Team:NRP-UEA-Norwich/Results/Result
RESULTS
To measure the optical density (OD) at 590 nm of both the wildtype MR-1 Shewanella oneidensis and double knock out (∆hydABC,hyaABC) strain when cultured 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 wildtype MR-1 Shewanella oneidensis strains in M72 media under anaerobic conditions. Refer to respective protocols for the preparation of M72 media with the additional 'Additional Growth Additions' (AGA). Protocol 'Gas Chromatography Preparation' 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.
Table 1. Cuvettes (stock bacteria solutions used to inoculate hungate tubes)
| Sample | Machine Reading | Scaled Value |
|---|---|---|
| MR1 | 0.40 | 1.60 |
| LS473 | 0.39 | 1.56 |
Table 2. Optical density readings for the S.oneidensis MR-1 and double knock out (∆hydABC,hyaABC) 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 increasing Optical Density. However, there is a spike in the growth of the double knock out (∆hydABC,hyaABC) bacterial strain during hour 5 and 6 before the OD drops again and levels out, as seen in table 2. By contrast the S.oneidensis MR-1 strain grows at a steadier rate over time before levelling out. This trend is shown in figure 1, which shows the double knock out (∆hydABC,hyaABC) strain peaking with an OD of 0.64, before levelling out after 20 hours with an OD of approximately 0.47. While the wildtype MR-1 strain shows a gradual increase in OD over time, but also levelling out over 20 hours with an OD of around 0.44.
Figure 1. Growth curve of Shewanella for wildtype MR-1 and double FeFe NiFe hydrogenase knock out (∆HydABC,HyaABc) strains over a 24 hour period kept under 30°C incubation . The cultural media is a negative control.
Table 3. Table displaying the chromatograph results, including the retention time, peak area and amount 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.
To demonstrate electrochemical hydrogen production using Shewanella oneidensis MR-1 under small scale lab conditions. This would be compared to the double knockout control (∆HydABC,HyaABC) which should not show reductive current readings (corresponding to continuous electron movement to the hydrogenase enzymes) since both FeFe and NiFe hydrogenases are not present. Since reductive current (and therefore hydrogen production) was not seen with just the bacterial cells added to the poised electrode, a mediator to help transfer electrons from the electrode to the enzymes was added. The mediator methyl viologen was used. The experiment was carried out under anaerobic conditions to preserve FeFe hydrogenase which is very sensitive to oxygen.
Refer to protocol 'Electrochemistry' preparation and use of the electrochemical cells.
Figure 2:Chronoamperometry to show the current over time in the electrochemical cell, and the difference between cells from the wildtype MR-1 and double knock out Shewanella oneidensis strains before and after the addition of the mediator methyl viologen at 1200 sec.
As shown by figure 2, the addition of the bacteria to the electrochemical cell caused a decrease in the current at roughly 600 sec, which then returned to just under 0µA until the mediator, methyl viologen, is added to each fuel cell at 1200 sec. This had little effect on the double knockout strain (∆HydABC,HyaABC) but caused a sudden drop in the current for the wildtype MR-1 strain, which begins to even out at -32µA but continues to slowly decrease over time.
This was a repeat experiment for the demonstration of electrochemical hydrogen production with Shewanella oneidensis MR-1 and our overexpression construct which contains the three FeFe hydrogenase subunits in the wildtype MR-1 strain, with the double knock out strain (∆HydABC,HyaABC) as a negative control. The aim of this experiment was to discover whether overexpression of FeFe Hydrogenase in Shewanella oneidensis MR-1 will have an effect on the current in our electrochemical system. We predicted the experiment overexpressing hydrogenases would demonstrate a larger reductive current compared to the wildtype, as the current corresponds to hydrogen production. As previously described, the experiment was initially conducted using just cell suspensions with the electrode, but when no reductive current was observed the mediator methyl viologen was added. The experiment was carried out under anaerobic conditions to preserve FeFe hydrogenase activity which is very sensitive to oxygen.
Refer to protocol 'Electrochemistry' for the general preparation and use of the electrochemical cells. In this experiment Shewanella oneidensis MR-1 and the FeFe overexpression construct overnights were prepared to inoculate 500ml of M72 media in durans (2% inoculum) and the antibiotic kanamycin (50µg/ml) was added to the FeFe overexpression culture. After inoculation both 500 ml durans were sparged for 10 minutes and 1mM arabinose (final concentration) was added to both cultures to keep the conditions comparable after 7 hours of growth (OD 0.3). This was used to induce expression in the FeFe overexpression construct strain and account for any effect on growth in the wildtype MR-1 strain.
Figure 3. Chronoamperometry of electrochemical cells for our three different cultures, wild type MR1, FeFe NiFe hydrogenase knock outs (∆HydABC,HyaABC) and our FeFe overexpression strain . The knockout strain acted as the negative control. Methyl viologen was added at 720sec.
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 FeFe overexpression construct. Each of the cultures were added at around 350 seconds and no changes in current were observed, this suggests the hydrogenases were not coupling to the electrode. After the addition of the mediator methyl viologen figure 3 shows how the double knock out strain did not change other than a temporary dip at 720 sec, due to the lack of hydrogenase expression, whereas the wildtype MR-1 strain dropped and levelled out around -25 µA. More promisingly, the FeFe overexpression strain, which has added arabinose to promote FeFe hydrogenase overexpression, has levelled out at a more negative current of around -33µA and continues to fall over time, demonstrating a larger reductive current compared to the wild type. This could suggest an increase in hydrogen production, supporting the hypothesis. There are however a number of caveats to this claim and this should be repeated/investigated in more detail. These three experiments were normalised to initial optical density (OD600) and a Bradford assay confirmed comparable amounts of protein in each bioreactor after the experiment.
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