Kagramian 1

Engineering an Electrochemical Cell For Shewanella oneidensis Using Previously

Calculated Results For Best Soil Condition
Michelle Kagramian michelle.kagramian@ocvts.org
Marine Academy of Technology and Environmental Science, NJ

Abstract:
People throughout the world are in need of light, warmth, and cooked meals. Using a renewable
source of energy fueled by soil located virtually everywhere can be much more advantageous as
opposed to pollution-inducing fossil fuels. In this experiment, Shewanella oneidensis bacteria
was used as the electrolyte in an electrochemical, microbial fuel cell made to act as a sustainable
source of energy. In this battery, copper acted as the anode while zinc acted as the cathode. The
anode of a battery was the negative electrode that released electrons after the electrolyte forced
the zinc to oxide. The casement of this cell was 3-D printed; the body chamber, which fit into
another larger chamber, used aluminum to be read by a voltmeter, model ELENCO M-1750.
Within the cell, there were six layers of zinc, soil, and copper, respectively. This demonstration
focused on the number of volts of electricity S. oneidensis would be able to produce when it was
put through six different volumes of water in three different types of soil, all in 35°C at 6.5 pH.
Three mL of water in potting soil produced the highest voltage output: 13.2v. Statistically, there
was a significant difference amongst the water treatments (ANOVA, P<0.05). However, the trend
indicated that soil with three mL water composition performed better than all other
combinations. Treatments with too great or too little of a water concentration were less effective.
This experiment allows for countries with electricity, or lack thereof, to utilize their ubiquitous
soil to create batteries using less expensive materials such as copper and zinc.

Introduction:
As humans, using batteries in more ways than one is part of our daily routines. We have
created multiple ways to form sources of renewable energy to power our electronics or machines:
harnessing the energy from the light of the sun or using wind power to create a turbine. Although
they are clean sources of energy, their downside is that they both rely on the weather to operate at
all times. Creating a battery that would depend on soil would be much more efficient.
Shewanella oneidensis, a soil residing bacteria, generates electrical currents after
reducing heavy metals (Meysman, 2017). The most common pathway is the Mtr pathway
of S. oneidensis MR-1 where electrons from the membrane move into the surface through a
redox reaction; the electrons are then collected by the acceptors (Hu, 2021). They survive in
different temperatures because of their psychrotolerant ability (Yin, 2011). Another factor that
affects the efficiency of this bacteria is the pH of the soil. The average pH level in soil is about
 Kagramian 2

6.5. Since S. oneidensis is able to function in a variety of pH levels and thermal conditions, then
the idea of using them throughout the world would become more probable.
Batteries store chemical energy and convert it into electrical energy (Australian Academy,
2018). In my previous research, I used zinc to capture the released electrons created by the
bacteria. The electrons were collected by the copper wire connected to each piece of zinc. In this
battery, the copper acted as the anode and the zinc acted as the cathode. The anode of a battery is
the negative electrode that releases electrons after the electrolyte forces the zinc to oxide. Those
electrons were transported through an external circuit made of aluminum to be captured by the
cathode made of copper (Walter, 2018). This total reaction is called the reduction-oxidation
reaction, more commonly known as the redox reaction (Australian Academy, 2018). The
casement of this cell was 3-D printed; within the cell, there were 6 layers of zinc, soil, and
copper, respectively. With each added layer of the anode, electrolyte, and cathode, the total volts
produced were measured using a Voltmeter, model ELENCO M-1750.
These batteries have the potential to be available for anyone, anywhere because of the
ubiquity of soil. Additionally, batteries that run on soil are more beneficial to the environment
because normal toxic, acid batteries eventually leak out into our water and into our earth. Having
a microbial fuel cell within a small battery casement will be much safer, especially since you
have the ability to reuse them with refillable properties. This project focused on engineering an
electrochemical cell; this is otherwise known as a battery.

Methodology:
Study site:
All containers with soil were kept in an incubation oven. They were kept within the
temperature range of 31°C-35°C. The soil was given 1-2 days in order for the bacteria to fully
grow. The containers had a pH level of approximately 6.5.
3-D printing the casement:
A. Created a 2-D drawing of the case and layers within the case (Figure 1).
B. Created a 3-D design of the case using online software, Tinkercad (Figure 2).
C. Using the Makerbot 3-D printer, printed out the body and lid of the case (Figure 3).
The inner case is 7.33” x 1.5” x 1.5” (18.62cm x 3.81cm x 3.81cm). The outer case
is 6.77” x 1.7” x 1.7” (17.2cm x 4.32cm x 4.32cm).
 Kagramian 3

Figure 2: A visualization of the anode (-) and cathode (+)

casements.

Figure 1: A drawing of the microbial

fuel cell created with labeling of the
components.

Figure 3: 3-D printed casement for the microbial fuel cell.

Preparing the soil samples:

A. There were 3 trials with 7 groups representing the 7 different amounts of water per
soil sample. The 3 trials consisted of potting soil, my backyard soil, and soil from
Cedar Bonnet (from the Edwin B. Forsythe Wildlife Refuge at Cedar Bonnet Island,
Stafford, NJ. Cedar Bonnet includes salt marsh and uplands habitats).
a. Using 16g of soil (measured by a VWE precision balance), the 1:2 ratio of
water to soil was used for the first test: 8mL of water (Figure 8).
b. After testing the 8mL, the 1:2 ratio was no longer applied. The control
trial was tested with each soil type and no water added. The next water
levels to be tested were: 4mL, 3mL, 2mL, 1mL. The water increased while
the amount of soil stayed the same.
B. To control the levels of pH in each sample, grounded limestone rocks were added to
initial soil to increase pH levels and aluminum sulfate was added to initial soil to
decrease pH levels.
C. The final pH of 6.5 was tested using a pH meter, Bluelab Soil pH Pen, once all of the
components in the soil were combined and set. While testing the pH levels, the
 Kagramian 4

temperature remained at ≅35°C using the incubator, as it is the best for bacterial
growth (Figure 9).
a. According to previously conducted research, the nanowires that create
electrons are made of lipids; the change in lipids that they experience
during cold temperatures (such as 16°C) negatively affects the creation of
electrical currents (Abboud, 2005). Therefore, in this experiment, an
average of 35°C was used. In order to adapt to the acidic environment, the
bacteria have to use proteins and lipids to form a tolerance (Yin, 201).
This weakens their ability to form electrons because under stressful
conditions they are more focused on staying alive rather than producing
electricity. Therefore, in this experiment, an average of 6.5 pH was used.

Figure 9: Storing the soil samples in the

incubation oven for 24 hours in order to
let the bacteria fully develop and adjust to
Figure 8: The use of an analytical scale (balance the water and temperature.
+/- 0.0001 g) to measure 16g of soil.
 Kagramian 5

Recording data:
I. To keep track of each container, all soil samples were recorded for their pH level,
thermal level, and water level, a chart was made.
II. The 3-D printed casement consisted of two parts: the body and the lid. At the bottom
of the body there was a hole in the shape of a negative sign (“-”) to represent the
anode. The lid had a plus (“+”) to represent the cathode. These holes allowed for the
aluminum to have contact with the anode and cathode to be able to reach the
voltmeter.
Constructing the fuel cell:
I. An aluminum wire was placed through the holes on the lid and the body of the casement.
Starting from the bottom layer in the negative casement, aluminum was tied to 2 zinc
washers (12 washers in total per trial and specific water level).
A. Next, the soil with its specific amount of water was added. To cover the initial layer
of zinc and aluminum 48g of soil was used. After this first layer, each soil layer to
follow was 16g.
B. Lastly, the copper sheets were cut into approximately 1x1 inch and layered on top of
one another on the soil layer (Figure 10). This layer of zinc, soil, and copper was
repeated 6 times for each test (6 layers within the case).
C. Each test was tested (and recorded) on how many volts of electricity it produced.

Figure 10: Cutting the copper sheet to fit into

the fuel cell after the zinc cathode and soil
electrolyte were added.
 Kagramian 6

Statistical analysis:
An ANOVA was conducted to determine any differences in electrical output amongst
water and soil concentrations. An alpha of 0.05 or less was used to determine significance for all
tests. Three T-tests were conducted to determine specific differences in electrical output amongst
the different soil types.

Results:
The results of this experiment showed evidence that the success of S. oneidensis does
have a correlation to the level of water and the type of soil it contains. The volts produced ranged
from <0.1 volts to 13.2 volts. An ANOVA test was run with a p-value of P=0.0469, meaning
there was a significant difference among the data. The ANOVA test showed that Trial A, potting
soil, produced the most amount of volts averaging a total of 8.43v; Trial B averaged 6.25v and
Trial C averaged 2.12v, meaning soil from Cedar Bonnet was the least conductive. Trial A also
had the largest total sum of volts (50.6v) when compared to the other trials. Trial B totaled 37.5v
and Trial C totaled 12.7v. Furthermore, within Trial A, a water level of 3mL generated the most
amount of voltage: 13.2v. Across every trial, 3mL of water was also the one to generate the most
amount of energy (Figure 4). Too little moisture (such as 1mL) had the worst results (Figure 4):
when no water was added (0mL), no more than 0.2v was produced (Figure 4). A T-test
comparing each trial to another showed that every trial had a significant difference (Figures 5-7).
This meant every sample set containing a different type of soil produced significantly different
energy amounts.
 Kagramian 7

Figure 4: Each of the three soil types (n=3) are tested with
various water levels, they were compared against each other to
determine if difference in soil composition affects S. oneidensis
appearance. Across every trial, 3mL of water was also the one to
generate the most amount of energy. An ANOVA was conducted
(uS/cm) to determine significance (⍺ = 0.05; p=0.0469; +/- 5%
error bars).

Figure 5: Comparison of the average conductivity (uS/cm) of

potting soil and backyard soil when different water amounts are
applied. A t-test was conducted to determine significance (⍺ =
0.05; p=0.0065; +/- 5% error bars).
 Kagramian 8

Figure 6: Comparison of the average conductivity (uS/cm) of

backyard soil and Cedar Bonnet soil when different water
amounts are applied. A t-test was conducted to determine
significance (⍺ = 0.05; p=0.0151; +/- 5% error bars).

Figure 7: Comparison of the average conductivity (uS/cm) of potting

soil and Cedar Bonnet soil when different water amounts are applied. A
t-test was conducted to determine significance (⍺ = 0.05; p=0.0113; +/-
5% error bars).
 Kagramian 9

Discussion:
A balanced ratio of nutritious water to the soil, such as 3mL to 16g, creates the optimal
moisture content for Shewanella oneidensis. S. oneidensis is anaerobic, meaning that they do not
require oxygen for their survival. These bacteria transfer their electrons to metal outside of their
cells as a way of respiration (Gescher, 2008). They breathe by reducing heavy metals such as
iron (Fe), lead (Pb), uranium (U), and magnesium (Mg) (Ghorbanzadeh, 2018). They gain energy
for their growth through Fe(III) or Mn(IV) reduction (Hu, 2021). Due to this fact, S. oneidensis is
often found in marine and freshwater sediments. S. oneidensis survives well in areas with a lot of
water because they have adapted to an oxygen-free environment and because they can swim with
the aid of a single polar flagellum (Venkateswaran et al., 1999). Of course, water is a key factor
in electron transportation, thus high water levels within the soil are better for the production of
electricity. The scientists also reported that the Shewanella grow more robust biofilms, with
greater microbe to surface interactions, when nutrient levels are poor, meaning, when they were
in Cedar Bonnet soil and they lacked nutrients, there was a greater biofilm which increases the
bacteria's ability to thrive off of nutrients most other bacteria would not be able to use. A biofilm
is a well-organized 3-dimensional (3D) matrix in which cells are covered in polymeric
substances; the films are made of electroactive components such as c-type cytochromes (c-Cyts)
and nanowires (Hu, 2021). Shewanella nanowires are the extensions of the outer membrane and
are the key players for long-distance electron transfer (Hu, 2021). This is why there was voltage
produced even in soil that is 80% sand. Shewanella biofilms are electrically conductive and are
an important factor in electron transport (“Nature Publishing,” 2014). Sand does not hold water
or nutrients very well (Le Bagousse-Pinguet, 2013). Although S. oneidensis can consume a wider
variety of nutrients when compared to other bacteria, when there are few nutrients available, it
becomes difficult to respire. Once the biofilm adhering the microbe to the metal is formed,
bacteria are required to transfer electrons from their cells to the metal for respiration
(Rosenbaum, 2012). The microbial electron exchange bioelectrochemical systems (BESs), is
called microbial extracellular electron transfer (EET). Exoelectrogens attach to the surfaces of
external electron acceptors (electrodes) (Hu, 2021). Potting soil contains the most amount of iron
nutrients; for this reason, S. oneidensis is most electroactive in the nutrient-rich potting soil. The
data collected is evidence that although S. oneidensis is able to conduct electricity in large and
 Kagramian 10

little amounts of water; some conditions, however, provide better support for the production of
electricity.

Conclusions:
Shewanella oneidensis is an anaerobic, exoelectrogenic bacteria that reduces heavy
metals and produces electrons which are transported by water to reach the designated electrode.
As the soil begins to lack water and nutrients, S. oneidensis’s ability to generate electricity
worsens. This soil-residing bacteria can be used to solve global problems such as climate change
and lack of electricity in some nations by converting organic waste into electricity as a renewable
source of energy. These batteries have the ability to be available for anyone, anywhere because
of the ubiquity of soil and the tolerance Shewanella species has to harsh environments.
Additionally, batteries that run on soil are more beneficial to the environment because normal,
toxic, acid batteries eventually leak out into our water and, consequently, the earth. There is an
endless amount of energy that can be put out through soil based batteries because the materials
used are relatively inexpensive and easy to acquire; economically, this battery is also efficient.
 Kagramian 11

References:

Australian Academy of Science. (2018, March 5). How A Battery works. Australian Academy of
Science. Retrieved from https://www.science.org.au/curious/technology-future/batteries

Chesterfield, L. (2017, July 19). Cathode materials and cathode/anode ratios. Products
Finishing. Retrieved from
https://www.pfonline.com/articles/cathode-materials-and-cathodeanode-ratios.

Do, M. H., Ngo, H. H., Guo, W. S., Liu, Y., Chang, S. W., Nguyen, D. D., Nghiem, L. D., & Ni,
B. J. (2018). Challenges in the application of microbial fuel cells to wastewater treatment
and energy production: A mini review. Science of The Total Environment, 639, 910–920.
https://doi.org/10.1016/j.scitotenv.2018.05.136

Gescher, J.S., Cordova, C.D. and Spormann, A.M. (2008), Dissimilatory iron reduction in
Escherichia coli: identification of CymA of Shewanella oneidensis and NapC of E. coli
as ferric reductases. Molecular Microbiology, 68: 706-719.
https://doi.org/10.1111/j.1365-2958.2008.06183.x

Ghorbanzadeh, N., Kumar, R., Lee, Sh. et al. Impact of Shewanella oneidensis on heavy metals
remobilization under reductive conditions in soil of Guilan Province, Iran. Geosci J 22,
423–432 (2018). https://doi.org/10.1007/s12303-017-0067-8

Hirose, A., Kasai, T., Aoki, M., Umemura, T., Watanabe, K., & Kouzuma, A. (2018, March 14).
Electrochemically active bacteria sense electrode potentials for regulating catabolic
pathways. Retrieved September 06, 2020, from
https://www.nature.com/articles/s41467-018-03416-4

Hu, Y., Wang, Y., Han, X., Shan, Y., Li, F., & Shi, L. (2021). Biofilm Biology and engineering of
Geobacter and Shewanella spp. for energy applications. Frontiers in Bioengineering and
Biotechnology, 9. https://doi.org/10.3389/fbioe.2021.786416

Kluepfel, O., Kluepfel, M., Lippert, B., & Williamson, J. (2012, October 20). Changing the pH
of Your Soil. Retrieved September 06, 2020, from
https://hgic.clemson.edu/factsheet/changing-the-ph-of-your-soil/

Le Bagousse-Pinguet, Y., Forey, E., Touzard, B. and Michalet, R. (2013), Disentangling the
effects of water and nutrients for studying the outcome of plant interactions in sand dune
ecosystems. J Veg Sci, 24: 375-383. https://doi.org/10.1111/j.1654-1103.2012.01462.x

Meysman FJR. Cable Bacteria Take a New Breath Using Long-Distance Electricity. Trends
Microbiol. 2018 May;26(5):411-422. doi: 10.1016/j.tim.2017.10.011. Epub 2017 Nov 21.
PMID: 29174100.

Nature Publishing Group. (2014). A biofilm is composed of attached microbial cells encased.
Nature news. Retrieved March 10, 2022, from
https://www.nature.com/scitable/content/a-biofilm-is-composed-of-attached-microbial-10
0450918/
 Kagramian 12

Pirbadian, S., Barchinger, S., Leung, K., Byun, H., Jangir, Y., Bouhenni, R., . . . El-Naggar, M.
(2014, September 02). Shewanella oneidensis MR-1 nanowires are outer membrane and
periplasmic extensions of the extracellular electron transport components. Retrieved
September 06, 2020, from https://www.pnas.org/content/111/35/12883

Rosenbaum, M. A., Bar, H. Y., Beg, Q. K., Segrè, D., Booth, J., Cotta, M. A., & Angenent, L. T.
(2012). Transcriptional analysis of Shewanella oneidensis MR-1 with an electrode
compared to fe(iii)citrate or oxygen as terminal electron acceptor. PLoS ONE, 7(2).
https://doi.org/10.1371/journal.pone.0030827

Venkateswaran K, Moser D, Dollhopf M, Lies D, Saffarini D, et al. 1999. “Polyphasic taxonomy

of the genus Shewanella and description of Shewanella oneidensis sp.” nov. Int. J. Syst.
Bacteriol. 49:705–24

Walter, M., Kravchyk, K. V., Böfer, C., Widmer, R., & Kovalenko, M. V. (2018, March 7).
Polypyrenes as high‐performance cathode materials for aluminum batteries. Wiley
Online Library. Retrieved from
https://onlinelibrary.wiley.com/doi/full/10.1002/adma.201705644

Wu, Y., Zeng, J., Zhu, Q., Zhang, Z., & Lin, X. (2017, January 4). PH is the primary determinant
of the bacterial community structure in agricultural soils impacted by polycyclic
aromatic hydrocarbon pollution. Retrieved September 06, 2020, from
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5209717/

Yin, J., & Gao, H. (2011). Stress responses of shewanella. Retrieved September 06, 2020, from
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3168786/