Introduction

Magnetite nano-particles are widely distributed mineral compounds found in various organisms including bacteria, bees, pigeons or even in human beings [1,2,3,4]. Membrane-enveloped magnetite, biosynthesized by magnetotactic bacteria (MTB), is referred to as magnetosomes [5, 6]. Although magnetosomes have great potential in bioengineering and biomedicine applications [7,8,9], the intracellular biomineralization mechanism of magnetosomes remains poorly understood. Since the discovery of MTB, it has been believed to be an optimal material for the study of biogenic magnetite biomineralization because of the simple structure of these organisms.

In most MTB, the magnetosomes are composed of magnetic magnetite (Fe3O4) or sulfide greigite (Fe3S4) [10, 11] enveloped by biological membrane [12]. Under the action of a skeleton-like protein MamK and its binding partner MamJ, single magnetosome are arranged in chains parallel to the long axis of the cell [13,14,15].

Many studies using genetic, biochemical and physicochemical approaches have been performed to reveal the biological control of magnetosome synthesis in MTB. A series of genes were identified to be involved in this process, the mam genes, comprising the magnetosome island (MAI), which is a large unstable genomic region spanning 80–150 kb in length in different MTBs [16, 17]. However, knowledge about the chemical route of iron during the magnetosome formation process is limited and how iron ions are incorporated into magnetite is still a matter of debate. Previous studies using Mőssbauer spectroscopy suggested that ferrihydrite is a precursor for magnetite formation [18, 19]. This was further confirmed in Magnetospirillum gryphiswaldense MSR-1, a type strain for MTB study, using Fe K-edge X-ray absorption near edge structure (XANES) and high-resolution transmission electron microscopy (HRTEM) analysis [20]. Real-time study about magnetosome biosynthesis using transmission electron microscopy (TEM) and X-ray absorption spectroscopy in MSR-1 revealed that full-sized magnetosomes formed within 15 min and immature magnetosomes contain a surface layer of hematite phase [21]. In mamXY gene cluster mutants of MSR-1, distinct types of α-Fe2O3 particles co-existed with magnetite, hinting that the transformation of hematite phase to magnetite phase is a biocatalysis process [22]. Magnetite formation from a phosphate-rich ferric hydroxide via nanometric ferric (oxyhydr) oxide intermediates was recently shown in Magnetospirillum magneticum AMB-1 [23]. Together, these studies showed that the formation of magnetite in organisms possibly occured via Fe (III) ion intermediates and their oxides, hydroxides or oxyhydroxides.

It was suggested in the previous study that a thermal unstable phase of ferric oxide, ε-Fe2O3, was found in a mutant of MSR-1, which raised the possibility of a new type of intermediate during biomineralization process [24]. In the present study, a kinetic analysis was performed to study the magnetosome maturation process from the early stage of its formation. The results showed that at each time point during maturation of magnetosomes after induction, magnetite co-existed with various iron-containing phases, including α-Fe2O3, ε-Fe2O3 and other undefined phases. This result indicated that mature magnetosomes form via different types of iron-containing intermediates, including some rare phases such as ε-Fe2O3.

Materials and methods

Bacteria and growth conditions

M. gryphiswaldense MSR-1 was cultured in SLM at 30 °C as described previously [25]. The medium contained (per liter double distilled water) 2.5 g sodium lactate, 0.4 g NH4Cl, 0.1 g yeast extract, 0.5 g K2HPO4, 0.1 g MgSO4 • 7H2O, 0.05 g sodium thioglycolate and 5 mL trace element mixture. The strains were cultured at 30 °C in 250-mL serum bottles containing 100 mL medium with shaking at 100 rpm. Antibiotics nalidixic acid (Nx) was applied at 5 μg/mL for MSR-1.

Cell magnetic response curve of MSR-1 strain

The coefficient of magnetism (Cmag) value of MSR-1 was calculated from measurements of the maximal and minimal optical density (OD600) using a UV-visible spectrophotometer (UNICO2100; UNICO Instrument Co., Shanghai, China) [26].

Transmission electron microscope measurement

The structural details of the nanoparticles in MSR-1 samples from different time interval were analyzed by the HRTEM method using a JEM-2100F, which was operated at 200 kV, and it was equipped with a field emission gun, ultra-high-resolution pole piece, and ultrathin window JEOL detector. HRTEM images were obtained with an OSIS CANTEGA CCD camera. The crystals’ structural parameters were obtained by Fast Fourier Transform (FFT) analyses.

Time-course experiment for the detection of the intermediate phase at the early stage of magnetosome formation

MSR-1 cells were cultured in 5.0 L of SLM medium in a 7.5-L bioreactor as described previously [27], until the relative dissolved oxygen concentration (DO) decreased to 1%. Then, 30 mL of 10 mM ferric citrate was added to the culture to induce the synthesis of magnetosomes. Samples for HRTEM observation were collected at 0, 5, 10, 15 and 25 min after induction and fixed immediately with 2.5% glutaraldehyde. After washing three times with double-distilled water, the cells were dispersed onto a copper grid for HRTEM analysis.

Results

For this study, in order to initiate the biomineralization of large amount of magnetite accurately, cultivation of M. gryphiswaldense MSR-1 was carried out in a 7.5-L bioreactor. After the relative dissolved oxygen decreased to 1%, ferric citrate was added to induce magnetosome biosynthesis, samples were taken at different time points and fixed in 2.5% glutaraldehyde to maintain their original state (Fig. 1). Then, cells were prepared for HRTEM analysis. From the result, even at the 0-min interval, the formation of several iron-containing nanoparticles was evident, though the size of the particles are very small. Then, the average diameter of magnetosomes grows with time passed (Fig. 2). During this time, the predominant phase composing the particles was magnetite, but other iron-containing phases, including α-Fe2O3 and ε-Fe2O3, were present (Fig. 3a and Fig. 4, the data of ε-Fe3O3 measured in this work and the theoretical data of ε-Fe2O3 and Fe3O4 (magnetite) are listed in Additional file 1: Table S1). The finding of α-Fe2O3 as an intermediate in magnetite biomineralization is in accordance with previously reports, but the existence of ε-Fe2O3, which is an unstable phase under normal conditions, has never been reported before. For this assay, a total of more than 400 particles were analyzed, and Fig. 5 shows the phase ratio of different iron species by induction time point, Fe3O4 (magnetite), ε-Fe2O3, and α-Fe2O3 (hematite) phases are denoted by subscripted M, ε, and H, respectively (Some of the data measured in this assay are listed in Additional file 1: Figure S1). At each induction time point before the formation of mature magnetosomes, iron oxides always co-exist and proportion of magnetite increased with time passed. This result indicates that various iron-containing precursors co-exist as intermediate phases at the initiation phase of magnetosome biomineralization. The HRTEM data are shown in Fig. 3a, Fig. 4 and the original images with high resolution are available in Additional file 1: Fig. S2. Other iron-containing phases will be discussed in detail in another paper.

Fig. 1
figure 1

Time dependence of relative dissolved oxygen and magnetic response (Cmag) of MSR-1 cultured in the SLM media. 60 μM ferric citrate was added when the relative dissolved oxygen decreased to 1% in the culture to induce the formation of magnetosome. Then samples were taken for certain interval after the inducing of ferric citrate. Thereafter they were collected for HRTEM observation

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Fig. 2
figure 2

The size distribution of magnetosomes at different time interval after the induction of ferric citrate

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Fig. 3
figure 3

Component analyses of intracellular iron oxide nanoparticles in MSR-1 wild type after the induction of ferric citrate for 5 min and 10 min by HRTEM. a. HRTEM analysis of intracellular iron oxide nanoparticles form samples, FFT analyses are shown below the corresponding HRTEM images. M, ε, and H subscripts shown in FFT pattern denote Fe3O4 (magnetite), ε-Fe2O3, and α-Fe2O3 (hematite) phases, respectively. The time shown beside the individual HRTEM image is the interval when the samples were taken out after the induction of ferric citrate. b. The schematic representation of crystal structures for magnetite, hematite and ε-Fe2O3

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Fig. 4
figure 4

Component analyses of intracellular iron oxide nanoparticles in MSR-1 wild type after the induction of ferric citrate for 15 min and 25 min by HRTEM. HRTEM analysis of intracellular iron oxide nanoparticles form samples, FFT analyses are shown below the corresponding HRTEM images. M, ε, and H subscripts shown in FFT pattern denote Fe3O4 (magnetite), ε-Fe2O3, and α-Fe2O3 (hematite) phases, respectively. The time shown beside the individual HRTEM image is the interval when the samples were taken out after the induction of ferric citrate. Yellow cycles are for eye guide

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Fig. 5
figure 5

Phase ratio of various iron containing nanoparticles synthesized by MSR-1 at each time interval after the inducing of magnetosome formation. The results are based on HRTEM analyses and showed that at the early stage of magnetosome formation, multiple iron oxide nanoparticles can co-exist in the cells besides magnetite, including hematite, ε-Fe2O3 and other iron containing phases. Some representative HRTEM images and their FFT analyses are listed in SI

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Discussion

Recent studies have confirmed the existence of α-Fe2O3 at the early stage of magnetosome formation and act as a precursor phase [20, 21, 23]. In this study, we discovered that a thermodynamically unstable iron oxide phase, ε-Fe2O3, co-existed with α-Fe2O3 at the early stage of magnetosome synthesis, thus demonstrating that both of these two iron oxide phases are intermediates during the maturation of magnetosomes.

α-Fe2O3, γ-Fe2O3, and Fe3O4 are the most frequent iron oxides present in bulk in nature (crystal structures of Fe3O4 and α-Fe2O3 are shown in Fig. 3b) [28]. By contrast, ε-Fe2O3 is a rare phase with scarce natural abundance due to its thermal instability [29, 30]. Recently, the natural occurrence of a ε-Fe2O3 phase in some plants has been reported [31]. The crystal structure of the ε-Fe2O3 phase is an orthorhombic non-centrosymmetric structure with Fe atoms occupying four distinct nonequivalent crystallographic sites, including one tetrahedral site and three different octahedral sites (Fig. 3b) [32]. In the context of nano-materials, this structure is interesting, perhaps indicating a critical role of high surface energy, a characteristic of most nanostructures, in the formation of the epsilon phase with diameters of approximately 20 nm.

Similarities between the crystal structures of magnetite and ε-Fe2O3 are shown in Fig. 3b. Both tetrahedral and octahedral coordination of Fe and O occur in the two iron oxides; the stacking pattern of the two is formed by octahedral layers alternating with tetrahedral layers, with the exception of ordered octahedral vacancies in ε-Fe2O3. These similarities facilitate the structural transformation of ε-Fe2O3 to magnetite. Therefore, the formation of magnetite-containing magnetosomes via a ε-Fe2O3 intermediate may be more facile, even though α-Fe2O3 is more thermally stable.

Conclusions

The chemical synthesis of highly crystalline magnetite requires harsh conditions, and the ability of organisms to form such particles rapidly under moderate conditions remains an interesting question. Based on our current data and evidence from previous studies, a new chemical route model of magnetite biomineralization in MTB is proposed (Fig. 6): ferric ion is taken up into the cell and stored as a phosphate-rich ferric hydroxide phase [23]. After dephosphorization, ferric ion is transferred to a magnetosome vesicle to form water- or hydroxyl-containing ferric oxide, followed by transformation into a variety of iron oxides that act as the precursors of mature magnetosomes, including Fe3O4, α-Fe2O3, ε-Fe2O3 and other phases that have not yet been defined. The mature magnetite crystals finally form from these intermediate phases.

Fig. 6
figure 6

Schematic chemical route of magnetite biomineralization in MSR-1. Magnetosome biomineralization occurs through the following steps: Iron uptake (store as a phosphate-rich ferric hydroxide phase), dephosphorization, transformation of iron ion into magnetosome vesicle, formation of magnetosome intermediates that composed of various kinds of iron oxides and formation of mature magnetosomes composed of magnetite. Gray circles in the figure refer to magnetosome membrane

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