SIGNIFICANCE, MECANISMS AND ENVIRONMENTAL IMPLICATIONS OF MICROBIAL BIOMINERALIZATION ......

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  • 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 mine Water pH~3

    20-60% As removed after 30 m of downflow

    Leblanc et al. Appl. Geochem. 1996 Casiot et al., Water Res. 2003 Casiot 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

    D iff

    ra ct

    ed in

    te ns

    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-oxidizers Fe2+ + As3+ + SO42- + H2O + O2

    + CO2 + CH2O

    Thiomonas sp.

    Amorphous Fe(III)-As(V)

    1 µm

    A. Ferrooxydans strain

    tooeleite

    1 µm

    Metabolic activity catalyzes the formation of minerals Colloque 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 these communities

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

  • Use of microscopy to look at microbial precipitates

    Synchrotron-based X-ray Spectromicroscopy STXM

    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

    monochromatic X-ray beam

    Objective = Fresnel lens

    Aperture

    He, air or vacuum

    x

    y

    Microscope chamber

    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

    bs or

    pt io

    n

    Amorphous As(V)- Fe(III) phase

    Tooeleite Fe6(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 biomineralization 2. Fossilization of microorganisms

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

  • However, most of the precipitates are extracellular…

    1 280 285 290 295 300 305

    Energy (eV)

    284.4 285

    287.6

    288.5 290.7

    Likely significant impact on sedimentation properties, maybe surface reactivity?

    5 μm Organic C map

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

    A bs

    or pt

    io n 284.4 eV: 1s-π* quinone

    285 eV: 1s-π* aromatics 287.6 eV : 1s-π* ketone 288.5 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., magnetotactic bacteria)

    Lethal:

    Disruption of membranes Limitation 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

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

  • 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

    pr ot

    ei n

    co nc

    en tr

    at io

    n (m

    g. m

    L -1

    )

    D issolved

    Fe(II) concentration, m M

    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 cell surface

    Extracellular mineralized filaments

    Outer membrane

    Plasmic membrane

    40nm

    40nm-thick layer = Periplasmic encrustation Colloque 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

    N um

    be r

    of p

    ix el

    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

    Extracellular precipitates

    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