Transcript Time-Dependent Analysis of Magnetite Nanoparticles in

Time-Dependent Analysis of
Magnetite Nanoparticles in
Magnetospirillum gryphiswaldense
Lars H. Böttger1,
Damien Faivre2, Dirk Schüler3,
Berthold F. Matzanke1
http://www.physik.uni-luebeck.de
Fe3O4 in a magnetosome
1 University
2
of Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany
Max Planck Institute of Colloids and Interfaces,Wissenschaftspark Golm, 14424 Potsdam, Germany
3 Microbiology,
Department of Biology, LMU München, Maria-Ward-Str. 1a, 80638 München, Germany
In recent years magnetic material has been found in several species, such as
termites [1], newts [2], pigeons [3], sea turtles [4] or magnetotactic bacteria [5].
In magnetotactic bacteria, magnetite crystals are surrounded by a lipid bilayer
membrane, termed magnetosome. The magnetosome membrane is derived
from the cytoplasmic membrane. The magnetosomes are aligned as a chain
within a filamentous structure of the cytoplasm [6, 7]. Together with an oxygen
sensing system, the magnetosome chain serves as a navigational device.
Optimum growth conditions are found in an environment of low pO2 which is
found in lower levels of ponds, lakes and rivers. Under microoxic conditions,
tremendous amounts of iron are transported into the magnetotactic bacterium
Magnetospirillum gryphiswaldense and are metabolized (up to 4% of the dry
weight [8]). Up to 100 cubooctahedral magnetite (Fe3O4) crystals per cell are
formed biosynthetically.
The unique crystalline and magnetic properties of magnetosomes have
brought them into the focus of multidisciplinary interest because they are used
in biotechnological applications [9] or as biomarkers for life on Mars [10].
Controlled growth in a fermenter
Fermenters allow control of several
parameters:
1.
2.
3.
4.
Temperature
pH
O2-partial pressure
Growth of magnetosomes without
biomass production
Figure 1:
Typical fermenter setup
To investigate the mechanism of magnetite-, magnetosome and magnetosome
chain formation, the bacteria were grown in a fermenter under conditions of iron
starvation. After harvesting the cells and reincubating them in a 57Fe-rich and Cpoor medium, biosynthesis of magnetite is induced and a time-resolved analysis of
the iron metabolite pattern can be achieved, which is not blurred by other metabolic
processes.
At defined times, cells were harvested, spun down in a centrifuge, washed and
approximately 108 cells were transferred to Mössbauer sample holders.
Typical Mössbauer spectrum of Magnetospirillum
with filled iron pools
Figure 2:
The spectrum is composed of 4 subspectra: two sextets representing magnetite A and B sites
(1:2 ratio), a Fe(II) high-spin doublet and a ferritin-like Fe(III) doublet.
Mössbauer spectra of time dependent 57Fe-accumulation
and iron metabolization
Figure 3:
Series of Mössbauer spectra taken of cells after the medium was incubated with 57Fe-citrate. The magnetite
contribution increases continuously during the accumulation period. Fe(II) remains at the same abolute levels
and the ferritin-like component is growing slowly. The hyperfine field of the magnetite components increase for
155 min after 57Fe induction, but show a decrease of about 0.6 T afterwards. The red line is for comparision.
Chain formation time, determined by Bhf
It has been shown by TEM that small magnetosomes containing a small
magnetite nucleus are formed initially. Their iron content is growing in a timedependent fashion. In a second step magnetosome chains are assembled.
Chain formation has been analyzed by 3 independent methods: TEM,
spectrophotometry and by analysis of the time-dependent changes of Bhf.
The hyperfine field Bhf of an isolated magnetosome nanoparticle can be
described by [11]:
kT
b
Bhf  Bd  B0 *  cos( ) T  Bd  B0 * (1  B )  Bd  B0 * (1  )
2V
t
Bd is the contribution of the demagnetizing field. B0 is the value of hyperfine field
that one would find in a macrocrystal. The cosine-term represents the thermal
average of the cosine of the angle between the easy axis of magnetization and
the actual magnetization. The cosine-term in the low temperature limit mainly
depends on the term kBT/2V with kB representing the Boltzmann constant, T the
temperature,  the anisotropic energy constant and V the particle volume. The
volume appears as time dependent term because of the ongoing growth of the
magnetosomes/magnetite crystals. Therefore, the constants can be combined to
a fit factor b and to the inverse of the time t.
Chain formation time, determined by Bhf
As soon as the nanoparticles are arranged in a chain of magnetosomes (see
figure 6) the demagnetizing field vanishes.
Using an integrated Gaussian distribution G(t) describing the fraction of
magnetic cells in a sample, one can describe the observed hyperfine field by:
b
b
Bhf (t )  (1  G (t )) * [B0 * (1  )  Bd ]  G (t ) *[B0 * (1  )]
t
t
t
1
1 t '  2
G (t )  
exp[  (
) ]dt '
2 
 2
The term µ represents the transition time after which half of the bacteria have
become magnetic, and the term  represents the half width of the Gaussian
distribution. A least square fit of magnetite A and B hyperfine fields yields the
factor b, the curves shown in figure 4 and a transition time of µ = 175 min.
The fit factor b allows to calculate the volume of particles and hence their
diameter (assuming a sphere, see figure 5):
3k BTt
d
b
3
A TEM series of the growth kinetics confirms both, the time of chain formation
and the size of the magnetosome-bound magnetite crystals (see figure 6).
Chain formation time of magnetite nanoparticles
determined by Bhf
Figure 4 Fit of the hyperfine fields using a
Gaussian distribution for magnetic cells. From
this fit the chain formation time of 175 min was
derived
Size of magnetite nanoparticles determined by Bhf
Figure 5:
Diameters of nanoparticles calculated
from Mössbauer Bhf-data (solid lines) in
comparison to diameters determined by
TEM data (black stars)
TEM of the induction series
0min
55min
100min
220min
340min
Figure 6:
Control of the chain formation in TEM: after 55 and 100 min isolated magnetosoms are visible. After 220 min
most of the magnetosomes have been arranged in chain fragments. In the sample after 340 min the chain was
fully formed. The arrows and ellipses emphasize the position of magnetosomes.
[1] Maher, Proc. R. Soc. Lond. B (1998) 265, 733-737
[2] Deutschlander, Borland and Phillips, Nature (1999) 400, 324-325
[3] Winklhofer, Holtkamp-Rötzler, Hanzlik, Fleissner and Petersen,
Eur. J. Mineral. (2001) 13, 659-669
[4] Lohmann, Cain, Dodge and Lohmann, Science (2001) 294, 364-366
[5] Blakemore, Science (1975) 190, 377-379
[6] Komeili, Li, Newman and Jensen, Science 311,242-245 (2006)
[7] Scheffel, Gruska, Faivre, Linaroudis, Plitzko and Schüler,
Nature 440,110-114 (2006)
[8] Lang, Schüler and Faivre, Macromol. Biosci. (2007) 7, 144-151
[9] McKay, Gibson, Thomas-Keprta, Vali, Romanek, Clemett, Chilier, Maechling
and Zare, Science (1996) 273, 924-930
[10] Schüler and Bauerlein, J. Bacteriol. (1998) 180, 159-162
[11] Mørup, Dumesic and Topsøe, Magnetic microcrystals, in Applications of
Mössbauer Spectroscopy, Cohen, 1980