1. General principles of biomineralization: Biologically induced mineralization
20 nm
Biologically induced magnetite nanoparticles
Ca HCO3-  CaCO3 + CO2 + H2O
Biologically induced mineralization involves the adventitious precipitation of inorganic minerals by reaction of extraneous ions with metabolic products extruded across or into the cell wall.
The mineral products are closely associated with the cell wall and crystallochemically heterogeneous.

2. General principles: Biologically controlled mineralization
uniform particle sizes
well-defined structures and compositions
high levels of spatial organization
complex morphologies
controlled aggregation and texture
preferential crystallographic orientation
higher-order assembly
hierarchical structures
Biologically controlled mineralization involves the specialized regulation of mineral deposition and results in functional materials with species-specific crystallochemical properties.

3. General principles: Site-directed biomineralization
Intercellular –
in the spaces between closely packed cells
Intracellular – bilayer lipid vesicles
enclosed compartments within the cell
H2O
0.5 mm
calcified coral
Extracellular –
on or within an insoluble macromolecular
framework secreted outside the cell
eggshell formation

4. General principles: Site requirements
Spatial delineation
– physical boundary for size and shape control
Chemical regulation
– for increasing ionic concentrations (ionic pumping)
Diffusion limited ion flow
– for controlling solution composition
Organic surface
– nucleation
General principles: Control mechanisms
Physiochemical
Spatial
Structural
Morphological
Constructional
Gene pool Bioenergetics Biochemical

5. Boundary-organized biomineralization
The delineation of biological environments is of key importance in boundary-organized biomineralization because it provides sites of controlled chemistry that are spatially defined.
Control functions:
spatial delineation – size, shape and organization of the mineral phase
diffusion limited ion flow – ionic activities, solution composition, supersaturation
mineral passivation – surface stabilization against dissolution and transformation
ion accumulation and transport – supplying chemicals to remote intra- and
extracellular mineralization sites
mineral nucleation – regulating interfacial energies
mineral transportation – moving mineralized structures to new construction sites.

6. Boundary-organized biomineralization; coccolith calcification
uniport
Ca2+
Ca2+
Ca2+
2HCO3 -
2HCO3 -
symport
HCO3 -
Coccoliths
CaCO3 CA OH -
CO2 H+
CO32-
H2O
CO2
Photosynthesis
+ Ca2+
Sugars
SO42-
SO42-
Polysaccharides
Golgi vesicle
Medium Cell
Light-dependent uptake: some CO2 fixed by photosynthesis  influence of light on calcification
Some calcification occurs in dark using stored metabolic energy.

7. Chemical control of biomineralization
Solubility
The solubility (S) of an inorganic salt depends on the balance between lattice energy (L) and ion hydration (H) ion pairing (IP) and complexation (C) in aqueous solution.
GS = GL - (GH + GIP + GC)
Lattice substitutions are important in controlling the solubility of biological apatite
Undersaturated
[Ca ] 2+
/M
HAP
FAP
Plaque fluid
Saliva
Supersaturated pH
100 10 1
0.1 5 6 7
Fluoride and tooth decay

8. Chemical control of biomineralization: Solubility product
MnXm(solid)  nM+(aq) + mX-(aq)
Ksp = {M+}n . {X-}m
The solubility product is a critical factor in determining the thermodynamic limit for the onset of inorganic precipitation. When the solubility product is less than the activity product (AP) of a solution then precipitation will occur until Ksp = AP.
Problems
Complexation with biological ligands (citrate/oxo/hydroxy)
Covalent polymers (silica)
Crystal size heterogeneity - Ostwald ripening
Kinetic effects organic sheaths etc

9. Chemical control of biomineralization: Supersaturation
Supersaturation is a measure to what extent a solution is out of equilibrium and represents the thermodynamic driving force for inorganic precipitation.
Relative supersaturation, Absolute supersaturation
SR = AP/Ksp SA = (AP – Ksp)/Ksp
The difference in chemical potential between the supersaturated solution and a solution at equilibrium with the solid is related to SR by
 = kT lnSR
Supersaturation is highly regulated in biology through the process of boundary-organized biomineralization.

10. Supersaturation control in spatial boundariesM n+
(n+1)+ MX
Matrix X - E1 E2
H2O MC A + B H
Direct mechanisms to increase S
ion pumping ( + redox)
ion complexation/decomplexation
enzymic regulation of anions
carbonic anhydrase (HCO3-)
alkaline phosphatase (HPO42-)
Indirect mechanisms
ionic strength - Na+ and Cl- transport
water extrusion – silica deposition? .
proton pumping – pH changes

11. Chemical control of biomineralization: NucleationG N * I B r r*
Homogeneous nucleation
The free energy of formation of a spherical nucleus, GN, is given by the difference between the surface (interfacial, I) and bulk (B) energies,
GN = GI - GB
GI = + 4r2
where  = interfacial free energy per unit surface area, and
GB = - 4r3 Gv
3 Vm
where Gv = per mole solid-liquid phase change, and Vm () is the molar (molecular) volume.
r* = critical size
GN* = activation energy
GN* = 1632 r* = 2Vm
3(kTlnSR) Gv
Rate; JN = Aexp(-GN*/kT)

12. Nucleation control in biomineralizationJN
SR*
 SR increase  GN* decrease  JN increase
GN*  (lnSR)-2
Need to control over catastrophic nucleation rate in pure solution at SR*
GN*  3
small reductions in  can have a marked effect on JN and r*
External substrate (dust etc) reduces  and hence increases JN at given value of SR.
Heterogeneous nucleation occurs at lower SR and under control if SR* is not breached.
GN* and JN determined by  and SR and biologically controlled by organic matrices and the membrane regulation of concentration gradients. JN A B C
SR* SR
Heterogeneous
Homogeneous
Extraneous particles with equal (A), variable (B) nucleation
efficiencies. (C) without extraneous particles).

13. Oriented nucleation (epitaxy)
  % mismatch
CaCO3//KBr
CaCO3//NaI
CaCO3 //KCl
CaF2//NaBr
CaF2//NaCl
CaF2//KBr A B C
(A) non-oriented, (B) mosaic with crystals aligned only perpendicular to the substrate, (C) iso-oriented array with 3-D crystallographic alignment.
Structural control in biomineralization involves the preferential nucleation of a specific crystal face or axis on the surface of an organic matrix.
Key concepts: molecular recognition, lattice matching, electrostatic, stereochemical and structural complementarity.

14. Chemical control of biomineralization: Crystal growth
Bulk diffusion
Surface adsorption
+ dehydration 2D
diffusion 1D
KINK
Crystal growth and termination are dependent on the level of supersaturation and occur through surface-controlled processes (active sites).
STEP
Rate of growth, JG = k (SA)x  
k = rate constant, SA = absolute supersaturation
Active sites - kinks, steps A B C
mass transport (diffusion-limited) very high values of SA (x = 0)
polynucleation (growth islands) high SA (x > 2)
layer-by-layer growth moderate SA (x = 1)
screw dislocation growth low SA (x = 2)
screw dislocation

15. Chemical control of biomineralization: Crystal growth inhibitors
apatite crystallization pH pH
calcite crystallization +
coccolith polysaccharides
7.4
8.0
t= 0
t = 4 min
Mg2+
7.2
7.5
control
control
7.0 F-
7.0 1 2 5 10 15
Time / h
Time / min
sea urchin
50 m
intercalation texture
Soluble additives bind at steps/kinks, inhibit growth, change composition, structure and form.

16. Chemical control of biomineralization: Crystal morphology
Crystal morphology (habit) is determined by the relative rates of growth of different crystal faces, with the slow growing surfaces dominating the final form.
010
110
100
Σ s{hkl}. A{hkl}
= minimum
CaCO3
control
HPO42-
Li+

17. Chemical control of biomineralization: Habit modification
[001]
[110]
CaCO3 +
[malonate]2-
CaCO polyaspartate
Molecular-specific interactions: electrostatic, stereochemical and structural matching modify the surface energy or mechanism of growth, or both.

18. Chemical control of biomineralization: PolymorphismJN
S (amorph)
S (crystal)
Amorphous
Crystal
Ksp
(crystal)
(amorph) X
Activity product
periodic order
Ostwald-Lussac law of stages
Crystallization proceeds along a series of structures with
decreasing solubility and increasing thermodynamic stability.
The structure of the critical nucleus is an important factor in controlling the crystallization pathway.
disordered/hydrated

19. Polymorphism and phase transformationsB G
Solution
(M+(aq) + X-(aq))
Amorphous
GN(B)
Gg(B) +
GT1
GN(A)
Gg(A)
GT2
GT3
Final mineral
(crystalline)
CALCIUM CARBONATE
CALCIUM PHOSPHATE
Amorphous CaCO3
Amorphous CaP
Accelerators
and
inhibitors
Vaterite
Brushite
Aragonite
Octacalcium phosphate
Calcite
Hydroxyapatite
amorphous silica - biologically stable
The chemical control of crystallization pathways that involve a sequence of kinetic inhibition and phase transformation can result in a high degree of selectivity in crystal structure and composition.

20. Phase transformations - examples
CDL OE
OCP
HAP
Calcium phosphate
slow transformations of amorphous phase
octacalcium phosphate to hydroxyapatite
in situ solid state hydrolytic transformation
x1 OCP (d100 = nm)  x2 HAP (2d100 = nm
Calcium carbonate
fast transformations of amorphous phase
unless stabilized by organic sheaths
high Mg2+ level in ACC  high Mg calcites (30 mol%)
eg. sea urchin spicules
Traces of OCP precursor are
left as a central dark line
in enamel HAP crystals.
1m
Larval sea urchin spicule
- early growth stage

21. Phase transformations - iron oxides
Solid state transformation
pH 7, 80 C
Dissolution
Reductive dissolution
Rapid
Slow
-low Fe
-low O2
-[H] agents
-complexation
-Fe2O3
FERRITIN
Fe2O3·nH2O
FeIII(aq) FeII(aq)
Magnetite
Amorphous Ferrihydrite Goethite
ferrihydrite  magnetite (bacteria, chitons) A1 A2
1.00 C
Intensity B
0.95
0.90
0.85
57Fe Mössbauer
-10.0
-5.0
0.0
5.0
10.0
Velocity (mm/sec)
FeIII O OH
+ FeII OH+(aq)
FeII + H+ + H2O
Fe3O4
+ H+ + H2O
[FeIIIFeII(O)x(OH)y]n+