Magnetism in living magnetically-induced bacteria

Abstract

Artificial magnetically-induced bacteria were prepared by labelling probiotic bacteria Lactobacillus fermentum with maghemite nanoparticles. Most bacteria remained alive after magnetic labelling and exhibited good viability. We have used this unique living magnetic material to address an issue never posed before: how does the magnetism of a living magnetic system vary while life proliferates. Monitoring of magnetic properties of this living system confirmed a magnetic dilution during proliferation. During bacteria division, the number of magnetic nanoparticles was equally shared among the daughter bacteria. Chain-like assemblies fixed into a LR-white resin with the application of a magnetic field enhance the anisotropic magnetic properties of this system.

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Introduction

Nature is a school for nanomaterials scientists. A promising alternative to fabricate functional inorganic materials may be to follow the strategies used by Nature.^1,2 Living magnets are one of the most exciting biomaterials offered by Nature. Magnetotactic bacteria are magnetite (Fe₃O₄)- and/or greigite (Fe₃S₄)-producing bacteria that are found both in freshwater and marine environments. They inhabit the oxic–anoxic transition zone under the microaerophilic conditions required for their growth.^3–6 Magnetite crystals are actively formed through biological mechanisms in intracellular organelles called magnetosomes.^3,7,8 They display a narrow size distribution and are in the magnetically stable single-domain range. Magnetosomes are assembled in chains inside the cell and provide the microorganism with a permanent magnetic dipole, which allows magnetotactic bacteria to feel the geomagnetic field and then to use it for orientation, navigation and migration.⁹

This living system represents an ideal framework to address a conceptual question: how does magnetism of a magnetic system progress with life. Recently, a time-resolved magnetic study was carried out on the biomineralisation process of the magnetotactic bacteria.

Magnetospirillum gryphiswaldense and indicated a dynamic transformation from the initial antiferromagnetic ferrihydrite to a ferrimagnetic magnetite phase.¹⁰ However, no data are available concerning the progress of magnetic properties during bacterial growth, i.e. proliferation, degradation, etc.

We recently reported that the magnetic properties of natural magnetobacteria can be roughly reproduced by following a biomimicry approach. Probiotic bacteria Lactobacillus fermentum and Bifidobacteria breve were used as bioplatforms to densely arrange superparamagnetic nanoparticles on their external surfaces, thus obtaining the so-called artificial magnetic bacteria (AMB).¹¹

Unlike magnetotactic bacteria, AMB offer extraordinary possibilities for manipulation because they are probiotics that can be produced at a large scale, show excellent viability^12,13 and their magnetic properties can be tuned by modulating parameters, such as the number of nanoparticles per bacteria, culture media, etc.

In this report, we evaluate how growth and proliferation of these artificial magnetic bacteria affect their magnetic properties.

In addition to answering this conceptual question, the influence of proliferation of living magnetic systems on their magnetic properties is crucial in a future scenario in which living magnets are used in different biomedical applications, such as magnetic resonance imaging (MRI), hyperthermia and drug delivery.^14–18

Experimental section

Grafting maghemite nanoparticles to bacteria

Lactobacillus fermentum were grown in MRS at 37 °C with orbital agitation for 24 h. The optical density was measured at 600 nm to obtain a concentration of 1 × 10⁹ CFU ml⁻¹. After 24 h, a 10 ml bacterial culture in MRS medium was collected at 3000g for 10 min. Then, 66.6 μl of a maghemite solution (0.95 M) was added and the solution was diluted to 10 ml with distilled water at pH 2. Bacteria labelled with maghemite nanoparticles (artificial magnetic bacteria) were collected at 100g for 20 min. Supernatant was discarded and 10 ml of fresh MRS medium was added and cells were left to grow again, and the medium was changed every 24 hours. Samples were taken at different time points: 15 h (sample 1), 18 (sample 2), 39 h (sample 3), 49 h (sample 4), and 62 h (sample 5). Each sample was freeze-dried overnight and then lyophilized.

Inclusion of AMB in LR-white resin

After 24 hours, a 10 ml culture of L. fermentum in MRS medium was collected at 3000g for 10 min. Then 66.6 μl maghemite per ml culture was added and the sample was dissolved in 10 ml of dH₂O, pH 2. Afterwards, the sample was centrifuged at 100g for 30 min. Once cells were collected, the supernatant was discarded and 5 ml of LR-white resin was added. Subsequently, samples were included in capsules, and 3 days of incubation under ultraviolet light was needed for the resin to polymerize. Capsules were cut into ∼0.5 μm slices.

Electron microscopy

For Transmission Electron Microscopy (TEM) grid preparation, a drop of AMB samples was placed onto a carbon-coated Cu grid (200 mesh). Samples 1 to 5 were embedded in epoxy resin. Electron micrographs were taken with a Philips CM-20 HR analytical electron microscope operating at 200 keV.

Magnetic measurements

Magnetic measurements were performed on lyophilized samples using a magnetometer (Quantum Design MPMS-XL-5) equipped with a SQUID sensor. The temperature was varied between 2 and 300 K, according to a classical zero-field-cooled/field-cooled (ZFC/FC) procedure in the presence of a weak applied magnetic field (5 mT), and the hysteresis loops were obtained at 5 K and 300 K in a magnetic field from 5 T to −5 T.

Results and discussion

We have prepared the AMB system and we have isolated a batch of samples after bacterial proliferation at different times. Specifically, once the maghemite nanoparticles were attached to the bacterial surface, fresh medium was added to boost the cells' proliferation. Samples were taken at different times (Scheme 1) corresponding to different bacterial generations (BG): 0 h (0), 15 h (26 BG) (1), 18 h (31 BG) (2), 39 h (67 BG) (3), 49 h (84 BG) (4), and 62 h (106 BG) (5) (see Experimental section). In all samples, labelling of the magnetic nanoparticles produced no negative effects on cell viability.

Scheme 1 A visual scheme of the preparation steps.

Fig. 1 shows typical TEM images of these samples after being included in epoxy resin. A decrease in the number of magnetic nanoparticles per bacteria is seen as bacteria proliferate.

Fig. 1 TEM images of thin epoxy resin sections of samples of AMB: control (t = 0) (a), after 15 h (26 BG) (b), 18 h (31 BG) (c), 39 h (67 BG) (d), 49 h (84 BG) (e) and 62 h (106 BG) (f).

The cellular proliferation of AMB could result either in a symmetric distribution of nanoparticles between each of the two new bacteria or in an asymmetric situation, in which one of the two new bacteria take all the nanoparticles (Scheme 2).

Scheme 2 Symmetric and asymmetric distribution of magnetic nanoparticles between bacteria while dividing.

A careful analysis by TEM of samples 1–5 revealed that the presence of nude bacteria was scarce and not significant. This suggests that cell division takes place according to the symmetric option.

The magnetism of samples resulting from the extreme symmetric and asymmetric mechanisms should be drastically different. We analysed the magnetic properties of lyophilized powders of samples 1–5.

The magnetic properties of these samples corresponded to those expected for magnetic assemblies of randomly oriented superparamagnetic nanoparticles with variable dipolar interactions,¹⁹ depending on the amount of nanoparticles per bacterium.²⁰

Hysteresis loops of powdered samples were recorded at 5 K and at 300 K and the obtained magnetic values are summarised in Table 1. Hysteresis at 300 K (Fig. 2) reached saturation at 1 T for samples 1, 2 and 3, with negligible coercive fields and remanent magnetisation values on increasing maghemite nanoparticles dilution due to bacterial growth. The AMB are magnets at room temperature except for samples 4 and 5.

Table 1 Variation of the reduced remanence (M_R/M_S), coercive fields (H_C) and blocking temperatures (T_B) of samples 1 to 5 at 300 K and 5 K

	MR/MS 5 K	HC 300 K [Oe]	HC 5 K [Oe]	TB^d [K]	(MR/MS)∥^a 5 K	(MR/MS)⊥^b 5 K	HC∥,⊥^c 5 K [Oe]
a MR/MS of the 1D bacteria–maghemite assemblies when applying a magnetic field parallel to the direction of the alignment.b Applying a magnetic field perpendicular to the direction of the alignment.c HC parallel and perpendicular to the direction of the alignment.d In brackets TB values obtained from ac measurements (Fig. 5).
1	0.22	9	175	90 (55)	0.42	0.39	320
2	0.2	9.3	176	76 (50)
3	0.06	7.6	174	70 (46)
4	0.01	0	105	58
5	0.006	0	52	0

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Fig. 2 Hysteresis loops of samples 1 (black line), 2 (red line) and 3 (blue line), at 300 K.

Hysteresis loops at 5 K (Fig. 3) were open below T_B with slightly lower coercivities when compared to the usual maghemite nanoparticles of 10 nm.²¹ The coercive field and reduced remanence ratios M_R/M_S increased with the amount of magnetic nanoparticles per bacterium (Table 1). In any case, AMB did not show a single-domain behaviour (M_R/M_S < 0.5) as is typically found in magnetotactic bacteria.

Fig. 3 Hysteresis loops of samples 1 (black line), 2 (red line), 3 (blue line), 4 (pink line) and 5 (green line) at 5 K.

Zero-field-cooled (ZFC) and field-cooled (FC) magnetisation measurements were performed for all samples (Fig. 4). The observed irreversibility between ZFC and FC is attributed to the characteristic blocking–unblocking process of the particle magnetic moment when the thermal energy is varied. The temperature of the maximum in the ZFC magnetization, the so-called blocking temperature T_B, determines the temperature below which the particle moment appears blocked within the time-scale of the experiment. The ZFC magnetisation curves of all the samples exhibited a maximum that moved to lower temperatures as the bacteria proliferated (Table 1).

Fig. 4 ZFC/FC curves for samples 1 to 5. Arbitrary units are used for clarity.

To investigate in more detail the nature of the irreversibility observed in the ZFC/FC curves, we measured the temperature dependence of the ac susceptibility at different frequencies. Fig. 5 shows the imaginary part of the ac susceptibility (χ′′) of samples 1, 2 and 3. These samples showed a single maximum near the T_B, whose position and amplitude were sensitive to the applied ac magnetic field frequency. The position shifted to higher temperatures as the frequency increased. This shift is clear evidence of superparamagnetism. We estimated a value of χ′′ maxima at ca. 55 K for sample 1, 50 K for sample 2 and 46 K for sample 3 measured at the lowest possible frequencies corresponding to T_B values (values shown in brackets, Table 1).

Fig. 5 Temperature dependence of the out-of-phase (imaginary) component χ′′(T) of the magnetic susceptibility for samples 1, 2 and 3 at different excitation frequencies and taken in a zero external magnetic field.

For superparamagnetic particles, thermal energy flips the magnetisation between the two states by overcoming an anisotropy energy barrier. The relaxation time, τ, is given by:

where τ₀ is a time constant that is usually in the range 10⁻⁹ to 10⁻¹¹ s for a superparamagnetic system; E_a is the effective anisotropy energy; K_B is the Boltzmann's constant; T is the blocking temperature of the particle.^21,22 Taking the log of eqn (1) gives the equation:

Fitting T_B with frequencies using eqn (2), we obtained τ₀ values of 10⁻¹³ s for sample 1, 4.9 × 10⁻¹⁰ s for sample 2 and 2.3 × 10⁻⁹ s for sample 3, indicating superparamagnetic behaviour for 2 and 3 and a deviation from this behaviour for 1.²³ Since T_B depends on the frequency, it can be checked that the particles are truly non-interacting by verifying the dependence of T_B on the measurement time as given by the Néel–Brown theory. Departures from this theory indicate interparticle interactions by, for example, dipole–dipole interactions.²⁴ For non-interacting particles, χ′ vs. T curves for various particle concentrations are identical when properly normalised. Deviation from this behaviour indicates that interparticle interactions are important. Fig. 6 shows that χ′ normalised curves for 1–3 do not superimpose, indicating the existence of dipolar interactions.

Fig. 6 Comparison between χ′(T) measured at 10 Hz for samples 1 (black line), 2 (red line) and 3 (blue line). Susceptibility curves have been normalised to their respective maximum values.

The existence of magnetic dipole–dipole interactions between nanoparticles is a suitable tool for achieving ordered – or at least assembled – magnetic structures.¹¹ In fact, this kind of system can be assembled by application of an external magnetic field.^25–30 This possibility is of interest in the context of materials for recording and sensing applications since these materials require to be nanostructured.³¹

In this context, we explored the possibilities of building supra-magnetic nanostructures from sample 1 in the presence of an external magnetic field. 1D AMB assemblies were formed and fixed into a LR-white resin with the application of a magnetic field of 1.2 T during the polymerization process to freeze the pictures. Bacteria aligned by following the external magnetic field lines and formed a chain-like nanostructure.^25–30 TEM images (Fig. 7) show the formation of long 1D nanostructures with a width corresponding to 3–4 adhered bacteria. The bacteria seem to stick to each other along the chain through the magnetic nanoparticles. Certainly, the exo-polysaccharides (EPS), where the maghemite nanoparticles are adsorbed, assist the contact between bacteria, since one of the roles of EPS is the formation of the biofilm. Interestingly, the presence of magnetic nanoparticles into the EPS gives rise in sample 1 to the formation of aligned magnetic biofilms.^32–36

Fig. 7 TEM micrographs of a thin resin section of sample 1, showing the 1D nanostructure formation after a magnetic field is applied. Maghemite particles surround the external surface making the bacteria align in a chain-like structure.

Hysteresis loops of the 1D AMB assemblies were measured when applying a magnetic field parallel or perpendicular to the direction of the alignment.

Magnetisation curves clearly show the change in the shape of the hysteresis loop with the orientation of the applied field (Fig. 8). For the two different field configurations, the reduced remanences are designated by (M_R/M_S)_∥ and (M_R/M_S)_⊥, respectively. When the field is parallel to the direction of the alignment, the hysteresis loop is squarer than that of the randomly oriented artificial magnetic bacteria (Fig. 8, left). Moreover, an increase in the reduced remanence (M_R/M_S) and coercivity values occurs (Table 1), especially when the field applied is parallel to the alignment.^37–42 The hysteresis curves are similar to those observed for whole magnetotactic bacteria.⁴⁰

Fig. 8 Magnetisation curves at 5 K that were recorded for sample 1 fixed in a thin section of resin. Top: hysteresis loop for randomly oriented (blue) and parallel-oriented with the direction of the 1D alignment. Bottom: hysteresis loops either parallel (red line) or perpendicular (black line) to the direction of the 1D alignment.

Such changes in the magnetisation curves of the 1D AMB compared to randomly oriented nanoparticles are due to the long-range dipole–dipole coupling, with a collective “flip” of the magnetic dipole.⁴³ The reduced remanence of the γ-Fe₂O₃ nanoparticles that are randomly oriented is 0.22. This value is lower than that calculated for single-domain particles (0.50). This clearly indicates that γ-Fe₂O₃ nanocrystals are characterised by uniaxial anisotropy.⁴³ The increase in the M_R/M_S ratio and coercive field is a measure of the strength of the magnetic anisotropy.

Conclusions

In conclusion, we have shown how proliferation and growth affect the magnetic properties of artificial magnetic bacteria. They behave as a superparamagnetic system with blocking temperatures that decrease as the bacteria growth, showing a magnetic dilution effect. We have shown how chain-like nanostructuration imposed by the external maghemite nanoparticles enhances the anisotropic magnetic properties of this system in a way similar to magnetosomes inside the magnetotactic bacteria.

Acknowledgements

This work was funded by MINECO and FEDER (project CTQ2015-64538) and Junta de Andalucía (project CTQ2011 P11-FQM-8136).

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