200 nm 1 μm200 nm

SIGNIFICANCE, MECANISMS AND ENVIRONMENTAL IMPLICATIONS OF MICROBIAL BIOMINERALIZATION

PROCESSES

Colloque Minéralogie environnementale - Académie des sciences - 14-15 septembre 2009

[As] ≤ 1mg/l

[As]= 300 mg/l

1 m

Acid Mine drainage: Carnoules (France)

Pb-Zn abandonned mineWater pH~3

20-60% As removed after 30 m of downflow

Leblanc et al. Appl. Geochem. 1996Casiot et al., Water Res. 2003Casiot et al., Sci Total Environ 2005

Colloque Minéralogie environnementale - Académie des sciences - 14-15 septembre 2009

1 m

As(III) tooeleite

As is trapped by the formation of diverse As-Fe-rich minerals (up to 22% As)

Amorphous As(V)-Fe(III)

2L-ferrihydrite

Schwertmannite

10 30 50 70 90

Diff

ract

ed in

tens

ity

Co K α 2θ (�)

* sample holder

Qz

**

*

tooeleite

As(III)/Fe = 0.60

As(III)/Fe = 0.42

As(V)/Fe = 0.72

schwertmannites

As(III,V)/Fe = 0.16

As(III,V)/Fe = 0.30

As(III,V)/Fe = 0.07

2L-ferrihydrite

As/Fe = 0.01

tooeleite

amorphous + to

amorphous

0.20

Morin et al., ES&T 2003

Colloque Minéralogie environnementale - Académie des sciences - 14-15 septembre 2009

Microbes indirectly impact the formation of minerals by redox transformations

Fe2+ + As3+ + SO42- + H2O + O2 Fe(III)-As(III) phase (Tooeleite)

Fe(III)-As(V) amorphous phase

Fe oxidizers

As- and Fe-oxidizersFe2+ + As3+ + SO4

2- + H2O + O2

+ CO2 + CH2O

Thiomonas sp.

AmorphousFe(III)-As(V)

1 µm

A. Ferrooxydans strain

tooeleite

1 µm

Metabolic activity catalyzes the formation of mineralsColloque Minéralogie environnementale - Académie des sciences - 14-15 septembre 2009

1 m

As(III) tooeleite

Diversity of minerals results from the diversity of bacteria

Amorphous As(V)-Fe(III)

2L-ferrihydrite

Schwertmannite

X-Ray Diffraction and XAS: (e.g. Morin et al. ES&T 2003; Morin et al. Am Min 2007)

Spatial and temporal variation of the mineralogy of sediments

Bruneel et al. (2006) AEM

Microbial diversity:→ Low diversity→ Species able to oxidize Fe/ As→ Spatial and temporal variations of thesecommunities

Colloque Minéralogie environnementale - Académie des sciences - 14-15 septembre 2009

Use of microscopy to look at microbial precipitates

Synchrotron-based X-ray SpectromicroscopySTXM

Transmission Electron Microscopy

Need to characterize both organics and minerals at the submicrometer scale

Colloque Minéralogie environnementale - Académie des sciences - 14-15 septembre 2009

Scanning Transmission X-ray Microscope

detector

sample

monochromaticX-ray beam

Objective= Fresnel lens

Aperture

He, air or vacuum

x

y

Microscopechamber

280 285 290 295 300

XANES C K-edge

Photon energy (eV)

Images with 20 nm resolution

High spectral resolution XAS By tuning incident energy : study of C, Fe, As

speciation

Synchrotron= source of high brightness X-ray radiations

500 nm

Colloque Minéralogie environnementale - Académie des sciences - 14-15 septembre 2009

Mapping As redox in Carnoules sediments at the 50-nm scale

2 μm

STXM image

2 μm

As map

2 μm

As(III)As(V)

Energy (eV)

1320 1330 1340 1350 1360 1370

1329.9eV1326.5eV

X-r

ay A

bsor

ptio

n

Amorphous As(V)-Fe(III) phase

TooeleiteFe6(AsO3)4SO4(OH)4,2H2

O

XANES As L3-edge

As(III)

As redox map

2 μm

STXM bacteria map

Difficult to evidence a correlation between As redox and specific microorganisms

Benzerara et al. GCA 2008

Colloque Minéralogie environnementale - Académie des sciences - 14-15 septembre 2009

TEM on thin section

400 nm

Bacterial cell

Bacteria cell walls provide nucleation sites for the precipitation of Fe-As minerals

500 nm

200 nm

1. Diverse patterns of biomineralization2. Fossilization of microorganisms

Colloque Minéralogie environnementale - Académie des sciences - 14-15 septembre 2009

However, most of the precipitates are extracellular…

1280 285 290 295 300 305

Energy (eV)

284.4285

287.6

288.5 290.7

Likely significant impact on sedimentation properties, maybe surface reactivity?

5 μmOrganic C map

STXM at the C K-edge shows the presence of pervasive organic carbon polymers

Abs

orpt

ion 284.4 eV: 1s-π* quinone

285 eV: 1s-π* aromatics287.6 eV : 1s-π* ketone288.5 eV: 1s-π* carboxylic

290.7 eV: 1s-π* Fe-carboxylics

Benzerara et al. GCA 2008

Colloque Minéralogie environnementale - Académie des sciences - 14-15 septembre 2009

100 nm

Abundance of mineralized vesicles in Carnoules: An overlooked biomineralization process ?

400 nm

TEM on thin sectionTEM

Bacterial cell

1 μm

SEM

Colloque Minéralogie environnementale - Académie des sciences - 14-15 septembre 2009

What do we learn?

Microbes and mineral precipitation:

indirect role via metabolic activity

nucleation surfaces: cell wall, extracellular polymers, vesicles

What needs to be further studied?

How bacteria deal with mineral precipitation?

→ Is it lethal? → Beneficial? → Are there adaptative responses to mineral precipitation?

Why are there different biomineralization patterns?

What are the molecular mechanisms of these biomineralization processes?

Colloque Minéralogie environnementale - Académie des sciences - 14-15 septembre 2009

Precipitation of minerals and Bacteria

Advantageous:

Production of H+ used for energy generation

Protection against antibiotics/radiation/dessication/toxic metals…

Magnetic sensing (e.g., magnetotacticbacteria)

Lethal:

Disruption of membranesLimitation of nutrient transfers

Some microbes develop strategies to limit precipitation within the cell

100nm

From Nicolas Menguy (IMPMC)From Jennyfer Miot (IMPMC) From Xavier Chatellier (CAREN, Rennes)

Colloque Minéralogie environnementale - Académie des sciences - 14-15 septembre 2009

EXPERIMENTAL APPROACH

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Experimental Fe biomineralization by neutrophilic anaerobic bacteria

Dilemma: source of energy but production of a highly insoluble compound

Fe(III)Fe(II)bacteria

pH = 7

4 Fe2+ + 11 H2O + CO2 4 Fe(OH)3 + 8 H+ + CH2O2 Fe2+ + 5 H2O + NO3- 2 Fe(OH)3 + 4 H+ + NO2

-

Encrusts

1 µm

BoFeN1

Does not encrust

1 µm

SW2

Colloque Minéralogie environnementale - Académie des sciences - 14-15 septembre 2009

Fe biomineralization by BoFeN1 (encrusting cells)

0

10

20

30

40

50

60

70

0

1

2

3

4

5

6

0 50 100 150

prot

ein

conc

entr

atio

n (m

g.m

L-1

)

Dissolved

Fe(II) concentration, mM

Time, hours

Biomass

Fe(II) concentration

t = 0 t = 3d t = 6d

• Where is Fe(II) oxidized?• Where does Fe(III) precipitate?

Colloque Minéralogie environnementale - Académie des sciences - 14-15 septembre 2009

Extracellular and periplasmic Fe-precipitates

40nm-thick layer of iron minerals

Globules at the cellsurface

Extracellularmineralized filaments

Outer membrane

Plasmicmembrane

40nm

40nm-thick layer = Periplasmic encrustationColloque Minéralogie environnementale - Académie des sciences - 14-15 septembre 2009

3h 6h

1 day 3 days 6 days

2µm 2µm

1µm 2µm 2µm

Fe(II)Fe(III)

Temporal evolution of iron redox state (STXM)

Both bacteria AND extracellular precipitates are eventually oxidized!Miot et al. GCA 2009

Colloque Minéralogie environnementale - Académie des sciences - 14-15 septembre 2009

0 0.2 0.4 0.6 0.8 1

Num

ber

of p

ixel

s

Fe(III)/Fe(total)

3 h

1 jour

3 jours

6 jours

0.18

0.28

0.27

0.39

0.80

0.50

0.94

0.55

50

Miot et al., Geobiology (2009)

Bacteria

Extracellularprecipitates

Iron gets oxidized on/in the cells

Quantitative evolution of iron redox vs. time

Colloque Minéralogie environnementale - Académie des sciences - 14-15 septembre 2009

100 nm

200 nm

Cell encrustation vs. Time:Cryo-TEM on cryo-sections

Precipitation starts on the plasmic membraneThen extends and fills the entire periplasm

Fe-oxidation likely occurs within the periplasm

1

2

3

200 nm

Colloque Minéralogie environnementale - Académie des sciences - 14-15 septembre 2009

ProteinsFe

Proteins are preserved in the encrusted periplasm

1 μm

Interesting for the one looking for fossils in old rocks

Colloque Minéralogie environnementale - Académie des sciences - 14-15 septembre 2009

Biomineralization of Fe by neutrophilic Fe-oxidizing bacteria

• Where is Fe(II) oxidized?→ probably within the periplasm. Sometimes outside the cells but…

• Where does Fe(III) precipitate?→ clearly within the periplasm then at the surface of the cells

Colloque Minéralogie environnementale - Académie des sciences - 14-15 septembre 2009

Is encrustation of BoFen1 cells lethal?

% of encrusted cellsvs. time

Number of viable cellsvs. time

Biomass vs. time

+Fe

No-Fe

+Fe

Colloque Minéralogie environnementale - Académie des sciences - 14-15 septembre 2009

SW2 does not encrust

TEM SEM

Colloque Minéralogie environnementale - Académie des sciences - 14-15 septembre 2009

0

C map Fe mapcell

filaments

Fe precipitates on extracellular filaments

1 μm

Miot et al. AEM 2009

Colloque Minéralogie environnementale - Académie des sciences - 14-15 septembre 2009

Fe-redox gradients along the filaments

1 µm

Fe(II)

500 nm

Fe(II)

cell

medium

0 0.5 1 1.50.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

Fe(I

II)/

Fe t

otal

Distance, µm

cell medium

Colloque Minéralogie environnementale - Académie des sciences - 14-15 septembre 2009

SW2

Adsorption

NucleationLimited growth

lipo-polysaccharidicfiber

Extracellular biomin

BoFeN1 Polysaccharidicfilament

Fossilization of proteinsBiomin in

periplasm

Nucleationlimited growth

Fe-biomineralization by neutrophilic Fe-oxidizing bacteria

Different patterns of biomineralization. Is there a genetic basis?

Extracellular biomin

Colloque Minéralogie environnementale - Académie des sciences - 14-15 septembre 2009

Bacterial biomineralization is not limited to Fe-minerals, AMD or modern Earth…

Stromatolitesmodern and fossil (up tp 3.5 Ga)

Banded Iron FormationPrecambrian (up to 3.8 Ga)

Pathogenic aortic calcification

Colloque Minéralogie environnementale - Académie des sciences - 14-15 septembre 2009

→ Need for coupling mineralogy/geochemistry and (molecular) biology tools

Conclusions

What is the quantitative importance of bacterial biomineralization?

What do biominerals tell us about past environments?

Are there adaptations for/against biomineralization?

Colloque Minéralogie environnementale - Académie des sciences - 14-15 septembre 2009

Georges Ona-NguemaJennyfer MiotGuillaume MorinFrançois Guyot

Corinne CasiotOdile Bruneel

Anniet Laverman

François Farges

Gordon E. Brown, Jr

Andreas KapplerMartin Obst

Co-auteurs et remerciements

Institut de Minéralogie et de Physique des Milieux Condensés (IMPMC)

Paris

Hydrosciences Montpellier

Sisyphe, Paris

MNHN, Paris

University Stanford, USA

University Tuebingen, Germany

Colloque Minéralogie environnementale - Académie des sciences - 14-15 septembre 2009