Team:NRP-UEA-Norwich/Results/Result
RESULTS
Date: 18th - 19th August 2016
The aim of the experiment is to measure the optical density of both the MR-1 shewanella oneidensis strains (MR1 and LS473) when stored anaerobically and quantify the amount of hydrogen in the headspace gas using gas chromatography.
As the hydrogenase of interest within our bacteria is oxygen sensitive we grew the MR1 Shewanella oneidensis strains in M72 media and in anaerobic conditions. Refer to protocol 3.1 for the preparation of M72 media with the additional AGA (additional growth additions). Protocol 3.1 also covers the purging procedure to removal almost all the oxygen within the hungate tube headspace and how often each OD should be taken during the 24 hour incubation period. The last step is to measure the hydrogen content of the headspace gas which is detailed in protocol 3.2.
Table 1. Cuvettes (stock bacteria solutions)
| Sample | Machine Reading | Scaled Value |
|---|---|---|
| MR1 | 0.40 | 1.60 |
| LS473 | 0.39 | 1.56 |
Table 2. Optical density readings for the MR-1 and LS473 strains and media control over a 24-hour period.
| Sample | Time (Hours) | Δ optical density | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| 0 | 1 | 2 | 3 | 4 | 5 | 6 | 22 | 23 | 24 | ||
| Media (1) | 0.05 | 0.07 | 0.05 | 0.06 | 0.04 | 0.04 | 0.05 | 0.05 | 0.05 | 0.06 | 0.01 |
| Media (2) | 0.04 | 0.04 | 0.04 | 0.04 | 0.04 | 0.03 | 0.04 | 0.04 | 0.03 | 0.04 | 0.00 |
| MR1 (1) | 0.08 | 0.11 | 0.15 | 0.19 | 0.23 | 0.27 | 0.30 | 0.43 | 0.43 | 0.44 | 0.36 |
| MR1 (2) | 0.07 | 0.10 | 0.14 | 0.20 | 0.23 | 0.25 | 0.29 | 0.46 | 0.44 | 0.43 | 0.36 |
| LS473 (1) | 0.07 | 0.11 | 0.16 | 0.30 | 0.45 | 0.64 | 0.62 | 0.48 | 0.47 | 0.47 | 0.40 |
| LS473 (2) | 0.09 | 0.12 | 0.20 | 0.29 | 0.48 | 0.63 | 0.64 | 0.49 | 0.48 | 0.49 | 0.40 |
These results are as expected as all bacteria show a trend of growing Optical density. However, there is a spike in the growth of the LS473 bacteria during hour 5 and 6 before the OD drops again and levels out, as seen in table 2. By contrast the MR1 bacteria grow at a steadier rate over time before levelling out. This trend is shown in figure 1, which shows the LS473 strain peaking with an OD of 0.64, before levelling out after 20 hours with an OD of approximately 0.47. While the MR1 wildtype strain shows a gradual increase in OD over time, but also levelling out over 20 hours with an OD of around 0.44.
Fig 1. Growth curve of both the Shewanella Oneidensis MR1 and LS472 strains over time with a media control.
Table 3. Table displaying the chromatograph results, including the retention time, peak area and volume of H2 gas for each headspace.
| Sample | Molecule | Retention time/ minutes | Peak area/mV.s | H2 volume/nmol |
|---|---|---|---|---|
| Air (test sample) | O2 | 0.660 | 576.268 | N/ |
| N2 | 0.752 | 1838.930 | N/A | |
| Media (1) | O2 | 0.668 | 71.540 | N/A |
| N2 | 0.756 | 2343.100 | N/A | |
| Media (2) | O2 | N/A | N/A | N/A |
| N2 | 0.748 | 2391.000 | N/A | |
| MR1 (1) | H2 | 0.444 | 26.886 | 2.95 |
| N2 | 0.744 | 2315.476 | N/A | |
| MR1 (2) | H2 | 0.456 | 32.455 | 3.97 |
| N2 | 0.760 | 2424.841 | N/A | |
| LS473 (1) | O2 | N/A | N/A | N/A |
| N2 | 0.752 | 2460.850 | N/A | |
| LS473 (2) | O2 | N/A | N/A | N/A |
| N2 | 748 | 2232.375 | N/A |
The O2 peaks are missing where they would be expected because the amount of O2 was below the detection limit of the machine.
H2 quantities
Table 4. Table to show the total Hydrogen level in the syringe and hungate tube produced by repeat 1 and 2 of the wildtype strain of Shewanella Oneidensis.
| Sample | H2 in syringe/nmol | H2 in hungate tube/nmol |
|---|---|---|
| MR1 (1) | 2.95 | 137.67 |
| MR1 (2) | 3.97 | 185.27 |
The equation used to convert peak area into quantity is:
y = 5.472x + 10.73
Date: 7th – 8th September
The aim of this experiment was to demonstrate a working electrochemical fuel cell with our MR-1 Shewanella Oneidensis strain in simulated lab conditions. This would be compared to the double knockout control which should not influence the current in the cell since both hydrogenases have been removed. The electron transfer of the electrode to the microbial cells will be facilitated by the mediator methyl viologen. The experiment will be carried out completely under anaerobic conditions to preserve FeFe hydrogenase which is very sensitive to oxygen.
Refer to protocol 3.3 for the preparation and use of the electrochemical cells.
Fig 2. A Chronoamperometry to show the change in current change over time in the electrochemical cell, and the difference between cells from the MR-1 and LS473 Shewanella Oneidensis strains before and after the addition of the mediator methyl viologen at 1200 seconds.
As shown by figure 2, the addition of the bacteria strains to the electrochemical cell caused a dip in the current at roughly 600 seconds, which then returned to just under 0 µA until the mediator, methyl viologen, is added to each fuel cell at 1200 seconds. This had little effect on the Knockout strain LS473 but caused a sudden drop in the current for the wildtype MR-1 strain, which eventual begins to even out at -32 µA but continues to slowly decrease over time.
Date: 5th - 7th October
This is a repeat experiment for the demonstration of a working electrochemical fuel cell with Shewanella Oneidensis MR-1 and our 8A5 construct which contains the three FeFe hydrogenase subunits in the Wildtype MR-1 Strain. The aim of this experiment is to discover whether the overexpression of the FeFe Hydrogenase in Shewanella Oneidensis MR-1 will have an effect on the current in our cells. The hypothesis is that the overexpression of hydrogenases will cause a more negative electrical current to pass through the cell compared to the wildtype strain without the construct, which will be initiated by the addition of the mediator Methyl Viologen. The experiment will be carried out completely under anaerobic conditions to preserve FeFe hydrogenase which is very sensitive to oxygen.
Refer to protocol 3.3 for the general preparation and use of the electrochemical cells. In this experiment Shewanella Onedensis MR-1 and the 8A5 construct overnights were prepared to inoculate 500 ml of M72 media in durans (2% inoculum) and 500 µl of the antibiotic kanamycin (50 µg ml-1) was added to the 8A5 culture. After inoculation both 500 ml durans were sparged for 10 minutes and 1 mM arabinose (500 µl) was added to both cultures after 7 hours of growth (OD 0.3) to induce expression in the 8A5 construct and account for any effect on growth in the wildtype.
Fig. 3. Chronoamperometry of Shewanella Oneidensis MR-1 compared to Hydrogenase double knockout and the 8A5 FeFe strain before and after the addition of the mediator methyl viologen at 720 seconds.
The results for this chronoamperometric analysis have been combined with the demonstration results gathered earlier for the double knock out strain and overlaid to show a clear comparison between the double knock out strain and the wildtype strains with and without the 8A5 construct. Figure 3 shows how the double knock out strain did not change with the addition of methyl viologen other than a temporary dip at 720 seconds, due to the lack of hydrogenase expression, whereas the wildtype MR-1 strain dropped and levelled out around -25 µA. However, the 8a5 strain, which has added arabinose to promote FeFe hydrogenase overexpression, has levelled out at a lower current of around -33 µA and continues to drop sharply over time.
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