As mentioned in the Collaborations page of this wiki, we first met Parisian iGEM teams in the beginning of June to discuss our own projects and to see if we could work together and on which aspects. We discovered that both the Ionis_Paris and our team were working on different microbial fuel cell (MFC) devices, but with different applications, and using the same biological principles: external electron transfer from the cytoplasm of an electroactive microorganism (Shewanella oneidensis MR-1) to generate an electric current. The initial collaboration with Ionis_Paris extended into a real partnership as we worked together throughout the year on a set of shared objectives related to both of our projects, as detailed below.

Microbial fuel cell (MFC) device developpement

The Ionis_Paris iGEM team was the first of our two teams to begin external electrons transfer experiments on their miniature microbial fuel cell prototype. They obtained good results and reached 200 mV using the S. oneidensis MR-1 as an electroactive microorganism. They shared with us this same stain and we were thus able to benchmark our MCF device as well. Our best measurement was 128 mV.

Following these initial tests, we met together several times between June and August to try to understand together those differences in measurements, optimize the electrical output of our MFC, and look for ways to properly improve our hardware.

Analyzing the differences in the electrical signal output

Differences in measurements may occur due to different MCF components, conditions of culture for S. oneidensis MR-1 in each MFC device, the design or the material used to build the MCF.

Indeed, as we both used the same software and 3D printer in order to design and build our MFC, the differences are less likely to come from this aspect of our project. We also used the same membrane (the Nafion™ 117) to separate the 2 electrode chambers.

However, the two teams used different types of electrodes. Specifically, we used carbon-brush electrodes based on titanium rods, while the Ionis_Paris iGEM team used iron electrodes. We chose titanium based carbon-brush electrodes because they are less vulnerable to corrosion in an aqueous environment and thus they are less toxic. In addition, carbon brush electrodes have demonstrated high efficiency yield when compared to other electrodes [1–4], while titanium wire success was already demonstrated in 3D printed MFC systems [5]. On top of all, for the high energy yield, carbon-brush electrodes are much cheaper, which resonates with our equity goals. According to our discussions, the differences we obtained may originate from this difference in the material used. The Ionis_Paris iGEM team used solid and we used semi-solid carbon fiber electrodes which are much softer and have a brush shape. This brush shape avoids the formation of bacterial biofilm, while the solid carbon fiber does not. However, a biofilm, which is a community of bacteria bound to each other, increases the electrical output and is known as one of the best ways to increase the signal when using an electroactive microorganism as Shewanella [6]. However, as our electrical brush electrodes occupied almost fifty percent of the MCF chamber space and the fact that we were using electron shuttles mediators (such as phenazine and canthaxanthin) we thought that we would not need biofilm formation.

The differences may also arise from the measurement conditions: we performed our measurements in aerobic conditions. When S. oneidensis MR-1 is in anaerobic condition, genes related to external electrons transfer such as the Mtr nanoconducts genes are overexpressed compared to those same genes in aerobic conditions. To help the Ionis_Paris iGEM team achieve anaerobic conditions, we shared with them the AnaeroGen™ Atmosphere Generation System (Thermo Fisher Scientific) that, according to supplier’s instructions, allows in a closed environment to achieve less than 1% of O₂ after 2 and a half hours of exposure.

Moreover, in order to limit the risks associated with semi-sterile conditions we made our measurements over a thirty minutes time span instead of days, which is the common time span used to measure the current production by Shewanella.

Improving the electrical signal output by using electron shuttle mediators

Electron shuttle mediators facilitate the transfer of electrons from the media to the anode by being involved in redox reactions.

The Ionis_Paris iGEM team did not initially plan to use any such molecules, but we did. We built parts to produce canthaxanthin and phenazine-1-carboxylate (PCA). This approach might be a very interesting way for them to further increase the electric power of their MCF.

To help the Ionis_Paris iGEM team increase their own electric output, we shared with them our engineered E. coli stains secreting PCA (BBa_K4432141 and BBa_K4432142 in the pSB1A3 backbone).

General variability?

Based on the data collected by our two teams and available evidence from literature, electrical current production by S. oneidensis MR-1 can be variable, depending on multiple factors, including concentration of key molecules inside the liquid medium [7] or the accumulation of bacterial end-products along the exchange membrane [8]. Interestingly, different current production is observed between different strains [9]. While this can be explained by differences in multiple biological factors, other variables such as end-molecules production and liquid medium composition are harder to identify.

Ionis_Paris iGEM team hosted in our lab

From the beginning of September, we hosted the Ionis_Paris iGEM team leader (Alexandre Trubert) in our lab to allow his team to execute one important experiment in the context of their project.

As mentioned, our both teams are working with S. oneidensis MR-1 to get an electrical output through a microbial fuel cell device (MFC). However, the Ionis_Paris iGEM team needed an electroporator to transform the S. oneidensis MR-1 cells, an equipment absent in their lab but available in ours.

The particular plasmid construction they wished to transform into S. oneidensis MR-1 cells contains the coding gene of the MtrC nanoconduct subunit protein under the control of a strong arabinose inducible promoter, of a strong ribosome binding sequence with high affinity for Shewanella’s ribosomes and of two terminators to avoid any leaking MtrC protein traces. MtrC is a membrane protein involved in the release of electrons from the outer membrane to the media and the overexpression of this particular subunit of the MtrCAB protein would significantly increase the electric output of their MFC.

We gave Alexandre Trubert access to our L1 laboratory and treated him as an Evry_Paris-Saclay team member :). He was allowed to use not only our electroporator, but also other required equipments like the shaking incubator, laminar flow hood and all the material he needed under the supervision of our team leader, Paul Weimer.

On his part, Alexandre Trubert helped us troubleshoot the leakage observed in our MFC.

Conclusions

Throughout our iGEM year, we worked in close contact with the iGEM Ionis_Paris team. We exchanged a lot of scientific information, designed and debugged experiments, discussed our results, and exchanged materials. It was an enriching experience that helped both teams obtain important results.

References

[1] 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.
[2] 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.
[3] 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.
[4] 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.
[5] 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.
[6] Liu T, Yu Y-Y, Deng X-P, Ng CK, Cao B, Wang J-Y, Rice SA, Kjelleberg S, Song H. Enhanced Shewanella biofilm promotes bioelectricity generation. Biotechnology and Bioengineering (2015) 112: 2051–2059.
[7] Jadhav DA, Ghadge AN, Mondal D, Ghangrekar MM. Comparison of oxygen and hypochlorite as cathodic electron acceptor in microbial fuel cells. Bioresource Technology (2014) 154: 330–335.
[8] Flimban SGA, Hassan SHA, Rahman MdM, Oh S-E. The effect of Nafion membrane fouling on the power generation of a microbial fuel cell. International Journal of Hydrogen Energy (2020) 45: 13643–13651.
[9] Páez A, Lache-Muñoz A, Medina S, Zapata J. Electric power production in a microbial fuel cell using Escherichia coli and Pseudomonas aeruginosa, synthetic wastewater as substrate, carbon cloth and graphite as electrodes, and methylene blue as mediator. Laboratory scale. Tecnología y ciencias del agua (2019) 10: 261–282.