A Preliminary Report on the Isolation and Identification of Magnetotactic bacteria from Iran Environment

Many organisms have been identified that are able to sense the earth’s magnetic field. Certain bacteria are also geomagnetically sensitive and are known as Magnetotactic bacteria. For the first time, in 1975, Richard Blakemore observed certain mud bacteria whose swimming direction could be manipulated by magnetic field (Schüler et al., 1999). The Magnetotactic bacteria are Gram negative, motile, aquatic and heterotrophs. They synthesize intracellular enveloped magnetic grains termed magnetosomes (Blakemore et al., 1982). Magnetotactic bacteria form chains of magnetite (Fe3O4) nano-crystals in microaerophilic environments. Each species of Magnetotactic bacteria has a special shape of magnetosomes, but almost all of the magnetosomes are on the theoretical transition lines for single-domain-to-multidomain behavior and superparamagnetic-to-single-domain behavior (Thomas-Keprta et al., 2000). Membrane of magnetosomes consists of a bilayer containing phospholipids and proteins (Gorby et al., 1988). The exact role of these magnetosome-specific proteins has not been elucidated, but it has been suggested that they have specific functions in iron accumulation, nucleation of minerals; redox and pH control (Grünberg et al., 2001).
Some commercial applications have been suggested for bacterial magnetosomes such as hyperthermia, magnetic targeting of pharmaceuticals, cell separation and contrast-enhancement agents in magnetic resonance imaging (MRI) (Stephens et al., 2006). Magnetotactic bacteria navigate along the magnetic field, this process is known as magnetotaxis and may be very useful in robotics. They can move through porous mediums and carry something controllably. Despite the efforts of numerous researchers through improvement of isolation methods, only a few strains of Magnetotactic bacteria are available in pure culture including: Magnetospirillum magnetotacticum, M. gryphiswaldense, M. magnetotacticum AMB-1 and MGT-1, Vibrios MV-1 and MV-2, coccus MC-1, Marine spirillum MV-4, and RS-1 (Li et al., 2007).
In this research, attempts were made to identify and isolate freshwater and marine magnetotactic bacteria from Iran. For this reason, the isolates were characterized by optical microscope and scanning electron microscope (SEM). Furthermore intracellular magnetosomes have been studied by transmission electron microscope (TEM) and magnetic behavior of the magnetosomes was characterized by alternating-gradient-force magnetometer (AGFM).

MATERIALS AND METHODS

Culture medium: The culture medium contained 10 ml of vitamin solution, 5 ml mineral solution, 2 ml 0.01M ferric quinate, 0.45 ml 0.1% resazurin, 0.68 g KH2PO4 , 0.12 g NaNO3 , 0.37 g tartaric acid, 0.37 g succinic acid and 0.05 g sodium acetate in 1.0 l of distilled water. The pH of the medium was adjusted to 6.8 with NaOH and autoclaved at 121°C for 15 min. For preparation of solid medium, 12 g/l agar was added to the above liquid medium.
Vitamin solution contained 2 mg biotin, 2 mg folic acid, 10 mg pyridoxine hydrochloride, 5 mg thiamine.HCl, 5 mg riboflavin, 5 mg nicotinic acid, 5 mg calcium D-(+)-pantothenate, 0.1 mg vitamin B12 and 5 mg p-aminobenzoic acid in 1.0 l of distilled water. Mineral solution consisted of (g/l) 1.5 nitrilotriacetic acid, 3 MgSO4.7H2O, 0.5 MnSO4.H2O, 1 NaCl , 0.1 FeSO4.7H2O, 0.1 CoCl2.6H2O, 0.1 CaCl2, 0.1 ZnSO4.7H2O, 0.01 CuSO4.5H2O, 0.01 AlK(SO4)2.12H2O, 0.01 H3BO3 and 0.01 Na2MoO4.2H2O in distilled water. Ferric quinate (0.01M) contained 0.27 g FeCl3 and 0.19 g quinic acid in 100 ml distilled water (Martins et al., 2007).

Isolation method: Samples with mud and water were collected from the Caspian Sea (53° 17’ E, 36° 50’ N) and Karkheh River (48° 2’ E, 32° 50’ N). They were then left undisturbed in dim light at room temperature for one month. From these samples, the magnetotactic bacteria were separated using a magnet as shown schematically in Figure 1. The water under the magnet, which supposed to contain the magnetotactic bacteria, was collected with the help of a pipette and inoculated on solid medium; the oxygen was removed by nitrogen stream and then the plates were sealed. After two days, three colonies from each plate were transferred into liquid medium and incubated at 30ºC for three weeks. Resazurin in the culture medium acts as an oxygen indicator turns pink in the presence of oxygen and colorless in its absence. When the growth medium was colorless, a small magnet was kept near the falcons to gather magnetic particles. In some falcons, it was possible to see magnetic particles. These particles might have been released from dead bacteria. Each of the samples that contained magnetic particles was supposed to have isolated magnetotactic bacteria.

Microscopic methods: For thin section preparation, the bacteria were grown in 2.5-liter jars, then centrifuged at 10000 g for 5 min, and washed twice with distilled water. Supernatants were discarded, and the pellet was fixed with 2% glutaraldehyde solution in water for 3 h and then postfixed in 1% osmium tetroxide. Thin sections were cut in an ultramicrotome and uranyl acetate stained samples examined with a Zeiss EM 900 (50KV) transmission electron microscope.
For SEM sample preparation, a few drops of cultured medium were dried on a glass and washed with distilled water without staining. The samples were examined with a VEGA\\TESCAN scanning electron microscope.

Sample preparation for AGFM: Since magnetotactic bacteria synthesize magnetosomes in microaerophilic conditions (Lang and Schüler, 2006), the isolated bacteria were incubated at 30ºC in anaerobic jars for one month till it became colorless. Then biomass of the cultured bacteria was centrifuged at 10000 g for 7 min and washed three times with distilled water. The pellet was freeze dried and used for AGFM analysis.

RESULTS

Microscopic analysis: Gram staining results indicated that the isolated bacteria were Gram negative. SEM images, presented in Figures 2 A,B, showed the freshwater and marine bacteria are both rod-shaped. Elemental maps of iron are shown in Figures 3 A,B. The concentrated positions of iron and chains of particles containing iron have been shown in these Figures. TEM results, shown in Figures 4 A,B, revealed that the sizes of magnetosomes are about 10 to 20 nm. Higher magnification images showed that these particles were elongated in one direction and therefore they had shape anisotropy properties. Magnetosomes have magnetic anisotropy properties and their magnetic field properties depend on the direction.
Magnetic analysis: Through optical microscopic observations and changing the position of a magnet near the samples, it was revealed that the isolated species are both South-seeking and North-seeking. In addition, AGFM analysis, Figures 5 A,B, on extracted magnetosomes showed superparamagnetic-to-single-domain behavior.

DISCUSSION

Magnetotactic bacteria are able to distinguish between south and north poles of magnet and migrate along the geomagnetic field; this behavior is known as magnetotaxis. Normally, the polarity of magnetic bacteria living in the northern hemisphere is North-seeking and South-seeking in the Southern hemisphere (Hanzlik et al., 2002). In addition, magnetotactic bacteria near the geomagnetic equator are both South-seeking and North-seeking (Frankel et al., 1981). The polarity of isolated bacteria was investigated and they were attracted to both poles of magnet. So, they can be categorized in the third group.
In elemental map analysis, electron beam had high energy levels, so electrons can penetrate the sample and the characterized area is bigger than the beam diagonal. This phenomenon makes the identification of the exact place and shape of magnetosomes inaccurate. Therefore, it was hard to put an accurate estimate on the dimensions of particles via elemental analysis and the images had a low resolution. Nevertheless the elemental maps of iron showed that iron rich particles were arranged in chains.
TEM images indicated the formation of magnetosomes. Shape and arrangement of these particles are accurately displayed in Figure 4. The shape and chain arrangement of these particles are evidence for synthesis of magnetosomes by the isolated bacteria.
Micron size or larger particles of magnetite at room temperature has a permanent magnetic moment but magnetite nanoparticles below a certain size have no magnetic remanence and show superparamagnetic behavior (Neuberger et al., 2007). In magnetic nanoparticles, thermal fluctuations cause the magnetic moment to wander relative to the crystallographic axes (Cullity and Graham, 2009). But Magnetosomes have shape anisotropy properties and the elongated directions of particles are along the easy magnetization direction of the crystals. This superposition of easy axes and elongated direction enhances anisotropy constant and therefore magnetosomes show remanence and superparamagnetic-to-single-domain behavior. Also AGFM analysis show coercivity of the samples is about 120 G and configurationally is in agreement with L. Han et al. (2007). These evidences imply that the isolated bacteria are magnetotactic and their magnetosomes reveal superparamagnetic-to-single-domain behavior.

Acknowledgments

This research was supported by Tarbiat Modares University. We are very thankful of Mrs. Teymuri for her helpful comments on isolation of these bacteria and Iran nano-technology initiative council that provided the grant for the electron microscope analyses.