hawaii 2013

4. Future ocean: Nitrogen fixation
Trichodesmium IMS101
N2 fixation
Growth rate
Laboratory results suggest changes in nitrogen fixation  changes in new nitrogen added to the ocean in future
Source: Barcelos e Ramos et al. (2007), Global Biogeochemical Cycles 21 (2):GB2028.
Species
Response of
diazotroph culture
References
Trichodesmium
erythraeum ↑
Barcelos e Ramos et al. (2007), Hutchins et al. (2007), Levitan et
al. (2007), Kranz et al. (2009, 2010)
Crocosphaera
watsonii
↑ (+Fe)
↔ (-Fe)
Fu et al. (2008)
Nodularia
spumigena ↓
Czerny et al. (2009)
Here is a brief summary of the results of some previous laboratory-based experiments. Under culturing conditions, it appears that there will be changes in nitrogen fixation and diazotroph physiology due to ocean acidification. But what about the bigger picture?
Crocosphaera – Fu et al. (2009)
A. Paul
C-MORE Hawaii 2013

5. Formulation of research questions
Specific tracing of newly-fixed diazotrophic N in a natural plankton community.
What is the fate of newly-fixed nitrogen in the ocean?
Little known about
nitrogen fixation and where nitrogen ends up in the ocean, …
Will net nitrogen fixation increase in a future acidified ocean?
Will the distribution of newly- fixed nitrogen change?
Observe accumulation of label in N pools over time and in different pCO2 treatments.
... even less known about the influence of future
global change …
Based on the fact that we know relatively little about nitrogen fixation rates, let alone where the newly-fixed nitrogen ends up in the system currently, some research questions were formulated to gain some insight into these processes. What is the fate of newly-fixed nitrogen in the ocean? Will this distribution change in the future due to ocean acidification? How might changes in diazotroph metabolism be reflected in a natural plankton community?
How might changes in diazotroph metabolism be reflected in natural plankton communities?
Identify changes in flow of newly-fixed nitrogen in future due to ocean acidification.
... and how this might affect the plankton communities
which form the basis
of the marine foodweb.
A. Paul
C-MORE Hawaii 2013

6. Experimental approach
Bottle incubations  rates of nitrogen fixation
Mesocosm enrichment experiment  cycling of newly-fixed
nitrogen
A. Paul
C-MORE Hawaii 2013

7. Diazotrophic organisms (eg. unicellular/filamentous cyanobacteria)
Nitrogen fixation rates
Bottle incubations
Isotope tracers (15N and 13C ) can be used for nitrogen and carbon fixation rate measurement in plankton communities and algae cultures under controlled conditions. N2
Incubation:
Add a known amount of 15N2 to the sample
Incubate for 24 hours
Termination of incubation by filtration
Analyse the change in δ15N
nitrogen fixation N2
Diazotrophic organisms
(eg. unicellular/filamentous cyanobacteria)
Nitrogen is taken up by diazotrophic organisms and is fixed, initially as ammonium within the cell, with some also released extracellularly which can be used in primary production by phytoplankton. Isotopes are commonly used for measuring carbon and nitrogen fixation, usually in bottle incubations either as laboratory cultures, or samples from the field. This provides important information on rates, but does not give any information on how this material filters down the foodweb from these organisms i.e through zooplankton grazing to stimulate secondary production or removal of organic material from the surface ocean though sinking particle flux.
A. Paul
C-MORE Hawaii 2013

8. bacterial remineralisation
Tracing newly-fixed nitrogen
Large-scale incubations: mesocosms
13C and 15N tracers used previously in streams, estuaries and mesocosms
Use of 15N as stable isotope enrichment specific for newly-fixed diazotrophic nitrogen in a large experimental setting
15N N2 N2
15N
bacterial remineralisation
15N
DON release
15N
15N
15N
DON
DIN
Secondary production
POM
grazing
DON release
15N
zooplankton
surface ocean
Higher trophic levels
For this, larger-scale incubations are required which enables the sampling of more diverse pools of organic material. This has also been done in the past in streams, estuaries as well as mesocosms. The idea for this project was to use a specific tracer of the diazotrophic nitrogen in the foodweb – labelled nitrogen gas (30N2). Enrichment of the material in a particular pool in 15N indicates that newly-fixed nitrogen has ended up in this pool. To my knowledge this is the first time that this tracer has been used in a large experimental setting.
15N
sinking organic/
sedimenting material
sediment
A. Paul
C-MORE Hawaii 2013
15N = stable isotope tracer of newly-fixed diazotrophic nitrogen

9. Case study: Baltic Sea
Site of experiment
28/6/2012
Diazotrophic cyanobacteria are an important annual nitrogen source in the Baltic Sea.
two key species
Nodularia sp.
Aphanizomenon sp.
extensive annual summer blooms
Finland
Sweden
Estonia
BALTIC SEA
Source: Seija Hällfors
The Baltic Sea Portal
Diazotrophic cyanobacteria are an important annual source of nitrogen for the foodweb and for primary production in the Baltic Sea to balance the excess of phosphate and nitrogen loss processes.
Each year extensive cyanobacteria blooms form in this region with two key species: Nodularia and Aphanizomenon. The light green visible in the sea near Sweden shows these cyanobacteria blooms as taken from a satellite photo taken during the experiment.
Source: M. Kahru,
Source: Seija Hällfors
The Baltic Sea Portal
A. Paul
C-MORE Hawaii 2013

10. Experimental design
Kiel Off-Shore Mesocosms for future Ocean Simulations (KOSMOS)
Water column
(~ 17m deep)
Seven mesocosms containing a natural phytoplankton community
pCO2 was manipulated at beginning of experiment to between 240 μatm and 1600 μatm
Experiment length ~ 8 weeks during June – August (summer)
Volume = ~ 55000L
In Kiel we are lucky to have the mesocosm infrastructure ‘KOSMOS’ allowing large scale experimentation and manipulation of CO2 using natural plankton communities including zooplankton. The experiment in the Finnish archipelago took place during the summer and covered a pCO2 range from the present day to an extreme future scenario.
Sediment trap
A. Paul
C-MORE Hawaii 2013
Source: GEOMAR

11. Experimental design
Kiel Off-Shore Mesocosms for future Ocean Simulations (KOSMOS)
1) Addition of isotope label to 5 mesocosms (initial pCO2 range µatm)
15N(aq)
2) Regular sampling from mesocosms
Water column
(~ 17m deep)
We enriched five of the mesocosms with the 30N2 and followed the experiment by sampling and analysis of the samples by mass spectrometry. The following preliminary results which I will show are taken from the sinking organic material which collected in the sediment trap at the bottom of the mesocosm.
3) Samples from different N pools taken for analysis by mass spectrometry
Sediment trap
A. Paul
C-MORE Hawaii 2013
Source: GEOMAR
Photos: A. Paul

12. Preliminary results Result:
15N addition
High pCO2
Low pCO2
Intermediate pCO2
CO2 manipulation
Result:
Higher pCO2 mesocosms had more 15N label detected in sinking material.
This figure shows the enrichment (reported in per mil relative to the isotope ratio in atmospheric nitrogen) in the sediment trap material over the experiment. The pCO2 was manipulated between day 0 and day 4. The isotope was added first on day 22 because there was little sign of cyanobacteria for the first half of the experiment, with a second addition on day 26. As you can see here, the enrichment generally increases over time with overall higher enrichment in the higher pCO2 treatments. This could be interpreted as a larger contribution of diazotrophic nitrogen to the sedimenting material at higher pCO2. Surprisingly, despite differences in the enrichment, there was no observed difference in total sediment weigh between treatments. This has been reported in previous mesocosm experiments using a carbon isotope tracer and is evidence for increased regeneration of diazotrophic material in the system.
Sediment material sampled, processed and sub-samples provided by Tim Boxhammer
Larger contribution of diazotrophic nitrogen to sedimenting material in higher pCO2 mesocosm
No difference in total sediment weight between CO2 treatments. Also observed in previous mesocosm experiments using 13C (de Kluijver et al. 2013) and suggests a regenerative system.
A. Paul
C-MORE Hawaii 2013

13. Preliminary results
15N addition
High pCO2
Low pCO2
Intermediate pCO2
Why was diazotroph nitrogen content in sinking material higher at higher pCO2 ?
more labelled nitrogen was fixed at higher pCO2
increased recycling and reincorporation of diazotrophic N at higher pCO2
increased zooplankton grazing enriched the zooplankton material (eg. faecal pellets, detritus) in labelled nitrogen
Although there is a considerable amount of data to yet be analysed, there are some ideas that may explain these results. It could be that one or more of these processes explain these results and hopefully the rest of the samples will give a fuller picture.
The most obvious idea is that more of the label was added to the higher pCO2 mesocosms due to more nitrogen fixed. It could also be that as for previous mesocosm experiments for carbon, there was increased microbial turnover and regeneration of the diazotrophic material at higher pCO2. Higher zooplankton numbers or grazing rates may have enriched zooplankton material in the 15N label which sunk out during the experiment.
Alternatively, the same amount of nitrogen may have been fixed in all mesocosms but the difference between treatments can be explained by changes in the phytoplankton productivity on the available nutrients.
A. Paul
C-MORE Hawaii 2013

14. Acknowledgements Primary Advisor: Prof. Dr. Ulf Riebesell
Secondary Advisor: PD Dr. Hermann Bange
Biological Oceanography group
Jan Czerny
Tim Boxhammer
Kai Schulz
Mathias Haunost
Michael Sswat
Lennart Bach
Andrea Ludwig
Dana Hellemann
The KOSMOS team and all experiment participants in Tvärminne 2012
Staff at Tvärminne Zoological Station, Finland
Photo: Maike Nicolai

16. CO2 (aq) H2CO3(aq) HCO3-(aq) + H+(aq) CO32-(aq) + 2H+(aq)
Global change: Ocean acidification
Marine carbonate system
CO2 (g)
CO2 (aq) H2CO3(aq) HCO3-(aq) + H+(aq) CO32-(aq) + 2H+(aq)
A. Paul
C-MORE Hawaii 2013

17. Preliminary data interpretation