1. Exploring iron-organic ligands and the microbial
utilization of iron in the ocean
Ocean color US CZCS European satellites and now modis satellie
Used to assess ocean producttivity (just like on land) bit that is not what is being measured
Measures light leaving the ocean (looks up and down), turns this into color, which is turned into chlorophyll (photosynthetic pigment, into biomass into productivity
Done by proxy. There is a finite depth to water leaving radience, and this may miss chorolyll. I was in the hole in the ocean and the chlorohyll may was at m.
Areas of high and low productivity
Agouron 2015 Lecture 3

2. Distribution of nitrate in surface waters
Primary productivity in most of the ocean is limited by macronutrient availability, such as P and N. (Cells will keep growing until they run out of something)
In some regions, which are often characterized by higher concentrations of these macronutrients, other things such as metal concentrations limit growth.
One major goal is to understand and predict changes in the distribution of marine species. (E.G. resource competition theory)
High Nutrient Low Chlorophyll regions (HNLC)
Agouron 2015 Lecture 3
Horn et al., EPSL, 2011

3. The Biological Roles of Iron
Photosynthesis
Oxidative stress
Respiration
Nitrogen fixation
DNA replication
Agouron 2015 Lecture 3

4. Iron as an essential, limiting micronutrient
John Martin of Moss Landing Marine Lab noticed
during a study of the N. Pacific HNLC region that
chlorophyll increased after an Asian dust event
Agouron 2015 Lecture 3

5. Fe (nm kg -1)
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
Depth (km)
King of trace metals at the moment is Fe. Respiration, N fixation, etc. Has a nut profile, with surface conc pM, rising to 0.6 nM in the deep ocean where it stabilizes. We don’t know why this is it is not clear if there is a gradient in Fe concentrations between the Atlantic and Pacific as we see for other nutrients- but based purely on the solubility of Fe(O)(OH) Fe should be several orders of magnitude lower concentration in the deep ocean. This has been explained as a scavenging profile with Fe +/- particles, most common explanation is the stabilization by organic matter.
Agouron 2015 Lecture 3
K. Coale et al.

6. Fe (nm kg -1)
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
Fe + H2O +Organic Matter (ligands, “L”) 
Fe’(H2O)+ FeL +Fe(O)(OH) + others
Depth (km)
King of trace metals at the moment is Fe. Respiration, N fixation, etc. Has a nut profile, with surface conc pM, rising to 0.6 nM in the deep ocean where it stabilizes. We don’t know why this is it is not clear if there is a gradient in Fe concentrations between the Atlantic and Pacific as we see for other nutrients- but based purely on the solubility of Fe(O)(OH) Fe should be several orders of magnitude lower concentration in the deep ocean. This has been explained as a scavenging profile with Fe +/- particles, most common explanation is the stabilization by organic matter.
Agouron 2015 Lecture 3
K. Coale et al.

7. Fe cell requirements for Synechococcus
and Prochlorococcus
Percent maximum growth rate
The is a plot on loan from Ann Thompson who is a student in Panny Chisolm and Mak Saito’s labs- a study she did on Fe requirements of proclholorcoccus. Bottom axis is plotted (Fe) conc, on the vertical axis is % maximum growth rate. If somehting is fully nutrient replete it will grow at 100%. IN red is the Syn data of Sunda and Huntsman, you can see that for Fe, once you get below conc of 100 pM growth rates begin to decline sharply, and this is the conc of Fe in many surface waters. Pro needs can grow well at lower concentrations either due to a lower cell requirement for Fe, or better uptake due to a smaller cell size or other mechanisms.
Anne Thompson Ph.D. Thesis, 2009
Agouron 2015 Lecture 3

8. Iron Limitation: Model of Small Phytoplankton Growth Limitation
Here we have an example of this type of modeling – where cellular quotas for various nutrients and metals was compared to environmental concentrations to determine which factor they would run out of first. This estimate suggests that ~1/3 of the ocean is limited by the availability of iron (Iron is necessary for many biological functions, but its concentration in the ocean is often very very low)
In reality, Fe as a nutrient is much more complex. More sophisticated and accurate models need to take into account the fact that not all iron is available to all organisms. It is the same as modeling all ‘organic carbon’ as one nutrient, or separating it into different fractions with different labilities such as sugars and proteins and lipids and refractory substances.
But… What fraction of iron is available to which organisms?
Agouron 2015 Lecture 3
Moore et al., Global Biogeochemical Cycles, 2004

9. “Give me a half tanker of iron and I will give you an ice age.”
Iron Limitation:
Model of Small Phytoplankton Growth Limitation
“Give me a half tanker of iron and I will give you an ice age.”
–John Martin
Here we have an example of this type of modeling – where cellular quotas for various nutrients and metals was compared to environmental concentrations to determine which factor they would run out of first. This estimate suggests that ~1/3 of the ocean is limited by the availability of iron (Iron is necessary for many biological functions, but its concentration in the ocean is often very very low)
In reality, Fe as a nutrient is much more complex. More sophisticated and accurate models need to take into account the fact that not all iron is available to all organisms. It is the same as modeling all ‘organic carbon’ as one nutrient, or separating it into different fractions with different labilities such as sugars and proteins and lipids and refractory substances.
But… What fraction of iron is available to which organisms?
Agouron 2015 Lecture 3
Moore et al., Global Biogeochemical Cycles, 2004

10. Agouron 2015 Lecture 3

11. Artificial Ocean Iron Fertilization
Agouron 2015 Lecture 3

12. Qualitative Nature of the Ligand Pool: Electrochemistry
1. Add Fe
2. Compete with known ligand
Natural ligands vs. SA
Current
Potential
3. Measure with electrochemistry
Agouron 2015 Lecture 3
Randie Bundy

13. Marine Iron Ligands
Concentration of Fe ligands nearly always exceeds dissolved Fe.
Conditional stability constant LogKFeL between
Suggests that >99% of dissolved Fe is complexed.
----- Meeting Notes (2/12/13 13:28) -----
Electrochemical measurements tell us what concentration of ligands are there and how strong they are - don't tell us what they are or where they come from.
Agouron 2015 Lecture 3
Boye et al., Marine Chemistry, 2006

14. How do microbes acquire iron from seawater ?
Fe (III) -Ligand S
S-Fe (III)
Ligand
light
S + Fe(II)
How do cells take up Fe? Lots of different ways. Fe is at a premium in many environments. We think of it as being added to the oceans by continental erosiaon, but it is often at a premium in soils, pathogens, Siderophores.
Fe (II)
S-Fe (III)
Siderophore (S)
Fe (III)
Agouron 2015 Lecture 3

15. Examples of siderophore structures from marine bacteria
Agouron 2015 Lecture 3
From JM Gauglitz, 2011

16. Iron fertilization is very controversial
Agouron 2015 Lecture 3

17. Steps for investigating metal ligands
Step 1 – DOM extraction from sample
Step 2 – Find metals associated with DOM
Step 3 – Characterize these compounds.
Agouron 2015 Lecture 3

18. Step 1: Solid Phase Extraction
Simplify matrix and preconcentrate organics
Agouron 2015 Lecture 3

19. Step 2: LC-ICPMS a b c d e f g h i j k l ICP-MS M-L M+
Agouron 2015 Lecture 3

20. Siderophore variability across an eddy
Sea Surface Height
56Fe LC-ICPMS: Station 2
56Fe LC-ICPMS: Station 2
Agouron 2015 Lecture 3

21. Siderophore variability across an eddy
Sea Surface Height
56Fe LC-ICPMS: Station 3
Agouron 2015 Lecture 3

22. Siderophore variability across an eddy
Sea Surface Height
56Fe LC-ICPMS: Station 4
Agouron 2015 Lecture 3

23. Step 3 ESI-MS ESI-MS spectra are extremely complex! ESIMS M-L M-L+
Agouron 2015 Lecture 3

24. Search for Fe isotope pattern
Δmass = 1.995
Δintensity = 0.06
56Fe
54Fe
Agouron 2015 Lecture 3

25. How do microbes get iron?
Agouron 2015 Lecture 3

26. Rapid and efficient discovery of siderophores
In the cyanobacterium Synechococcus 7702
Agouron 2015 Lecture 3

27. MS/MS
Agouron 2015 Lecture 3

28. Siderophores from Alteromonas
56Fe LC-ICPMS
Identified as marinobactins
54Fe and 56Fe LC-ESIMS
Marinobactin A’
Marinobactin A
Marinobactin B
Marinobactin C
Marinobactin D
Marinobactin E
Agouron 2015 Lecture 3

29. Siderophore variability across an eddy
Sea Surface Height
56Fe LC-ICPMS: Station 4
Amphibactins
New siderophore
m/z
Agouron 2015 Lecture 3

30. Siderophores in the GEOTRACES Program
Agouron 2015 Lecture 3

31. Subtropical North Pacific (Hoe-Phor)
2nd separation on collected fractions
Agouron 2015 Lecture 3

32. Subtropical North Pacific (Hoe-Phor)
----- Meeting Notes (2/12/13 13:04) -----
Mention that in Mn we don't see anything, but in Co we do.
Agouron 2015 Lecture 3

33. 1.) Add << 57Fe citrate
Future plans – field experimental work
Incubation Time
Natural abundance
Equilibrium
1.) Add << 57Fe citrate
2.) Incubate
3.) Measure Fe Ratio
56Fe-Ligand + 57Fe
57Fe-Ligand + 56Fe
56Fe
(57Fe +56Fe) kd
REPLACE THIS SLIDE WITH A CONCEPTUAL IRON FLOW FROM HETEROTROPHS TO AUTOTROPHS
Sample
Dissociation of Fe-Ligand (kd) determines 56Fe loss rate
Agouron 2015 Lecture 3

34. 57Fe isotope exchange Amphibactins 56Fe (57Fe +56Fe) Unknown ‘Humics’
Incubation Time (Days)
[Total Dissolved] = 120pM Fe
[Humics] ~ 30 pM Fe
[Siderophores] ~ 7 pM Fe
Time (min)
Agouron 2015 Lecture 3

35. The experiment we really really want to do
1) Label natural ligands
2) Spike seawater
with labeled ligands
3) Flow sort cells
56Fe-Ligand + 55Fe
55Fe-Ligand + 56Fe
4) Measure where
The iron-55 is
Agouron 2015 Lecture 3

36. Future plans – other metals, P and S
‘Biomolecules’ appear to have a preference for one metal over others.
Ill-defined ‘humics’ seem to bind all metals.
Retention Time (min)
Agouron 2015 Lecture 3

37. Agouron 2015 Lecture 3

38. With thanks to: Rene Boiteau Randie Bundy and- Jess Fitzsimmons
Ed Boyle
Jim Moffett
Eric Webb
Penny Chisholm
NSF & CMORE
Rene Randie
Agouron 2015 Lecture 3

39. Each ESI-MS scan is complex
Agouron 2015 Lecture 3

40. Distribution of nitrate in surface waters
Primary productivity in most of the ocean is limited by macronutrient availability, such as P and N. (Cells will keep growing until they run out of something)
In some regions, which are often characterized by higher concentrations of these macronutrients, other things such as metal concentrations limit growth.
One major goal is to understand and predict changes in the distribution of marine species. (E.G. resource competition theory)
Agouron 2015 Lecture 3
Horn et al., EPSL, 2011

41. Exploring microbial iron acquisition in the ocean.
Agouron 2015 Lecture 3