Review of Literature (continued)

•Magnetospirillum strain J10 (magnetosome lacking mutant) and M. gryphiswaldense grew well in acetate-rich media, showing acetate was the limiting substrate. The dry weight (DW) biomass yields of Magnetospirillum strain J10 and M. gryphiswaldense were virtually identical at 12 g DW mol acetate-1 (Geelhoed et al, 2010).

•When sulfide was supplied in addition to acetate, the production of biomass increased (Figure 2). This was shown to be the optimal acetate and sulfide concentration.

•The supply of 10 mM acetate and 10 mM sulfide to the

cultures approximately doubled the biomass production compared with growth on 10 mM acetate alone, which shows that both Magnetospirillum strain J10 and M. gryphiswaldense are able to derive energy for growth from the oxidation of sulfide.

Introduction

•Magnetotatic bacteria are a unique group of microorganisms that are able to migrate either towards or away from geomagnetic field lines to find optimal conditions to grow.

• To achieve this amazing feat, the bacteria are able to synthesize unique organelles known as magnetosomes. These magnetosomes contain either magnetite (FeO4) or greigite (FeS4) which enable the bacteria to sense magnetic field lines by having these magnetosomes align parallel to the magnetic field line present (Bazylinsky, 1995).

•Bacteria use these magnetosomes to find optimal oxygen concentrations know as aerotaxis.

•Magnetotatic bacteria are able to form magnetic nanoparticles in specific microaerophilic oxygen concentrations. Any higher than this range will result in no production of magnetic nanoparticles (Heyen and Schuler, 2003).

•Magnetotatic bacteria are more efficient at making magnetic nanoparticles than chemical manipulations such as co-precipitation, thermal decomposition, microemulsion, and flame spray synthesis.

•Important applications of these magnetic nanoparticles in nanomedicine include as cancer treatments, stem cell tracking and therapy, gene therapy, and tissue engineering (Lin et al, 2010).

Magnetospirillum gryphiswaldense Cell Growth and Magnetosome Production Mediated by Mixotrophic Conditions

Review of Literature (continued)

Magnetospirillum are shown to be strongly influenced by the oxygen concentration in their environment.

When exposed to oxygen, the M. gryphiswaldense are shown to migrate away from the incoming oxygen gradient with various magnetic field strengths.

In a study done by Smith et al (2006), M. gryphiswaldense was shown to respond faster to the incoming oxygen gradient when the bacteria was able to sense a weak or strong magnetic field more efficiently than magnetosome-deficient bacterial strands DmagA1 (Figure 3).

Proposed Results

•Mixotrophic conditions have been demonstrated by Geelhooed et al (2006) to produce the greatest amount of Biomass and respiratory activity.

•Mixotrophic conditions should support higher magnetospirillum growth due to the higher biomass accumulation and respiratory activities brought by the media (Figure 4).

•Mixotrophic conditions should enable the bacteria to produce greater amounts of magnetosomes due to mixotrophic conditions providing the best carbon source in acetate and energy source in sulfide (Figure 5). Having optimal energy source and carbon source should increase the production of siderophores which are able to intake Fe(III) with the aid of energy in the form of ATP produced from sulfide in media.

Magnetosome Iron

concentration is measured

Colony forming units (CFU) is

measured

Bacteria stained with two different

dyes

Bacteria grown in

thioglycollate growth media with different treatments

Bacteria grown in same media

in oxygen-controlled fermentor

Magnetospirillum Growth Medium

(MSGM)

Control with magnetic field

stimulus

Control without magnetic field

Sulfide-rich media with magnetic field

Sulfide-rich media without magnetic

field

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stimulus

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field stimulus

Acetate-rich media with magnetic field

stimulus

Acetate-rich media without magnetic

field stimulus

Acetate and sulfide-rich media without

magnetic field stimulus

Acetate and sulfide-rich media without

magnetic field stimulus

SYBR Green II stains all cells, dead or alive, green.

When SYBR Green II is coupled with Propidium Iodide (PI), all dead cells are stained red.

All alive cells are counted by taking 0.1 mL from microaerophilic zone in media and transferring it to 2 mL tube containing buffer solution. Then, dilute the solution three times at 1/10. Take 0.1 mL of solution and spread on plate and count live green cells.

1.5 mL sample of bacteria is taken from microaerophilic zone and centrifuged in 2 mL tube. The pallet is dried at 60°C until it is at a constant weight. Iron content is measured using Inductively Coupled Plasma Optical Emission Spectrometry. The percentage of Iron in cells was calculated as the Iron content divided by the dry weight.

Objective

•The purpose of this study is to test the conditions of the magnetotactic bacteria Magnetospirillum gryphiswaldense to see if the bacteria are able to increase their growth as well as increase their development of magnetosomes.

•In addition, this research seeks to determine if the Magnetospirillum gryphiswaldense magnetosome production increases due to environment containing vital respiratory compounds.

Fig. 1 Effect of various constant dissolved oxygen tensions ongrowth and magnetite formation in oxystat cultures of M.gryphiswaldense. Growth and magnetism were determined after 22 hour cultivation in large-scale medium (LSM).

Review of Literature

•Magnetosome formation is influenced by the dissolved oxygen concentration of the media. As shown in Figure 1, the magnetosome development was optimized when the oxygen concentration was extremely low. Heyen and Schuler (2003) showed a dissolved oxygen tension pO2 at 0.25 mbar as being the optimal oxygen concentration for magnetosome development.

Fig. 2. Growth of Magnetospirillum spp. in different growthconditions.A. Biomass production of Magnetospirillum strain J10 and M.gryphiswaldense in heterotrophic (acetate), mixotrophic (acetateand sulfide) and autotrophic (sulfide) conditions in continuousculture at D = 0.07 h-1.B. Per cent increase in biomass production compared with the sumof heterotrophic growth on acetate and autotrophic growth onsulfide (Sum = Yield for heterotrophic growth (YH) x acetateconcentration + Yield for autotropic growth (YA) x sulfideconcentration).

Bryce Bendl, Department of Biology, York College of Pennsylvania

Proposed Methods

Figure 3. Experiment and model (shaded lines) of magneto-aerotacticresponse in an applied magnetic field and oxygen gradient. DmagA1 1 hi,WT 1 hi (150 G vertical field), WT 1 lo (10 G vertical field), and WT 1 no(0 G vertical field) initially showed an increase in density caused by aerotaxisand accumulation of cells at the level of the spectrometer window. Then as thepreferred oxygen concentration moved below the spectrometer window, mostof the cells followed, leaving a very low density of cells behind.

Literature Cited •Blakemore, R. P., Frankel, R. B. & Kalmijn, A. J. 1980. Southseeking magnetotactic bacteria in the southern hemisphere. Nature 236: 384–385•Geelhoed, J. S., Kleerebezem, R., Sorokin, D. Y., Stams, A. J., & Van Loosdrecht, M. 2010. Reduced inorganic sulfur oxidation supports autotrophic and mixotrophic growth of Magnetospirillum strain J10 and Magnetospirillum gryphiswaldense. Environmental microbiology, 12: 1031-1040.•Heyen, U., & Schüler, D. 2003. Growth and magnetosome formation by microaerophilic Magnetospirillum strains in an oxygen-controlled fermentor. Applied microbiology and biotechnology, 61: 536-544.•Lin, M. M., Kim, H. H., Kim, H., Muhammed, M., & Kim, D. K. (2010). Iron oxide-based nanomagnets in nanomedicine: fabrication and applications. Nano reviews, 1:4483 •Smith, M. J., Sheehan, P. E., Perry, L. L., O’Connor, K., Csonka, L. N., Applegate, B. M., & Whitman, L. J. 2006. Quantifying the magnetic advantage in magnetotaxis. Biophysical journal, 91: 1098-1107.• Zhang, F., Yu-Zhang, K., Zhao, S., Xiao, T., Denis, M., and Wu, L. 2010. Metamorphosis of Magnetospirillum Magneticum AMB-1 Cells. Chinese Journal of Oceanology and Limnology 28: 304-09.

AcknowledgmentsI would like to thank Dr. Thompson for all his support and wisdom which proved to be immeasurable in helping my ideas as well as progress with the research. In addition, I would like to thank Dr. Mathur for assisting me in suggesting appropriate media for microaerophilic bacteria and for assisting in my own Magnetospirillum growth experiments. Lastly, I would like to thank the whole York College of Pennsylvania Biology department for sculpting me into the best undergraduate I could be.

http://www.se.kanazawa-u.ac.jp/bioafm_center/images/MS-1-a.jpgZhang, F., Yu-Zhang, K., Zhao, S., Xiao, T., Denis, M., and Wu, L. 2010. Metamorphosis of Magnetospirillum Magneticum AMB-1 Cells. Chinese Journal of Oceanology and Limnology 28: 304-09.

Control Sulfide Acetate Acetate+sulfide SDS treated0.0

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Iro

n C

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Figure 5. Iron content present in Magnetospirillum gryphiwaldensegrown with different medium treatments with or without magnetic fieldstimulus (n=15). Tukey's multiple comparison test (p<0.05), bars withsame letter are not significantly different from each other.Error barsrepresent 95% confidence interval (95% CI).

a a a a a a

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Figure 4. Mean cololny forming units for Magnetospirillumgryphiwaldense diluted to 10-6 growing in different mediatreatments with or without magnetic field stimulus (n=15). Dunn'smultiple comparison test (p<0.05), bars with same letter are notsignificantly different from each other.Error bars represent 95%confidence interval (95% CI).

a, c a, c

a, b ,c a,b, c

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