Better Living through Biochemistry Enhanced Diffusive Flux as a Measure of Bioavailable Aquatic Iron

1
Better Living Through Biochemistry
Enhanced Diffusive Flux
as a Measure of
Bioavailable Aquatic Iron Concentration
By Brian Gaas
2
On the Menu

• Project Summary
• Justification
• Background
• Fe chemistry
• Biological Fe uptake
• Diffusive flux model
• Objectives
• Methods
• Genetic modification
• 55Fe uptake
• Flux calculation
• Conclusion

D.F.
55Fe
T.w.
3
Project Summary

• Determination of bioavailable Fe in marine
  systems
• Based on diffusion rate from medium to cell
  surface as function of concentration gradient

Q (D/l) (Cs Cm)
55Fe uptake
Hydrodynamic modeling
Genetic engineering
The Unknown

• Just add seawater and voila! Instant
  bioavailable Fe concentrations!

4
Who Ordered This?

• Iron (Fe) micronutrient, capable of limiting
  community plankton growth in HNLC regions
• Assays exist to determine total Fe concentration,
  speciation, and potential Fe limitation
• But, populations can differ in relation to Fe
  quotas
• To measure the amount of bioavailable Fe,
  irrespective of the Fe demand of the organism, a
  different method must be employed.

5
Place Settings Fe Chemistry

• Biological, physical and chemical processes all
  affect Fe biogeochemistry and speciation
• Almost all dissolved Fe is Fe (III)
• Fe(OH)x at surface ocean pH (8.2) and surface
  temperature (25C)
• Fe(OH)2 and Fe(OH)30
• Many confounding factors to determining Fe
  concentration
• Filter size
• Oxidation state change
• Fe hydroxide precipitation Fe(OH)x, Fe(OOH),
  Fe2O3
• Temperature, pH, light
• Organic complexes! 99.9 total Fe (III)

6
Marinobactin siderophore (peptide-based)
Hydroxamate siderophore (ferrichrome)
Humic acid
7
Place Settings Biological Use

• Fe deficiencies and excesses can limit
  phytoplankton growth
• Not all forms of Fe are available for uptake
• Fe (III) Fe(OH)x
• Colloidal iron has no direct biological use
• Not all Fe is taken up
• Internal Fe requirement
• Surface-bound transporters
• Siderophores
• Phytotransferrin
• Michaelis-Menten equation describe Fe uptake
  kinetics

8
Place Settings Flux Model

• Maximum diffusion 8 mean cell radius
• QR 4pR2JR 14.4pRD(?C)
• QR rate of transport to the cell
• JR nutrient flux at cell surface
• R mean cell radius
• D diffusivity constant
• ?C concentration difference between medium and
  cell surface
• Non-spherical shape of a centric diatom does not
  change the calculations significantly

9
Genetic engineering of T. weissflogii
Flux-derived Fe values compared to inorganic Fe
measurements
55Fe uptake kinetics
The Master Plan
Field testing
Cm -QR/(14.4pRD) Cs
Mass culturing of evil Super-Plankton for world
domination
Flux calculation for bioavailable Fe
10
Methods and Associated Madness
Part 1 Gene Jockeying
GATTACGCTCTCTAATGAGAGA
11
Methods and Associated Madness
Part 2 Nuke em All!
Section A incubation
24hrs


Low Fe AQUIL
55FeCl3
Modified T. w.
RadQUIL
Culture
Section B kinetics
Liquid scintillation counting


Uptake Rate
Saturation Curve
10min 3hr cell filtration
Ti(III) citrate EDTA
12
Methods and Associated Madness
Part 2, cont Glow-in-the-dark weissflogii?
Section C diffusion differences
L.S.C.


Uptake Rate
Culture
Diffusion ligand
Ti(III) citrate EDTA
Section D non-equilibrium control
L.S.C.


Uptake Rate
Culture
Strong ligand
Ti(III) citrate EDTA
13
Methods and Associated Madness
Part 3 Fluxing flocculations, Batman!
Cm -QR/(14.4pRD) Cs
CM bioavailable Fe concentration QR rate of
transport to the cell (flux) p
3.1415926535897932384626433832795 R mean
cell radius D diffusivity constant Cs
cell surface bioavailable Fe concentration
Unknown
55Fe uptake
Natural constant
Microscope
Literature
Genetically set to zero
14
In Conclusion

• Fe is an important element with wide-ranging
  effects, especially on oceanic biology
• Current methodologies do not give a sufficiently
  accurate measure of bioavailable Fe
• The 55Fe flux approach will be valuable for
  determining Fe limitation, as well as for
  understanding Fe-involved biogeochemical processes

15
To Inquire, or Not to InquireAny Other Questions?
?
16
Youve Gone Too Far!Go Back, Go Back!
17
Random Data Slides