1. Time-Dependent Analysis of Magnetite Nanoparticles in Magnetospirillum gryphiswaldense Lars H. Böttger 1, Damien Faivre 2, Dirk Schüler 3, Berthold F. Matzanke 1 http://www.physik.uni-luebeck.de 1 University of Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany 2 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 Fe 3 O 4 in a magnetosome

2. 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 pO 2 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 (Fe 3 O 4 ) 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].

3. Fermenters allow control of several parameters: 1.Temperature 2.pH 3.O 2 -partial pressure 4.Growth of magnetosomes without biomass production Controlled growth in a fermenter 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 57 Fe-rich and C- poor 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 10 8 cells were transferred to Mössbauer sample holders.

4. 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. Typical Mössbauer spectrum of Magnetospirillum with filled iron pools

5. Mössbauer spectra of time dependent 57 Fe-accumulation and iron metabolization Figure 3: Series of Mössbauer spectra taken of cells after the medium was incubated with 57 Fe-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 57 Fe induction, but show a decrease of about 0.6 T afterwards. The red line is for comparision.

6. It has been shown by TEM that small magnetosomes containing a small magnetite nucleus are formed initially. Their iron content is growing in a time- dependent 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 B hf. Chain formation time, determined by B hf The hyperfine field B hf of an isolated magnetosome nanoparticle can be described by [11]: B d is the contribution of the demagnetizing field. B 0 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 k B T/2  V with k B 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.

7. 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: 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): Chain formation time, determined by B hf 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).

8. Chain formation time of magnetite nanoparticles determined by B hf 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 Figure 5: Diameters of nanoparticles calculated from Mössbauer B hf -data (solid lines) in comparison to diameters determined by TEM data (black stars) Size of magnetite nano- particles determined by B hf

9. 0min55min100min220min340min 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. TEM of the induction series

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