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Bio-electric Interface:
Microbial half fuel cells
Methods
The half fuel cells were constructed using the following components provided by Matthew Knighton from Dr Bruce Ward’s lab:
- 250 ml or 500 ml glass bottle
- A standard plastic cap with two holes drilled for electrodes
- carbon weave electrode fixed to the cap with silicone sealant
- reference electrode "red rod" REF201 available for sale from Radiometer analytical
- Following the assembly, bottles were autoclaved (reference electrodes were instead sterilised with alcohol as they are temperature sensitive). Under sterile conditions, reference electrodes were dipped in alcohol, inserted into the cap of the bottles and sealed with silicon sealant. The half fuel cells were then filled with media, inoculated with bacteria and sealed with parafilm in order to ensure anaerobic growth. The bacteria were left to grow at room temperature. (Figure 1)
- Media used: standard LB or M9 (minimal growth medium) supplemented with 1% lactose, 0,4% glycerol or 0,4% sodium acetate.
- Measurements were obtained using a digital multimeter.
Results
- We have examined the behaviour of S. oneidensis and E. coli in different media using half fuel cells. We managed to obtain results using the following media: LB, M9 with glycerol and M9 with sodium acetate. The results are summarised in figures 2 and 3 below. We also performed a measurement for Citrobacter freundii to see whether it differs from other bacteria.
Figure 1: Our microbial half fuel cells with S. oneidensis and E. coli
Figure 2: Half fuel cells experiments 1 and 2, using LB medium for growth of S. oneidensis and E. coli. Experiment 1 (left) was performed using 500 ml of medium while experiment 2 (right) was performed using 250 ml of medium.
Figure 3: Half fuel cell experiments 3 and 4, using M9 medium for growth of S. oneidensis, E. coli and Citrobacter freundii. Experiment 3 (left) was performed using 250 ml of medium M9 with 0,4% glycerol while experiment 4 (right) was performed using 250 ml of medium M9 with 0,4% sodium acetate. In experiment 4, C. freundii was also tested.
Growth-based biosensor
We have designed and tested a growth-based arsenic biosensor with a direct electric output. In order to test the principle of this device, we have transformed E. coli JM109 with Edinburgh 2006's BBa_J33203 BioBrick (arsenic promoter with arsR repressor linked to lacZ' gene responsible for lactose degradation). We have then prepared 3 half-fuel cells with lactose medium (M9 with trace elements and thiamine + 1% lactose):
1) BBa_J33203 transformants in medium with sodium arsenate (100 parts per billion concentration)
2) BBa_J33203 transformants in medium without sodium arsenate
3) control, wild type E. coli in medium with sodium arsenate (100 parts per billion concentration)
Figure 4: Growth-based arsenic biosensor: change in voltage over time using E. coli transformed with BBa_J33203 + lacZ' and WT E. coli as control
The growth of cells and associated change in voltage was much slower compared to our previous experiments. This can probably be attributed to the lower temperature of incubation as the fuel cells were incubated at room temperature. Despite the slower growth rate, the results we have obtained are encouraging. BBa_J33203 transformed cells in the presence of arsenate show a faster drop in voltage compared to other samples. This is especially important compared to the BBa_J33203 cells in the medium without arsenate. These results show promising prospect for growth-based biosensors. With more sophisticated measurement methods it would be possible to connect our system to a computer which would allow for automated and quantitative analysis of the data, allowing for simple and automated contamination detection.
Current results are encouraging but background growth is still present in the media and therefore further experiments are necessary to optimise the growth parameters. One possible improvement includes the addition of the cscA BioBrick that we have designed this year. Using sucrose instead of lactose may reduce background growth and allow for tighter control of the system.
Acknowledgements
We would like to thank Dr Bruce Ward and Matthew Knighton for their help with the fuel cells and for lending us their lab equipment.
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