Team:Hong Kong HKUST/MFC Result

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  Overview PE Degradation Alkane Metabolism Alkane Results MFC Design MFC Result Experimental protocol Notebook Parts
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  Laccase Alkane Metabolism Alkane and MFC (FBA)
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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.

Figure 1. Growth curve of Shewanella MR-1 in LB under 30^oC aerobic condition From the graph, we obtained the stationary OD₆₀₀ is around 1.8-1.9, sustained for 28 hours. In addition, the doubling time was 0.981h.

Since stable cell population is desired for stable electricity generation in the MFC, we attempted to stimulate the anaerobic bacterial growth.

Figure 2 Growth curve of Shewanella MR-1 in 25^oC M9 anaerobic medium Stationary Shewanella MR-1 in LB was washed and transferred to M9 anaerobic medium at time zero. Although no significant cell growth was observed, the OD₆₀₀ stayed within 1.45-1.5 for 24 hours. Since a decreasing trend was observed, we decided to repeat the measurement but allowing aeration to the cell culture after shifting from LB to M9 for better cell adaptation to the new medium.

Figure 3 Growth curve of Shewanella MR-1 shift from M9 aerobic to M9 anaerobic condition.

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

Figure 4 The 1st MFC prototype

Figure 5 The circuit construction of 1st MFC prototype

Figure 6 The miniauture MFC prototype for experimental use

Figure 7 Cell potential measurement over 130 minutes.(compare with control)

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.

Figure 8 internal resistance against emf

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

Figure 10

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

Figure 11

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.

Figure 12 (From figure 10, the average efficiency of our MFC design is around 0.2-0.3.)

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].