Correlation of CTC reduction with growth rate in Escherichia coli and Temperature dependence of electron acceptor reduction in Geobacter sulfurreducens
Open Access
- Author:
- Hunt, Heather
- Graduate Program:
- Environmental Engineering
- Degree:
- Master of Science
- Document Type:
- Master Thesis
- Date of Defense:
- None
- Committee Members:
- John Michael Regan, Thesis Advisor/Co-Advisor
John Michael Regan, Thesis Advisor/Co-Advisor - Keywords:
- Geobacter
microbial electrolysis cell
CTC - Abstract:
- Understanding a microbial cell's metabolic state is critical to understanding its role in an environmental system, be it engineered or natural. This course of work presents two studies in bacterial metabolic activity. The first study centered on 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), which produces a fluorescent formazan upon reduction and is typically used to indicate a cell¡¯s viability status. CTC reduction was investigated in Escherichia coli and shown to correlate with growth rate, suggesting it may provide more insight into a cell¡¯s metabolic state. E. coli cells were cultured at different temperatures to induce different growth rates. Cells at these different growth rates were sampled at identical optical densities, incubated for varying lengths of time with CTC, and then either analyzed in a spectrofluorometric plate reader or counterstained with DAPI and visualized with epifluorescent microscopy. By assessing the change in the fraction of CTC(+) cells in the total cell population over incubation time, as well as the change in intensity of fluorescence of those cells, rates of CTC reduction were determined. Results demonstrated that overall CTC intensities and abundance increased with increasing growth rate. Moreover, CTC reduction rate increased relative to an increase in growth rate, most noticeably when determined from averages of single-cell intensities. The second study centered on the variance of electron acceptor reduction with respect to temperature in Geobacter sulfurreducens, a species that has received much attention due its ability to transfer electrons to electrodes. Understanding how G. sulfurreducens functions is critical to optimizing its role in energy production, particularly with regard to microbial electrolysis cells (MECs). The temperature dependence of G. sulfurreducens when reducing different electron acceptors was investigated. Experiments were carried out in both suspended cultures and MEC anode biofilms. In suspended-culture tests, G. sulfurreducens was grown with acetate as the electron donor and either fumarate or ferric citrate as electron acceptor at different temperatures (4C, 20C, 30C, and 37C). Fumarate reduction and ferric iron reduction were monitored. Cells from fumarate cultures were incubated with CTC and visualized with epifluorescent microscopy to further assess activity. In MECs, G. sulfurreducens was fed MEC medium containing acetate as an electron donor; a carbon paper anode served as the insoluble electron acceptor. Reactors were operated at 20C, 30C, and 37C, with voltage monitored across an external resistor (10 ohms). Voltage was applied (0.6 V) to promote hydrogen evolution at the cathode. Results from suspended-culture tests showed increasing fumarate reduction with increasing temperature, while ferric iron reduction and CTC reduction were greater at 30C compared to 20C and 37C. MEC operation at 37C resulted in the highest peak current (70 A/m3), hydrogen production (93.5 mL), hydrogen recovery (rH2 of 91%) and Coulombic efficiency (90%) when compared to reactors operated at 20C and 30C. These results suggest that 1) the temperature dependence of electron acceptor reduction by G. sulfurreducens depends on which electron acceptor is present, and 2) care must be taken when basing reactor operating conditions on results obtained from suspended-culture optimization.