Team:Hong Kong HKUST/Design
MFC DESIGN
OVERVIEW
Aside from plastic degradation and alkane metabolism, generation of electricity was another important focus of our iGEM project. For this module, we focused on generating a stable electrical current by utilizing Shewanella oneidensis MR-1 strain’s inbuilt extracellular electron transport mechanism. In order to better harness its electrogenicity, we housed a culture of the bacterium within a microbial fuel cell of our design, aiming at maximizing electrical output for a given amount of substrate.
While many other microorganisms are capable of electron transport by reduction of suitable substrate, we chose to work with Shewanella oneidensis MR-1 strain as it is a relatively well-studied facultative anaerobe capable of generating current by reducing a broad range of substrates, utilizing them as metabolites. While the processes behind this electron transport mechanism are not yet fully understood, it is known that a few outer and inner-membrane Cytochromes are responsible for the shuttling of electrons from within the cell body to the extracellular substrate. The multiple heme-centres present within these Cytochromes allows for efficient transport of electrons to the extracellular space.
According to literature[1], electrons are shuttled from the inner-membrane Cytochrome c molecule CymA to another Cytochrome c molecule, MtrA, present in the periplasm. They are then transported to the outer-membrane Cytochromes MtrC and OmcA. These outer-membrane cytochromes are in fact lipoproteins associated to the outer membrane and outer-membrane protein MtrB. By this arrangement, the outer-membrane Cytochromes are exposed to the extracellular space where they may reduce suitable substrates. This reduction may occur by direct contact between cell surface and substrate or may be mediated by Flavins[2] to reduce substrate far from the cell surface. Riboflavins can mediate such indirect reduction of substrate because of their conjugated double bonded structures, allowing for the small energy transitions required to carry electrons. Additionally, its large polar tail confers its significant solubility within the external medium to shuttle electrons to distant substrates.
Aside from its extensive electron transport pathways, Shewanella oneidensis MR-1 is particularly useful to our project from a microbial fuel cell perspective because of its ability to produce biofilms that facilitate close contact between the bacteria and the cell electrode. These biofilms are formed by an interconnected network of a type IV pili from the cell membrane that adheres to the solid substrate. The pili are conductive and function as nanowires that aid in the direct reduction of the substrate[3]. It is however worth noting that more robust biofilm formation occurs in anaerobic and low-nutrient conditions. This was an important factor to consider while designing our microbial fuel cell as we had to decide the nutrient concentration of the culture medium for optimal cell growth and biofilm formation while maintaining an oxygen-free environment.
Having gone through many prototypes, we designed our own microbial fuel cell taking inspiration from those of past iGEM teams such as the 2013 Bielefeld team[4]. We decided to employ 3D-printing as a fast and low-cost method to produce our own prototypes. For the body, we chose PLA (Polylactic acid) because it could not be degraded by Shewanella while known to be non-toxic to the bacterium. The anode compartment housed the culture medium containing Shewanella oneidensis MR-1 strain in anaerobic conditions. To achieve such an anaerobic environment, we initially considered flooding the chamber with inert nitrogen gas but later decided to use an anaerobic chamber due to limitations our lab faced with usage of nitrogen gas. For the anode material, we decided to use carbon cloth as it has a high effective surface area and provides a suitable surface for the bacteria to tether onto and initiate biofilm formation. Meanwhile the cathode compartment was aerobic in nature with Ferricyanide as the terminal electron acceptor and PBS buffer to maintain the pH constantly at 7 throughout the cell’s operation. We needed a proton-selective membrane to separate the anode and cathode compartments so that the circuit may be completed by the transfer of H+ ions from the anode to the cathode without allowing for the direct reduction of the terminal electron acceptor. For this, we chose to use a Nafion membrane due to its highly selective nature and efficiency in proton exchange.
With the microbial fuel cell assembled and the bacteria introduced into the anode chamber, we began a series of tests to determine our output potential. The results of which are elaborated here.
RESULTS
Being widely reported its natural ability to generate electricity, Shewanella oneidensis MR-1 was fed with lactate in the MFC to quantify the performance of our MFC device and as the experimental control of our engineered Shewanella system. While Shewanella can utilize different carbon sources such as lactate, pyruvate and acetate as carbon source, we chose lactate as carbon source because it has shown that lactate is an energy-favorable carbon substrate for this strain[1]. As described from literature[2], it is desirable to inoculate Shewanella culture which has entered early stationary phase. Therefore, we decided to characterize the stationary OD600 of Shewanella in LB by growth curve since we used LB to proliferate the cell. To get fuller understanding of the cell population of Shewanella in the designed condition of MFC, we also stimulated the anaerobic bacterial growth in M9 minimal medium.
Since stable cell population is desired for stable electricity generation in the MFC, we attempted to stimulate the anaerobic bacterial growth.
Stationary Shewanella MR-1 in LB was washed and transferred to M9 aerobic medium at time zero. At time t=30h, M9 medium was renewed. The drop of OD600 is likely due to the loss of cell during medium replacement. Similar to figure 2, the OD600 stayed at 1.79-1.84 with slight fluctuation. While no significant cell growth was observed again, the OD600 sustained for 24 hours after shifting to anaerobic condition when 20mM lactate was added.
MFC measurement
The control has the same construction as the sample except no cells were added. 20mM lactate was added as carbon source.Despite the fluctuation occured from time 20 to 35 minutes, the cell potential increased over time in general. The control indicated the difference in voltage is likely due to the presence of Shewanella.
Considered the set-up as right figured with the ideal voltmeter, no current would be present in the circuit to drive any resistive load, including the internal resistance. Thus, the potential across the MFC obtained from the above experiment could be defined as the standard electrode potential between the cell culture and the ferrocyanide, which is comparable to the literature value 0.256V [3,4].
We further connected the MFC with external variable resistor as the set-up as shown below.
Considered ε= I[R +r(ε)], where I is the current, R is the variable resistor, ε is the emf of the mfc and r(ε) is the internal resistance. By rearranging of the equation, 1I=(1ε)R+r(ε)ε, We could yield the following graph.
Therefore, it is possible to determine the internal resistance by plotting the above graph, given that the emf is a constant. However, emf is not a constant for mfc as well as the internal resistance. As known, the internal resistance of a microbial fuel cell is a function of emf itself. To determine its relationship, different emf with corresponding internal resistance was recorded over time. Since the above graph is only valid in constant emf, we could only approximate the constant emf by obtaining the current-resistance relationship in a very short period of time so that the change of the emf could be neglected.
The graph above shows the relationship of internal resistance and the output voltage of the microbial fuel cell across the first 90 minutes after it was built. 20mM lactate was added as the sole carbon source. The curve indicates that the internal resistance increases with the output voltage more rapidly in the early stage, but reaches its maximum as the voltage further increases.
Figure 9 and figure 10 show the terminal power output and the total work done of the mfc setup as mentioned before over 120 minutes time period, respectively. It is clear that the power was increasing over time.
MFC efficiency
Assuming the decrease of cell population in the growth curve was because of using up the 20mM lactate after 24 hours in the M9 anaerobic medium, we estimated the lactate uptake flux from the wet mass of cell, then we search for the theoretical electron generating flux through the FBA model described in figure 4 of modelling pages.
The wet mass was converted to dry mass by a factor of 0.3, assuming 70% water content in the mass. As described in modelling, the corresponding maximum DET flux was evaluated based on the assumption of 5% of biomass growth flux. The efficiency was evaluated by comparing the measured current with the maximum theoretical current.
Transformation
We first made the chemically competent cells and attempted to transform the Shewanella by chemical method. After several unsuccessful attempts by increasing both cell and DNA concentration, we turned to use electroporation for higher transformation efficiency. Instead of RFP in the competency kit plate, GFP is chosen as reporter for competency check to avoid confusion with pink Shewanella oneidensis MR-1. However, we cannot get any transformed colony yet. Literature also showed low transformation efficiency of pSB1C3-GFP in Shewanella, 4 colonies were observed on the transformed plate after electroporation[5]. We hypothesized that the competent cell concentration is not high enough to achieve observable transformed colonies on plate.
References:
[1]J. Myers and C. Myers, "Role for Outer Membrane Cytochromes OmcA and OmcB of Shewanella putrefaciens MR-1 in Reduction of Manganese Dioxide", Applied and Environmental Microbiology, vol. 67, no. 1, pp. 260-269, 2001.
[2]E. Marsili, D. Baron, I. Shikhare, D. Coursolle, J. Gralnick and D. Bond, "Shewanella secretes flavins that mediate extracellular electron transfer", Proceedings of the National Academy of Sciences, vol. 105, no. 10, pp. 3968-3973, 2008.
[3]M. El-Naggar, G. Wanger, K. Leung, T. Yuzvinsky, G. Southam, J. Yang, W. Lau, K. Nealson and Y. Gorby, "Electrical transport along bacterial nanowires from Shewanella oneidensis MR-1", Proceedings of the National Academy of Sciences, vol. 107, no. 42, pp. 18127-18131, 2010.
[4]"Team:Bielefeld-Germany/Project/MFC - 2013.igem.org", 2013.igem.org, 2018. [Online]. Available: https://2013.igem.org/Team:Bielefeld-Germany/Project/MFC. [Accessed: 30- Aug- 2018].