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 180-200 m. Areas of high and low productivity Agouron 2015 Lecture 3

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

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

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

Fe (nm kg -1) 0.2 0.4 0.6 0.8 1.0 1.2 1.4 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 50-100 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.

Fe (nm kg -1) 0.2 0.4 0.6 0.8 1.0 1.2 1.4 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 50-100 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.

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

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

“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

Agouron 2015 Lecture 3

Artificial Ocean Iron Fertilization Agouron 2015 Lecture 3

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

Marine Iron Ligands Concentration of Fe ligands nearly always exceeds dissolved Fe. Conditional stability constant LogKFeL between 10 -13. 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

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

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

Iron fertilization is very controversial Agouron 2015 Lecture 3

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

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

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

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

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

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

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

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

How do microbes get iron? Agouron 2015 Lecture 3

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

MS/MS Agouron 2015 Lecture 3

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

Siderophore variability across an eddy Sea Surface Height 56Fe LC-ICPMS: Station 4 Amphibactins New siderophore m/z 709.364 Agouron 2015 Lecture 3

Siderophores in the GEOTRACES Program Agouron 2015 Lecture 3

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

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

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

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

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

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

Agouron 2015 Lecture 3

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

Each ESI-MS scan is complex Agouron 2015 Lecture 3

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

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