For making Electricia Coli possible, we designed 2 microbial fuel cells (MCF) (mini and large) using the online CAD (computer aided design) software Onshape.

Microbial fuel cell (MCF) is a generic term used to describe a chamber-based bacterial incubator, in which electricity can be generated, collected and detected. In order to meet requirements, such devices usually contain an electrode, fluid inlets and outlets and most importantly a lid for maintaining anaerobic environment. In addition, a protein-exchange membrane (PEM) is introduced at the intersection of anode (bacterial) and cathode chambers. For the PEM we used the Nafion™ 117 [1] for its ubiquitous use and success in MFC applications. The PEM allows the free movement of protons (H⁺) which are generated during respiratory redox reactions.
Building anaerobic chambers is recommended when working with species such as Shewanella oneidensis, since it is a facultative anaerobe with increased electrogeneratory protein production, when it switches to anaerobic respiration. For our system, we built aerobic chambers, since E. coli, our bacterium of choice is a facultative anaerobe.

Design & Build

Electrodes

We chose titanium-based carbon-brush electrodes (Figure 8E) because they are less vulnerable to corrosion in an aqueous environment and thus less toxic. In addition, carbon-brush electrodes have demonstrated high efficiency yield when compared to other electrodes [2–5]. And titanium wire success was already demonstrated in 3D printed MFC systems [6]. When choosing the electrode, we also took equity-based device manufacturing into consideration. Furthermore, when compared to gold-based electrodes, carbon electrodes are much cheaper. Regarding the smaller MFC, we chose to experiment with flat-surface carbon-electrodes, well established in acupuncture therapies.

Mini-MFC

The Mini-MFC was designed in two parts, bottom and top chambers. It contained three threaded holes on each side, for the bolts. In addition, it had four threaded holes on the bottom side, from which, when bolts were added, carbon electrode was held in place by direct pressure. Dimensions and all design details of top chamber can be observed in the Figure 1. The top chamber contains male cap, which when combined with the female cap of the bottom MFC, seal is maintained of 5mm depth and 5mm thickness. This was ultimately the location for PEM, which was locked in place. Bottom MFC has a special compartment, where closing lid is situated (Figure 2) this part was later re-engineered to have our logo and served merely as a structural rigidity component. In Figure 2, we can also observe Seal-Inlet parts, which were added and glued to the chambers to create inlets. Inlets and outlets from both sides were 0.8mm in diameter, which was designed to fit the available tubing system. For both Mini-MFC and Large-MFC, same threaded holes, bolts & nuts were used. The latter were printed along with the rest of the MFC, but were designed to be 0.03mm shorter in diameter, than the threaded holes inside MFC, for easy-assembly. This number can vary between different printers, for as long as the mechanism works. Simulation of both chambers can be seen in Figure 3. Alas, this figure was generated without bolts & nuts and an electrode.

Large-MFC

For the larger MFC, the design was based on two major principles, safety system and carbon-brush electrode encapsulation. For proper carbon-brush encapsulation, the bottom sides of the chambers were designed to have trap features, along a “station” hole with a diameter of 4.03 mm and depth of 1.34 mm. The diameters of both the female (Figure 4) and male (Figure 5) chambers were based on the available electrode diameter. The length of the system was designed, so that the inlet would not come in direct contact with the brush. Both chambers had the same overall chamber width and threaded holes, which were included at the lateral wings.
Interestingly, since the female and male chambers are different in their design, so is their overall volume, but this must not come as an impedance to the overall system.







In order to achieve good printability, top and bottom layers of the male chamber were printed separately, which can be seen in Figure 6. All additional parts were glued using non-toxic solvent-free glue, which maintained good water isolation and was removable using pressure. For the larger chamber, PEM was designed to be attached to the protrusions of male chamber.

Finally, the MFC safety-box was built (Figure 6), which was designed around the base sketch of both chambers of the Large-MFC. The design featured a water drainage system placed directly under the MFC and a water trap. The latter contained a protrusion for locking the water sensor and holes for passing the cables.

MFC's control

MFC was controlled using IOT Arduino (MKR WiFi 1010) circuitry. Voltage & current was sensed using a precise INA3221 or INA219-module. Liquid inlet and outlet was maintained by 5V liquid pumps. System also contained a buzzer, and the button. Our MFC was controlled both in-person (using a button) and online on Arduino IOT Cloud. Safety was ensured by monitoring water leakage by water sensor (DC3-5V), which if spill was detected, would activate the buzzer and block the pumps. Onboard RGB LED was coded to inform an accident, or the in-person user, that the online user was engaged. All Arduino modules other than liquid pumps were controlled by Arduino board itself. Liquid pumps were powered by using a relay module, which was connected to the 9V battery. This would, in future, guarantee system's versatility, since our relay can directly convert the AC voltage and thus it can be powered separately using an additional wall-power-block. Although, for a more complex control, motor modules should be preferred. Modules and connections can be visualized in the Figure 7.

System control was simplified to one major button. Pushing this button would activate pumps for as long as the button is pushed. This way, in-place user was able to control the liquid flow. Online user, on the other hand, was limited to how much of the liquid can be pumped, to avoid the overflow. Specifically, the online pump activation lasted for approximately 2 seconds, followed by a five seconds cooldown. In order to avoid “clash” between in-place and Online users, RGB LED was set to change colors. In case of Online engagement, it was set to go blue to alert the in-place user. Importantly, color-change would also occur if the pumps were controlled, to visualize the pump activity.

Test

Preliminary tests were performed on INA3221 module setup, due to its precision and triple-monitoring capacity. This module studies the same current, by which it is powered. The voltage was tested using a potentiometer and regulated by increasing resistance. Our plan was to test this setup using a lower 0.08 volt setup, but, since the minimum working voltage was found to be 0.8 volts, INA3221 was proved to be inapplicable to MFC studies (Figure 8C). We still tried to upgrade the module by cutting the board circuit which was connecting the sensing site to the power source, and while such examples exist online, in our case the sensor corrupted. Consequently, all further experiments were based on the INA219 module, since it was by-design able to sense external current.



The MFC hardware was tested through the IOT website, where current was alternated along with voltage to test alternating power. While all online features were functioning properly, setting up multiple tests for system activity led to delayed response times (as the the loop-function would need to complete the tests, before it could respond to the outside input, such as button switch).



To improve the time-lag, all testing features were shortened. Using this setup, once recorded, it is possible to download generated data in CSV format for further analysis, after leaving the MFC for as long as required.

Successful in vivo tests were also performed using both S. oneidensis MR-1 cells and our E. coli engineered strains to generate and improve the electric current. These experimental results are presented in the Proof of concept page on this wiki.

Learn

Our microbial fuel cell (MFC) device was designed to be re-usable and easy to disassemble and reassemble for changing the most vulnerable part of the MFC, the protein exchange membrane (PEM). In addition, the MFC parts can be removed and replaced by printing them separately, effectively eliminating the need of complete replacement. While such a design has a lot of flexibility for users, certain issues still emerged during final assembly, along with few characteristics that could easily be upgraded.

First, safety features can be improved. For this part we considered a few options of how MFC should react given the detected disintegration of the chambers and emerging leaking. One option would be to include a chelating agent, that should be dumped into the bacterial chambers, if there is any leakage. Another option would be to have better sealed chambers, having a locking mechanism that activates in case of emergency to avoid further contamination. Finally, we also thought of including a shocking system that could eliminate all bacteria inside the chambers. While all the above options have their pros and cons, dumping a chelating agent may turn out to be the easiest to implement, because it would require an independent, off-grid pocket that can be easily triggered.

Next, we have found that inlets that were originally designed for the chambers can be upgraded. Either by adding more inlets/outlets for redundancy, in case there is any sort of clogging in major ones, or by introducing a separate inlet/outlet system. In such a system each liquid-flow can be controlled separately, which would provide an additional tool for optimizing the MFC system. For example, we could envision controlling both the cathode and anode chamber liquids at a separate rate or separate volumes. In the current setup, all pumps are connected to the same relay, which means that they’re controlled together at almost the same rate.

Apart from the aforementioned individual improvements the entire MFC design could also be improved. One of the major issues that emerged during our setup was the difficulty to seal the PEM between the chambers. Our solution was to use solvent-free removable water-sealing glue. While it was effective as treatment, it still adds extra steps between rebuilding the system. To improve upon that, the chamber locking mechanism could be better designed either by using more accurate printing devices (eliminating gaps between two chambers), or changing the existing design completely (currently it consists of two chambers coming together just like in the lock, where one has protrusions that seals in another chamber). Finally, gaps can be eliminated by using SLA-3D-printers.

While SLA- 3D printers have higher precision, they are more expensive and time-consuming to operate at the proof of concept development stage. However, the latter may not impede manufacturing if the system is minimized. Interestingly, as observed in the literature, minimizing the system should lead to both faster data-acquisition and reduced use of all kinds of resources. Minimizing the MFC will eventually lead to a more sustainable solution, since smaller MFC would be much easier to contain in case of an emergency. In addition, minimizing MFC size can give us space to introduce more chambers, permitting pluri-sensing of multiple markers in a single run. While the current MFC model is sufficient to serve as a proof of concept for our project, there are simple, easy-to-implement features that emerged during our setup that could yield better end-results. We hope that these observations would be useful for future iGEMers interested in improving similar setups.

Note

The Code for the Arduino can be found in our Github folder.

References

[1] Lehmani A, Turq P, Périé M, Périé J, Simonin J-P. Ion transport in Nafion® 117 membrane. Journal of Electroanalytical Chemistry (1997) 428: 81–89.
[2] Kakarla R, Min B. Evaluation of microbial fuel cell operation using algae as an oxygen supplier: carbon paper cathode vs. carbon brush cathode. Bioprocess and Biosystems Engineering (2014) 37: 2453–2461.
[3] Ma J, Ni H, Su D, Meng X. Bioelectricity generation from pig farm wastewater in microbial fuel cell using carbon brush as electrode. International Journal of Hydrogen Energy (2016) 41: 16191–16195.
[4] Sayed ET, Alawadhi H, Olabi AG, Jamal A, Almahdi MS, Khalid J, Abdelkareem MA. Electrophoretic deposition of graphene oxide on carbon brush as bioanode for microbial fuel cell operated with real wastewater. International Journal of Hydrogen Energy (2021) 46: 5975–5983.
[5] Liao Q, Zhang J, Li J, Ye D, Zhu X, Zhang B. Increased performance of a tubular microbial fuel cell with a rotating carbon-brush anode. Biosensors and Bioelectronics (2015) 63: 558–561.
[6] Papaharalabos G, Greenman J, Melhuish C, Ieropoulos I. A novel small scale Microbial Fuel Cell design for increased electricity generation and waste water treatment. International Journal of Hydrogen Energy (2015) 40: 4263–4268.