Riboflavin-mediated Motility in
Shewanella oneidensis MR-1, Revealed by
a Microfluidic Device
M.Eng Thesis
The Biofluidics Lab, Biological and Environmental Engineering, Cornell
University
Tiffany C Kuo
1/1/2014
Abstract: The motility of Shewanella Oneidensis (S. oneidensis), a facultative anaerobic capable of
electron donation to extracellular electron acceptors such as electrodes or metal oxides, is mediated in
both aerobic and anaerobic environments by the self-secreted electron shuttle riboflavin. Riboflavin acts
as an electron shuttle in the reduction of electron acceptors and is a putative chemoattractant. Studies in a
gradient-generating microfluidic device show that increasing concentrations of supplemented riboflavin
increases migration speed of cells. In addition, S. oneidensis motility increases with oxygen depletion.
Experiments on riboflavin chemotaxis are not conclusive, and require further studies. Quantifying
riboflavin-mediated motility would allow for a better understanding and optimization of S. oneidensis
electron donation within a bioelectrochemical system (BES).
Contents
I. Introduction to BES and S. oneidensis.................................................................................................... 1
The Role of S. Oneidensis in BESs ..................................................................................................... 1
Electron Transfer Mechanisms in S. oneidensis ............................................................................... 1
II. Motivation for S. oneidensis Cellular Transport Study.......................................................................... 2
III. The Role of Riboflavin in Electron Transfer.......................................................................................... 3
Riboflavin and Motility ..................................................................................................................... 3
Riboflavin and Current Generation ........................................................................................................... 4
IV. Prior Chemotaxis and Motility Experiments in S. oneidensis .............................................................. 4
V. Our approach – Microfluidic platform for S. Oneidensis motility and chemotaxis .............................. 5
S. oneidensis MR-1 Experimental Setup ........................................................................................... 6
Syto-9 stained S. oneidensis MR-1 Experimental Setup ................................................................... 6
VI. Materials and Methods ....................................................................................................................... 7
Riboflavin Gradient Calibration........................................................................................................ 7
Cell Culture ....................................................................................................................................... 8
Microfluidic Device ........................................................................................................................... 8
Pumping ........................................................................................................................................... 9
Riboflavin ......................................................................................................................................... 9
Anaerobic Condition Generation ...................................................................................................... 9
Microscopy ..................................................................................................................................... 10
VII. Analysis Method ............................................................................................................................... 10
VIII. S. Oneidensis MR-1 Cell Trajectory Paths under the Influence of Riboflavin .................................. 10
Trajectory Graphs for S. oneidensis MR-1 motility in Aerobic Conditions ..................................... 11
Trajectory Graphs for S. oneidensis MR-1 motility in Anaerobic Conditions ................................. 12
IX. Instantaneous Speed Data for S. oneidensis MR-1 motility in Aerobic and Anaerobic Conditions ... 13
X. Y-velocity Data for S. oneidensis MR-1 motility in Aerobic and Anaerobic Conditions ...................... 15
XI. Syto-9 stained S. oneidensis MR-1 Cell Trajectory Paths under the Influence of 55.6 nM/mm
Riboflavin in Aerobic and Anaerobic Conditions .................................................................................... 16
XII. Instantaneous Speed Data for Syto-stained S. oneidensis MR-1 motility in Aerobic and Anaerobic
Conditions ............................................................................................................................................... 17
XIII. Y-velocity Data for Syto-stained S. oneidensis MR-1 motility in Aerobic and Anaerobic Conditions
................................................................................................................................................................ 18
XIV. Conclusion........................................................................................................................................ 19
XV. Acknowledgements .......................................................................................................................... 20
XVI. References ....................................................................................................................................... 21
I. Introduction to BES and S. oneidensis
The Role of S. Oneidensis in BESs
Bioelectrochemical systems, also known as BESs, are systems within which bacteria grow and
are able to transfer electrons from organic materials such as wastewater into energy. A promising
technology, BESs can provide potential solutions for a host of high-impact problems, such as
alternative energy generation, production of expensive biomaterials, and biosensors for detection
of pathogens [1,2].
Shewanella Oneidensis (S. oneidensis) is one of the microbial organisms suitable for BESs, due
to its respiratory versatility. A facultative anaerobic bacteria originally isolated from lake Oneida,
S. oneidensis is capable of coupling the oxidation of organic substrates to the reduction of
extracellular electron acceptors. S. oneidensis is capable of acting as an electron donor to a wide
range of these external electron acceptors, which span from metal oxides to electrodes, the last of
which can be taken advantage of for current generation. [2,3,5-7,9]
Electron Transfer Mechanisms in S. oneidensis
There are two distinct extracellular electron transfer mechanisms that S. oneidensis uses: MET
(mediated electron transfer) and DET (Direct Electron transfer). Of the two mechanisms, MET is
dominant; 75% of extracellular electron transfer are attributed to this method. [8]
MET consists of the transport of electron by self-secreted electron shuttling molecules such as
flavins. These molecules are secreted by the cell and then diffuse to transfer electrons at electron
acceptor surfaces located externally of the cell. [5] Fig 1 below depicts the secretion of a
shuttling molecule, a flavin, and how it approaches and donates electrons to the electron acceptor.
e-
e- Extracellu
lar
electron
acceptor
Electron shuttling molecule
(flavin)
Fig.1. Mediated Electron Transfer Mechanism. The blue circle represents a cell, and the orange
box an extracellular electron acceptor such as an electrode. The blue arrow indicates diffusion.
The black arrows indicate electron transfer.
Page | 1
There are two ways for electron transfer to occur via direct contact with the extracellular electron
acceptor. The first is long, conductive bacterial nanowires, which have been suggested to be able
to transfer electrons to oxides at locations distal from cell surfaces.[10] The second is contact
with cytochromes on the S. oneidensis cell membrane. The electrons are transferred to the outer
membrane via the Mtr pathways, and the cytochromes there can then directly reduce substrates
when the cell comes in close proximity. [4] Fig 2 below diagrams both methods of DET.
Outer Membrane Cytochrome
eExtracell
- ular
electron
acceptor
Conductive Nanowires
Fig 2. Direct Electron Transfer Mechanism. There are 2 methods of direct
transfer:(1)Cytochrome-electron acceptor surface contact (2)Conductive nanowires.
II. Motivation for S. oneidensis Cellular Transport Study
Current studies of BESs have mainly been concerned with current production or the reduction of
electron acceptors by S. oneidensis. In contrast, we would like to take a more microscale,
mechanistic approach to studying BESs and S. oneidensis, with the long-term goal of eventually
producing a model of its cellular transport and the effects that S. oneidensis motility has on
electron transfer.
Most importantly, since S. oneidensis donates electrons to extracellular electron acceptors via the
two aforementioned mechanisms of direct transfer and mediated transfer, it is important to
quantify and characterize the motility of S. oneidensis within a bioreactor, so that we can
optimize how a cell may taxis towards or come into contact with an electron accepting surface.
Understanding motility would provide insight as to how S. oneidensis moves within its
environment in the process of seeking out electron acceptors and reducing them.
Page | 2
III. The Role of Riboflavin in Electron Transfer
Riboflavin is an exogenous electron shuttling molecule secreted by S. oneidensis. It mediates
electron transfer to extracellular electron acceptors. [9,11]
Riboflavin and Motility
Interactions at the electron acceptor-cell surface or shuttle molecule interfaces have long been a
topic of great interest in BES studies. However, the question still remains as to how S. oneidensis
is able to detect, approach, and reduce external electron acceptors in the first place. We believe
that riboflavin, apart from facilitating electron transfer by providing shuttling to reach distant
electron acceptors, may also mediate S. oneidensis motility, particularly toward electron
acceptors.
Riboflavin has been hypothesized to be also a chemoattractant that guides S. oneidensis’s
directional migration towards external electron acceptors in its environment. In this mechanism,
S. oneidensis secretes reduced riboflavin, which shuttles electrons to a distally located electron
acceptor surface, whereupon it is oxidized. The riboflavin, now in its oxidized form and a yellow
color, then serves as an attractant towards which S. oneidensis taxis. [9] Fig 3 below is a
representation of this mechanism.
Fig 3. Riboflavin as a Putative Mediator of S. oneidensis Motility towards Electron
Acceptors. The mechanism works in three steps: (1)Reduced flavins (colorless) secreted by
Shewanella diffuse through the medium. (2) The diffused reduced flavins are oxidized (yellow)
when they come in contact with electron acceptors such as insoluble MnO2 or Fe(OH)3. (3)
The oxidized flavins diffuse and become a tactic attractant that draw cells towards the
electron acceptor
Page | 3
In order to investigate if S. oneidensis drawn up the gradient of oxidized riboflavin near the
electron acceptor, the directional migration and overall speed of S. oneidensis MR-1 motility
without and with riboflavin needs to be characterized and quantified.
Riboflavin and Current Generation
Oxygenated cultures have been shown to produce higher current, which is attributed to higher
production of flavins such as riboflavin. [1] Riboflavin production has been found to be maximal
under micro-aerobic growth conditions with 5% dissolved oxygen. In contrast, when alternative
electron acceptors such as fumarate are present in anaerobic MR-1 cultures, no flavins are
synthesized. General consensus in prior literature holds that that aerobic conditions promote
higher flavin production, which is contradictory to the idea that flavin plays a MET role as an
electron shuttler under anaerobic conditions, in order to transfer electrons to electron acceptors.
[12]
IV. Prior Chemotaxis and Motility Experiments in S. oneidensis
S. oneidensis has been shown to chemotaxis towards a wide variety of electron acceptors with a
range of traditional motility assays. Some notable chemoattractants are nitrite, nitrate, TMAO
(Trimethylanime N-oxide), fumarate, DMSO (dimethyl sulfoxide), metals such as MnO2 and
Fe(OH)3, charged electrodes, and fumarate [3,6,7,9,14]
Traditional assays used to gauge motility behavior in S. oneidensis are agar plate assays such as
the plug-in-pond assay and the swarm plate assay, both of which gauge chemotaxis behavior by
observing cell motility in a plate culture over time.
Plug-in-pond assays consist of hard agar plugs containing a putative chemoattractant that are
placed in soft agar plates. Cells that display chemotactic behavior would accumulate around the
plug. An example of chemotaxis in a plug assay is depicted below, in fig 4. [7]
In swarm plate assays, bacteria are stabbed into soft agar with the chemoattractant. A gradient is
generated by metabolism of the chemoattractant by the bacteria; after consuming the
chemoattractant around them, cells swarm away from the location where they were stabbed into
the gel in search of more chemoattractant creating rings where the chemoattractant is depleted
and the bacteria are taxiing. [7]
Fig. 4. Taxis of S. oneidensis responding to Mn(II) in Plug Assays. Mn(II) was added to hard
agar at 20mM. (a) Absence of Mn(II); (b)Presence of Mn(II) [7]
Page | 4
However, these chemotaxis assays are not very quantitative, and consist of only visualizations of
the clustering of cells on the plates, as seen in fig 4. The previously mentioned swarm and plug
plate studies qualify the directional motility of the cells by observing their location on the culture
plate relative to where the chemoattractant is located, as seen in the larger ring in Fig 4(a) and
the more visible ring in (b), after set amounts of time. Additionally, such assays focus on the
overall shape of the culture and the entire colony of cells as a whole, rather than on an individual
cell level.
It is essential for S. oneidensis motility to be quantified in parameters such as directional velocity
and speed, rather than just observing cell location on a plate. Therefore, this study will use a
chemoattractant gradient generating microfluidic device, which can visualize cells in a
microchannel under a microscope, to quantify cell motility. As a system that provides quick
evaluation and high throughput, the microfluidic device is ideal for such experiments.
V. Our approach – Microfluidic platform for S. Oneidensis
motility and chemotaxis
Two sets of experiments were conducted on S. oneidensis in 3-channel microfluidic devices that
establish gradients via diffusion. Fig 5 below depicts the device. [13] Please refer to section VI
for more detail on the device and riboflavin gradient generation methods for aerobic and
anaerobic conditions, and VII for the data analysis.
Fig.5. Three-channel gradient generating microfluidic device. A chemical gradient diffused from
the source channel over the cell channel, and finally to the sink channel. [13]
To characterize and quantify the influence of riboflavin on motility, S. oneidensis MR-1 were
seeded into a three-channel microfluidic device and placed under supplemented gradients of
riboflavin of different concentrations and anaerobic or aerobic environments. The cells were
observed with time-lapse microscopy.
Page | 5
S. oneidensis MR-1 Experimental Setup
S. oneidensis motility under the influence of 4 concentrations of riboflavin: 0 nM/mm, 55.6
nM/mm, 111 nM/mm, and 222 nM/mm were evaluated under both aerobic and anaerobic
conditions. These particular concentrations of riboflavin were used because it is similar to
riboflavin’s nanoscale secretion of riboflavin. [12] Additionally, Li et al., 2012 also used similar
concentrations in plate experiments on the increased cellular migration rate and reduction of
MnO2 in the presence of riboflavin, and found that riboflavin was detected in tactic bands at
average concentrations of about 100nM. [9] S. oneidensis MR-1, the wild type, was used in all
experiments, and visualized with brightfield microscopy.
Both aerobic and anaerobic conditions were chosen due to concerns of S. oneidensis being
unmotivated to migrate towards electron acceptors or riboflavin in aerobic environments.
Oxygen is the terminal electron acceptor that gives the highest energy yield for S. oneidensis. [14]
Therefore, it may be likely that the presence of oxygen as an electron acceptor would be
sufficient and would override any need for riboflavin to indirectly transfer electrons, by
extension decreasing S. oneidensis response towards riboflavin.
The 8 conditions were assessed with triplicate trials for each, for a total of 24 experiments. Table
1 below shows the different type of trials that were used.
Table 1. Triplicate Trials of S. oneidensis MR-1 motility under the influence of riboflavin and
oxygen. The riboflavin concentrations used were 0nM/mm, 55.6 nM/mm, 111 nM/mm, 222
nM/mm.
Condition No.
Riboflavin
Oxygen Condition
Number of Trials
Concentration
(Aerobic or
conducted for each
[nM/mm]
Anaerobic)
experiment
1
0
Aerobic
3
2
55.6
Aerobic
3
3
111
Aerobic
3
4
222
Aerobic
3
5
0
Anaerobic
3
6
55.6
Anaerobic
3
7
111
Anaerobic
3
8
222
Anaerobic
3
Syto-9 stained S. oneidensis MR-1 Experimental Setup
As seen in later sections, 55.6nM/mm exhibited consistent chemotaxis and increased speed in
two out of the three triplicate trials for aerobic conditions. It also was consistent in the anaerobic
trials as well. Therefore, we decided to study 55.6nM/mm further in more aerobic and anaerobic
experiments. Again, triplicate trials were performed for each condition. However, instead of
using brightfield microscopy, fluorescence microscopy was used. The MR-1 cells were stained
with a green Syto-9 nucleic acid stain. Table 2 below shows the experiments done on Syto-9
stained S. oneidensis MR-1.
Page | 6
Table 2. Triplicate Trials of Syto-9 stained S. oneidensis MR-1 motility under the influence of
riboflavin and oxygen. The riboflavin concentrations used were 0nM/mm, 55.6 nM/mm, 111
nM/mm, 222 nM/mm.
Condition No.
Riboflavin
Oxygen Condition
Number of Trials
Concentration
(Aerobic or
conducted for each
[nM/mm]
Anaerobic)
experiment
1
0
Aerobic
3
2
55.6
Aerobic
3
3
0
Anaerobic
3
4
55.6
Anaerobic
3
VI. Materials and Methods
Riboflavin Gradient Calibration
The riboflavin gradient calibration was conducted by diffusing a very high concentration of
riboflavin, 40uM. Riboflavin is innately fluorescent with an absorbance maximum at 450 nm and
a fluorescence emission maximum at 525 nm, which fortunately allows for imaging with a green
fluroescent Chroma technology 41001 filter, which has an excitation of 455-500 and emission of
510-560. Fig 6 below depicts the calibration curve.
Riboflavin Gradient
Relative fluorescence
1.0
0 min
2 min
4 min
6 min
8 min
10 min
12 min
14 min
16 min
18 min
20 min
0.8
0.6
0.4
0.2
0.0
0
400 650
1050 1300
1700
Distance (mm)
Fig. 6. Riboflavin Gradient Calibration Curve. The time over which riboflavin diffusion was
oberved was 30 minutes. The imaging was done with a green fluorescent filter, and the timelapse video analyzed in ImageJ.
Page | 7
As seen from Fig 6., the riboflavin gradient are established in the center cell channel within 10
minutes. Correspondingly, riboflavin and media are pumped into the side microfluidic channels
for that amount of time in order to develop the gradient first, before seeding the S. oneidensis
cells.
Cell Culture
A S. oneidensis MR-1 colony was placed in a plastic culture tube of 2.5 mL LB medium, and
then incubated at 30°C overnight in a hot water bath shaker for around 15 hours. The following
day, the cells were diluted down to OD600 of 0.2 and incubated at 30°C in the hot water bath
shaker for around two and a half hours until the culture reached a density around OD600 0.9.
After washing twice by centrifuging at 1200 rpm for 3 minutes and discarding the supernatant,
the cells were resuspended in fresh M4 media at an OD600 of 0.8 in a 1mL eppendorf tube. The
resuspended cells were then gently pipetted with a micropipet tip into the microfluidic device’s
center channel for visualization.
Microfluidic Device
The agarose gradient generating microfluidic device used for evaluating S. oneidensis motility
was assembled as according to a prior protocol from Dr. Mingming Wu’s lab. Fig 7 below
depicts the top view of the microfluidic device.
Fig 7. 3-Channel Microfluidic Device. The device is placed on a microscope stage.The tubes
coming out of the inlet and outlet holes carry the media and riboflavin on the left and right
channels respectively.
A plastic spacer was placed on a silicon wafer patterned with the 4 sets of devices with a three
microchannel configuration. 0.3g of agarose powder (Fisher Scientific BP1356-100) was mixed
Page | 8
with 12mL PBS (Phosphate Buffered Saline 1X Solution, Fisher Scientific BP2438-4) and
dissolved in a microwave oven for 45 seconds into a 3% agarose solution. The liquid was poured
quickly while it was still hot onto the wafer fenced in by the spacer, and prodded at with a
micropipet tip until there were no bubbles. A glass slide was pressed on top of the wafer and
spacer to mold the agarose gel into the 4 sets of microchannels.
After a minute and the gel solidified, the glass slide was removed and gel and spacer were lifted
off the wafer. The gel was punched with holes for inlets and outlets with a plastic hole punch of
0.012 inch diameter. (Small Parts, Inc. Hypo Tube S/S 304-RW) The gel was then soaked in M4
medium for a hour. The M4 medium recipe was kindly shared by Dr. Angenant’s lab. The gel
and spacer were then placed on a second glass slide, with the channel sides facing the glass. The
gel was squirted with a little PBS to keep it damp, and the glass was placed on a metal bottom
holder. A plexiglass top plate was then placed on top of the gel and screwed into place, with the
inlet and outlet holes matching up to those punched in the gel.
The inlets and outlets were then overfilled by pipetting 70mL of PBS inside, in order to check for
leakages in the channels or failure to match the inlet and outlet holes on the plexiglass to those
punched in the gel. After testing, the inlet and outlet holes can be plugged with PDMS plugs. The
entire device can be stored covered and alongside two bottle caps of water for humidity inside a
refrigerator.
Pumping
The inlet and outlet holes of the microfluidic device were connected to Cole Parmer Pharmed
BPT tubing of 0.51mm diameter. Microtips cut from the tips of micropipet tips (Cole Parmer Gel
Loading Tips) were attached to the tubing to fit it in the inlet and outlet holes. A peristaltic pump
(Watson Marlow 205U) pumped the riboflavin and media sources into the microfluidics channel
at a flow rate of 0.7RPM.
Riboflavin
55.6nM/mm, 111 nM/mm, and 222 nM/mm were chosen as the riboflavin concentrations. The
solutions of each concentration were made from a stock solution of 40uM riboflavin, with 5uL of
the stock per mL of M4 media. The riboflavin was kindly supplied by the Angenant lab.
Anaerobic Condition Generation
To generate the anaerobic condition in the microfluidic device, the media and riboflavin solution
were degassed of oxygen by bubbling with nitrogen gas in small glass bioreactors (provided by
the Shuler lab) that were connected with to a gas tank with air-tight tubing (Pharmed BPT tubing
of 0.081 inch diameter). After half an hour of degassing, the media and riboflavin were pumped
into the device with a peristaltic pump.
Apart from degassing in bioreactors, a transparent plastic cover connected to the same nitrogen
stream as the bioreactors was used. The plastic cover was placed completely over the
microfluidic device on the microscope platform, filling the space in between with nitrogen gas in
Page | 9
order to better maintain the anaerobic environment after the degassed media and riboflavin were
pumped in. This also prevented oxygen from the ambient environment from infiltrating in.
Fig 8 on the right depicts the setup of the
connections to the microfluidic device. The
bioreactors are on the right side, connected to
the nitrogen gas line along with the cover,
which is over the microfludic device on the
microscope stage on the left.
Fig 8. Entire Experimental Setup. The
microfluidic device is connected to tubing that
pumps the degassed media and riboflavin from
the bioreactors into the side channels.
Microscopy
To visualize the S.oneidensis MR-1 cells, brightfield microscopy was used, at 20x magnification.
The light microscope and mounted camera used was the Olympus IX51. For the Syto-9 stained
MR-1 cells, the green fluorescent Chroma Technology 41001 filter was used, which has an
excitation of 455-400 nm and an emission of 510-560 nm.
VII. Analysis Method
The time-lapse videos taken of the center cell channel was taken at a rate of 30 frames per
second for 500 frames, with the software program IPLab. The resulting video files were
processed in ImageJ.
100 cells for each trial were manually tracked with ImageJ’s manual tracking plugin, for both
MR-1 and Syto-9 stained cells. In order to prevent tracking one cell twice, the screen was
divided into quadrants and then into smaller blocks, within which cells originate from and then
are tracked. The resulting text file of x and y coordinates that ImageJ puts out are then processed
with a Matlab program provided by Beum Jun Kim, called SpeedGate, which automatically
calculates the average speed, the x and y velocities, and the x and y persistence lengths.
VIII. S. Oneidensis MR-1 Cell Trajectory Paths under the
Influence of Riboflavin
As aforementioned, the riboflavin concentrations that were used were 0 nM/mm, 55.6 nM/mm,
111nM/mm, and 222nM/mm. For aerobic conditions, the microfluidic device was place in an
aerobic environment, exposed to ambient air. For anaerobic conditions, the microfluidic device
was exposed to nitrogen gas underneath the plastic cover and with the inputted riboflavin and
Page | 10
media in the source and sink channel respectively degassed in a bioreactor before being pumped
into the device. The following data is from both aerobic and anaerobic trials.
Trajectory Graphs for S. oneidensis MR-1 motility in Aerobic Conditions
Trajectory maps of an individual S. oneidensis cell’s swimming path within the microfluidic
channel are ideal for depicting directional motility. All cells have been normalized to begin their
path at the origin, to allow for better visualization of direction along the axes. The SpeedGate
trajectory graphs of 100 cell tracks for aerobic conditions are depicted below in Fig 9.
Page | 11
Fig. 9. S. oneidensis MR-1 Trajectories under Aerobic Conditions. Graphs were made in Matlab
with SpeedGate program. Axes are micrometers. The direction of the riboflavin gradient are
indicated by the yellow arrow. (a) S. oneidensis motility in Control conditions (0 nM/mm
Riboflavin) b. 55.6nM/mm Riboflavin; (c) 111nM/mm; (d) 222 nM/mm.
As seen from Fig 9 above, the directionality of S. oneidensis migration under the influence of
riboflavin is difficult to distinguish in most of the cases, as the trajectories are proceeding in all
directions.
Trajectory Graphs for S. oneidensis MR-1 motility in Anaerobic Conditions
Fig 10 below displays the trajectory data for S. oneidensis under the influence of the four
different concentrations of riboflavin under anaerobic conditions.
Page | 12
Fig. 10. S. oneidensis MR-1 Trajectories under Anaerobic Conditions. Graphs were made in
Matlab with SpeedGate program. Axes are micrometers. The direction of the riboflavin gradient
are indicated by the yellow arrow. (a). S. oneidensis motility in Control conditions (0 nM/mm
Riboflavin) b. 55.6nM/mmRiboflavin; (c) 111nM/mm; (d) 222 nM/mm.
IX. Instantaneous Speed Data for S. oneidensis MR-1 motility in
Aerobic and Anaerobic Conditions
The instantaneous speed for S. oneidensis under the influence of the four concentrations of
riboflavin in aerobic conditions were calculated and depicted below in fig 11(a) below, which is
charted with representative data from one of the triplicate trials. The instantaneous speed gives a
reliable indicator of cellular motility. Unpaired t-tests were used to determine the statistical
significance of the speed differences between the speed displayed by S. oneidensis for a certain
concentration of riboflavin vs. that of the control. 2-tailed ANOVA, as shown in Fig 11(b), was
used to distinguish whether riboflavin or oxygen concentration had an effect on motility. All ttests and 2-way ANOVA was conducted in Graphpad Prism.
Page | 13
Fig 11. Graph of Instantaneous Speeds of S. oneidensis MR-1 under the influence of 0, 55.6, 111,
and 222 nM/mm Riboflavin. The units of speed are um/s, and the riboflavin concentrations are
[nM/mm]. The chart and the 2-way ANOVA and t-test were completed using Graphpad Prism.
The black stars indicate that differences in speed between a trial with a certain concentration of
riboflavin and the control are statistically significant. The blue stars indicate that the difference
in speed between the anaerobic control and aerobic control trials are statistically significant.
As seen from Fig 11, we can observe that the speed change with riboflavin addition does occur
for both aerobic and anaerobic conditions. In aerobic conditions, the instantaneous speed is
directly correlated with increasing riboflavin concentration, as could be seen from the
increasingly upward trend. 222nM/mm riboflavin seems to have a chemorepulsive effect, though
that can be attributed to the data for the 222nM/mm riboflavin trials being inconsistent; as
aforementioned, the 55.6nM/mm triplicate trials were the only concentration to consistently
show a chemotactic trends towards riboflavin.
Interestingly, riboflavin’s role in motility is bi-phasic under anaerobic conditions, as can be seen
from the increase in speed at the 55.6 nM/mm concentration, and the subsequent dropping of
speed with the two other higher concentrations. In addition, speed generally increases with
oxygen depletion.
Page | 14
X. Y-velocity Data for S. oneidensis MR-1 motility in Aerobic and
Anaerobic Conditions
The velocity in the direction of the Y-axis was evaluated because the riboflavin gradient was
originating from that direction. Therefore, the Y-velocity data can be a good indicator of
chemotaxis towards riboflavin. Fig 12 below depicts the Y-velocity data in representative
aerobic and anaerobic trials for 100 cells each. Like the instantaneous speed data, t-tests and
ANOVA were conducted in Graphpad Prism.
Fig 12. Graph of Y-velocities of S. oneidensis MR-1 under the influence of 0, 55.6, 111, and 222
nM/mm Riboflavin. The units of speed are um/s, and the riboflavin concentrations are [nM/mm].
The chart and the 2-way ANOVA and t-tests were completed using Graphpad Prism. The black
stars indicate that differences in directional velocity between a trial with a certain concentration
of riboflavin and the control are statistically significant.
As seen from Fig 12 above, all concentrations of riboflavin promote chemotactic behavior in S.
oneidensis MR-1 under aerobic conditions. Additionally, the response increases mildly with the
first two increases in riboflavin, but is highest for the highest concentration of riboflavin, 222
nM/mm.
Page | 15
However, under anaerobic conditions, a similar biphasic response to that of instantaneous speed
in anaerobic conditions is observed. The chemotaxis is clearly highest with 55.6nM/mm
riboflavin, and decreases with 111nM/mm and 222 nM/mm concentrations.
XI. Syto-9 stained S. oneidensis MR-1 Cell Trajectory Paths
under the Influence of 55.6 nM/mm Riboflavin in Aerobic and
Anaerobic Conditions
As 55.6nM/mm riboflavin triplicate trials with MR-1 cells showed consistent motility increase
and chemotaxis behavior, we looked more closely at the effects of 55.6nM/mm riboflavin on
motility, though this time in Syto-9 stained cells.
Fig 13 below shows the trajectory paths of the Syto-stained MR-1 under the influence of
0nM/mm and 55.6 nM/mm riboflavin in both aerobic and anaerobic conditions.
Page | 16
Fig. 13. Syto-9 stained S. oneidensis MR-1 Trajectories under Aerobic Conditions. Graphs were
made in Matlab with SpeedGate program. Axes are micrometers. The direction of the riboflavin
gradient are indicated by the yellow arrow. (a) motility in aerobic control conditions (0 nM/mm
Riboflavin) (b) 55.6nM/mm Riboflavin in aerobic conditions; (c) motility in anaerobic control
conditions (0 nM/mm Riboflavin); (d) 55.6nM/mm Riboflavin in anaerobic conditions
While directionality is non-obvious from these plots, the track length for each cell is noticeably
shorter. This can be partially attributed to how it is more difficult to manually track the Syto-9
stained fluorescent cells that move out of focus, which dissipate into a bright circular halo
instead of only becoming fainter as in brightfield. Alternatively, Syto-9 staining may impact the
motility of the MR-1 cells or only stain less motile cells, leaving the faster cells unmonitored.
XII. Instantaneous Speed Data for Syto-stained S. oneidensis MR1 motility in Aerobic and Anaerobic Conditions
Fig 14 below depicts the instantaneous speed for Syto-stained S. oneidensis MR-1 under the
influence of 0nM/mm and 55.6 nM/mm riboflavin under aerobic and anaerobic conditions.
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Fig. 14. Graph of Instantaneous Speeds of Syto-9 stained S. oneidensis MR-1 under the
influence of 0 and 55.6 nM/mm Riboflavin. The units of speed are um/s, and the riboflavin
concentrations are [nM/mm]. The chart and the 2-way ANOVA and t-test were completed using
Graphpad Prism. The black stars indicate that differences in speed between a trial with a certain
concentration of riboflavin and the control are statistically significant.
As seen from the prior figure, speed decreases with the addition of 55.6 nM/mm riboflavin in
aerobic conditions but slightly decreases in anaerobic conditions. The former is contrary to the
prior unstained MR-1 experiments, and may be attributed to the staining having a detrimental
effect on cellular motility, at least under aerobic conditions. However, as seen in the previous
experiments, speed increases with depleted oxygen, whether or not 55.6nM/mm riboflavin was
present.
XIII. Y-velocity Data for Syto-stained S. oneidensis MR-1
motility in Aerobic and Anaerobic Conditions
Fig 15 below depicts the Y-velocity data in representative aerobic and anaerobic trials for 100
Syto-stained cells each. Like the instantaneous speed data, t-tests and ANOVA were conducted
in Graphpad Prism.
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Fig 15. Graph of Y-velocities of Syto-9 stained S. oneidensis MR-1 under the influence of 0 and
55.6 nM/mm Riboflavin. The units of speed are um/s, and the riboflavin concentrations are
[nM/mm]. The chart and the 2-way ANOVA and t-tests were completed using Graphpad Prism.
The black stars indicate that differences in y-velocity between a trial with a certain
concentration of riboflavin and the control are statistically significant.
As seen from the figure, Syto-stained MR-1 shows directional migration towards a 55.6 nM/mm
riboflavin gradient in both aerobic and anaerobic conditions. Additionally, the ANOVA analysis
indicates that riboflavin does have an effect on cellular velocity in the y direction.
XIV. Conclusion
This study has found that speed increases with riboflavin supplementation, particularly for 55.6
nM/mm. This increase applies to both anaerobic and aerobic conditions. Additionally, speed
increases with oxygen depletion, seeing as trials with anaerobic conditions overall had greater
speeds than those of aerobic conditions.
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Additionally, chemotaxis was observed in both anaerobic and aerobic environments with
riboflavin concentration of 55.6 nM/mm. Directional migration was inconsistent for other
concentrations, which are inconclusive.
Several steps to take in the future include quantifying the oxygen content within the microfluidic
device, to ensure that the anaerobic condition is consistently maintained throughout the duration
of experimentation. Moreover, the effects that Syto-9 staining have on S. oneidensis cells must
be characterized. As aforementioned, it is possible that it negatively impacts motility, which
accounts for the unusually slow speeds witnessed in the stained cells, but far faster motility in the
unstained cells. Another possibility is optimizing the staining protocol, so that more cells are
stained and the stain would not selectively stain only immobile cells, which can also explain the
slow speeds.
Regarding future riboflavin experiments, the ∆bfe mutant strain of S. oneidensis is needed. The
strain knocks out flavin export, [8] therefore reducing the impact that self-secreted riboflavin has
during the chemotaxis studies with supplemented riboflavin. In the current experiments, it is
possible that the riboflavin supplemented to the cells, which already secrete riboflavin of their
own, leads to an excessive concentration in the environment; this may lead an underestimation of
motility changes if cells are not incentivized to move in a saturated environment.
Lastly, a correlation between motility and current generation must be drawn. Teravest, 2013
indicates that current generation is greatest under aerobic conditions, which can be attributed to
higher flavin production. [1] Yet, we observe that motility and chemotaxis towards riboflavin are
highest under anaerobic conditions, when current production is not as optimal. There are several
ways to interpret this; firstly, it is possible that increased speed and directional motility may be
unrelated to current generation. Alternatively, an explanation for the discrepancy would be that
in oxygenated environments, since more flavin is secreted, S. oneidensis may not have to be as
mobile as in anaerobic environments. Direct contact methods of electron transfer may not be as
necessary when there are so many electron shuttles in the secreted flavin to conduct the electron
transport. Moreover, the large surrounding amounts of oxygen in an aerobic environment may
also act as an electron acceptor, reducing the need for S. oneidensis to move to a more favorable
area with more electron acceptors. Overall, anaerobic conditions, which lack oxygen as an
electron acceptor, may induce more motility in S. oneidensis because they are required to seek
out alternative electron acceptors.
XV. Acknowledgements
I would like to thank Dr. Mingming Wu for her constant support and BJ Kim for the Matlab
SpeedGate program as well as lots of teaching and help over the duration of this project. I would
also like to thank Injun Chu for his work with generating an oxygen gradient calibration curve,
and Alyssa Fiore for her S. oneidensis growth curve. I also greatly appreciate the help from
Michaela Teravest, Eric Holmes and the Angenant Lab, which provided the recipe and
ingredients for M4 media as well as the GFP strain of S. oneidensis. I also thank Dr. Winans for
the Cyto stain.
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XVI. References
1. Teravest , 2013. Biological Limitations of Shewanella Oneidensis MR-1 in bioelectrical
systems. Cornell University.
2. Pant et al., 2012. Bioelectrochemical systems (BES) for sustainable energy production
and product recovery from organic wastes and industrial wastewaters. RSC Adv 2:12481263.
3. Harris et al., 2012. Shewanella oneidensis MR-1 chemotaxis proteins and electrontransport chain components essential for congregation near insoluble electron acceptors.
Biochem Soc Trans 40:1167-1177.
4. Coursolle et al., 2010. The Mtr respiratory pathway is essential for reducing flavins and
electrodes in Shewanella Oneidensis. Journal of Bacteriology 192(2):467-474.
5. Brutinel and Gralnick, 2011. Shuttling happens: soluble flavin mediators of extracellular
electron transfer in Shewanella. Appl Microbiol Biotechnol 93:41-48.
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acceptors in Shewanella oneidensis. Molecular microbiology 73(2):278-290.
7. Bencharit and Ward, 2005. Chemotactic responses to metals and anaerobic electron
acceptors in Shewanella oneidensis MR-1. J Bacteriol 187(4):5049-5053.
8. Kotsloski and Gralnick, 2013. Flavin electron shuttles dominate extracellular electron
transfer by Shewanella oneidensis. Mbio 4(1):1-4.
9. Li et al., 2012. Soluble electron shuttles can mediate energy taxis toward insoluble
electron acceptors. Environ Sci Technol 46:2813-2820.
10. Gorby et al., 2006. Electrically conductive bacterial nanowires produced by Shewanella
oneidensis strain MR-1 and other microorganisms. PNAS 103(30): 11358-11363.
11. Marsili et al., 2008. Shewanella secretes flavins that mediate extracellular electron
transfer. PNAS 105(10):3968-3973.
12. Roy et al., 2012. A study of the flavin response by Shewanella cultures in carbon-limited
environments. RSC Advances, 2:10020-10027.
13. Cheng et al., 2006. A hydrogel-based microfluidic device for the studies of directed cell
migration. Lab Chip 7:763-769.
14. Von Canstein et al., 2008. Secretion of flavins by Shewanella species and their role in
extracellular electron transfer. App Environ Microbiol 74(3):615-623.
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