Team:LZU-China/wetlab4

From 2014.igem.org

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" " http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd"> LZU-China 2014

We constructed a pollutant substrate(PNP) bio-sensor coupling riboflavin synthetic gene cluster, the genetically modified E.coli can secrete riboflavin when added PNP in MFC anode medium.Riboflavin is a efficient redox mediator as well as a stimulator of MFC. We created a novel MFC devices and built a quantitative monitor system of PNP via measuring voltage increment.

DESIGN AND CREATE MFC DEVICES

MFC Assembly and Operation.

Generally speaking a MFC has two electrodes, an anode and a cathode, a proton exchange membrane and a circuit pathway linking the two electrodes for the electron transfer from anode to cathode.

Figure-11. Schematic diagram of a typical two-chamber microbial fuel cell(Du et al., 2007)

The catholyte in all experiments was an unstirred 50 mM potassium ferricyanide solution in 100 mM phosphate buffer (pH=7.4), which was continuously open to air. The MFC design with carbon brush electrodes is shown in Figure 12. Brush anodes were made of carbon fibers (Haoshi carbon fiber, Lanzhou, Gansu, China) cut to a 3cm length and wound using a simple brush manufacturing system into a twisted core consisting of two titanium wires.

Figure-12. Components of MFC devices we previously used.

We designed a kind of cube-like two chamber MFC device, reference others’ single-chamber researches(Logan, Cheng, Watson, & Estadt, 2007). The cube was like a symmetrical 5×5×5cm polymethyl methacrylate cube, each chamber has two hole to aerate or load medium, each cylindrical chamber has 30ml with 4cm diameter, the blue print was shown in figure 13 . The white latex rubber gaskets were applied between every component of MFC devices to seal (figure 14).

Figure 13. Blue print (3 views) of one MFC component design.

Figure-14. A blank MFC device without carbon brush.

Microbiological Methods

It’s reported that Shewanella, a well-known star of electricity-producing bacteria family, can secretes riboflavin that mediate extracellular electron transfer (Marsili et al., 2008) , additionally, this amazing bacteria can produce electrically conductive pilus-like appendages called directly transfer electric charge by touching relative remote anode material(Gorby et al., 2006) . So we buy standard bacteria strain, Shewanella oneidensis strain MR-1, from China Center of Industrial Culture Collection, CICC.

Luria-Bertani (LB) broth was inoculated with S. oneidensis strain MR-1 and incubated aerobically at 30 °C for 5 days with shaking at 200 rpm. After assembling the MFC (described below) devices, 2 mL of MR-1 culture was transferred to a sterile MFC anode chamber, as well as 28ml sterile anode medium and lactate was added (20 mM) to ensure excess electron donor. Anode medium contained (per liter): 0.46 g NH4Cl, 0.225 g K2HPO4, 0.225 g KH2PO4, 0.117 g MgSO47H2O, 0.225 (NH4)2SO4, plus 10 ml of a mineral mix (containing per liter: 0.1 g MnCl24H2O, 0.3 g FeSO47H2O, 0.17 g CoCl26H2O, 0.1 g ZnCl2, 0.04 g CuSO45H2O, 0.005 g AlK(SO4)212H2O, 0.005 g H3BO3,0.09gNa2MoO4, 0.12 g NiCl2, 0.02 g NaWO42H2O, and 0.10 g Na2SeO4). Medium was adjusted to pH 7, sparged with oxygen-free N2, anode was sealed with white latex gasket.

REFERENCE

Du, Z., Li, H., & Gu, T. (2007). A state of the art review on microbial fuel cells: A promising technology for wastewater treatment and bioenergy. Biotechnol Adv, 25(5), 464-482. doi: 10.1016/j.biotechadv.2007.05.004
Gorby, Y. A., Yanina, S., McLean, J. S., Rosso, K. M., Moyles, D., Dohnalkova, A., . . . Fredrickson, J. K. (2006). Electrically conductive bacterial nanowires produced by Shewanella oneidensis strain MR-1 and other microorganisms. Proc Natl Acad Sci U S A, 103(30), 11358-11363. doi: 10.1073/pnas.0604517103
Logan, B., Cheng, S., Watson, V., & Estadt, G. (2007). Graphite fiber brush anodes for increased power production in air-cathode microbial fuel cells. Environ Sci Technol, 41(9), 3341-3346.
Marsili, E., Baron, D. B., Shikhare, I. D., Coursolle, D., Gralnick, J. A., & Bond, D. R. (2008). Shewanella secretes flavins that mediate extracellular electron transfer. Proc Natl Acad Sci U S A, 105(10), 3968-3973. doi: 10.1073/pnas.0710525105
Rabaey, K., & Verstraete, W. (2005). Microbial fuel cells: novel biotechnology for energy generation. Trends Biotechnol, 23(6), 291-298. doi: 10.1016/j.tibtech.2005.04.008