Transcript Magnetospirillum strain J10 and M

Magnetospirillum gryphiswaldense Cell Growth and Magnetosome
Production Mediated by Mixotrophic Conditions
Figure 3. Experiment and model (shaded lines) of magneto-aerotactic
response 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 aerotaxis
and accumulation of cells at the level of the spectrometer window. Then as the
preferred oxygen concentration moved below the spectrometer window, most
of the cells followed, leaving a very low density of cells behind.
Objective
• Magnetotatic bacteria are more efficient at making
magnetic nanoparticles than chemical manipulations
such as co-precipitation, thermal decomposition,
microemulsion, and flame spray synthesis.
• 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.
• 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).
With magnetic field
Without magnetic field
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• 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.
•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.
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• 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.
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• 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).
•Mixotrophic conditions should support higher
magnetospirillum growth due to the higher biomass
accumulation and respiratory activities brought by the
media (Figure 4).
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• Bacteria use these magnetosomes to find optimal
oxygen concentrations know as aerotaxis.
•Mixotrophic conditions have been demonstrated by
Geelhooed et al (2006) to produce the greatest amount
of Biomass and respiratory activity.
Co
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• 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).
• 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).
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• 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.
Proposed Results
Review of Literature (continued)
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Introduction
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.
Colony Forming Units (cells/mL)
http://www.se.kanazawa-u.ac.jp/bioafm_center/images/MS-1-a.jpg
Bryce Bendl, Department of Biology, York College of Pennsylvania
Media Treatment
Figure 4. Mean cololny forming units for Magnetospirillum
gryphiwaldense diluted to 10-6 growing in different media
treatments with or without magnetic field stimulus (n=15). Dunn's
multiple comparison test (p<0.05), bars with same letter are not
significantly different from each other.Error bars represent 95%
confidence interval (95% CI).
Proposed Methods
Review of Literature
0.5
Bacteria grown
in same media
in oxygencontrolled
fermentor
Fig. 2. Growth of Magnetospirillum spp. in different growth
conditions.
A. Biomass production of Magnetospirillum strain J10 and M.
gryphiswaldense in heterotrophic (acetate), mixotrophic (acetate
and sulfide) and autotrophic (sulfide) conditions in continuous
culture at D = 0.07 h-1.
B. Per cent increase in biomass production compared with the sum
of heterotrophic growth on acetate and autotrophic growth on
sulfide (Sum = Yield for heterotrophic growth (YH) x acetate
concentration + Yield for autotropic growth (YA) x sulfide
concentration).
When exposed to oxygen, the M. gryphiswaldense are
shown to migrate away from the incoming oxygen gradient
with various magnetic field strengths.
Fig. 1 Effect of various constant dissolved oxygen tensions on
growth and magnetite formation in oxystat cultures of M.
gryphiswaldense. Growth and magnetism were determined
after 22 hour cultivation in large-scale medium (LSM).
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 magnetosomedeficient bacterial strands DmagA1 (Figure 3).
Bacteria
stained with
two different
dyes
Colony forming
units (CFU) is
measured
Magnetosome
Iron
concentration
is measured
Control with
magnetic field
stimulus
Control without
magnetic field
Sulfide-rich media
with magnetic field
Sulfide-rich media
without magnetic
field
Review of Literature (continued)
Magnetospirillum are shown to be strongly influenced by
the oxygen concentration in their environment.
Bacteria grown
in
thioglycollate
growth media
with different
treatments
SDS in media with
magnetic field
stimulus
Magnetospirillum
Growth Medium
(MSGM)
SDS in media
without magnetic
field stimulus
Acetate-rich media
with magnetic field
stimulus
Acetate-rich media
without magnetic
field stimulus
Acetate and sulfiderich media without
magnetic field
stimulus
Acetate and sulfiderich media without
magnetic field
stimulus
b
With magnetic field
Without magnetic field
Iron Content In Cells (%)
• 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.
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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.
Control
Sulfide
Acetate Acetate+sulfide
SDS treated
Medium Treatment
Figure 5. Iron content present in Magnetospirillum gryphiwaldense
grown with different medium treatments with or without magnetic field
stimulus (n=15). Tukey's multiple comparison test (p<0.05), bars with
same letter are not significantly different from each other.Error bars
represent 95% confidence interval (95% CI).
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.
Acknowledgments
I 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.