AN ABSTRACT OF THE THESIS OF Robert G. Hall for the degree of Master of Science in Biological and Ecological Engineering presented on August 3, 2011. Title: Metabolic Engineering of Shewanella oneidensis MR-1 for Microbial Fuel

approved: Frank W.R. Chaplen Shewanella oneidensis MR-1 is a gram-negative, facultative anaerobic bacteria with the capability of dissimilatory metal reduction. The ability of the organism to reduce a wide range of solid metal-oxides during anaerobic respiration makes it an ideal candidate for the powering of microbial fuel cells (MFCs), which capture the electrons discharged by the organism as work energy. The transfer of electrons from S. oneidensis to an anode surface occurs either as the result of direct electron transfer from a proximal outer membrane (OM) surface, or via soluble shuttling compounds which ferry the electrons from distal OM surfaces. The concentration of these electron shuttling compounds in the media have a strong influence on the current produced in the MFC. The ability of S. oneidensis to produce soluble electron shuttling compounds de novo in the form of riboflavin and flavin mononucleotide (FMN) provides a metabolic target for the enhancement of current generation in an MFC. This study uses variable ion concentrations in the growth media to deregulate the riboflavin synthesis pathway in S. oneidensis and induce the overproduction of electron shuttling compounds. The increase in electron shuttle production is demonstrated to increase the current produced in an MFC. Additionally, the metabolic relationship between the rates of growth, electron shuttle production, and anaerobic respiration are explored using flux balance analysis (FBA). Statistically designed screening experiments were used to quantify the influence of Mg, Ca, K, NH4 , PO −3 4 , SeO −2 4 , and Na + concentrations in the growth media on the metabolic production rate of riboflavin and FMN by S. oneidensis. Mg was the only compound identified to be a significant factor in influencing the production of these electron shuttling compounds. A 5.75 mM reduction in Mg concentration (0.25 mM vs. 6.00 mM) correlated to an increase of 0.55 μM gAFDW−1 (grams ash-free dry cell weight) combined flavin concentration after 72 hours of anaerobic growth. The difference in electron shuttle concentration resulted from a 0.95 μM gAFDW−1 increase in FMN concentration and a 0.40 μM gAFDW−1 decrease in riboflavin concentration with the reduced Mg dose. The influence of Mg on electron shuttle production was highly sensitive to the presence of other compounds in the media. The addition of mineral and vitamin supplements to the media eliminated the significant influence of Mg on electron shuttle production rate. S. oneidensis current generation in an MFC was evaluated at different metabolic production rates of electron shuttling compounds. The concentration of Mg was used as the sole variable to influence these production rates. At three tested magnitudes of load resistances, the MFC receiving the low Mg treatment outperformed the high Mg treatment. The low Mg treatment produced 52% higher maximum current at 27800Ω resistance, 74% higher maximum current at 8250Ω, and 151% at 1100Ω. This corresponds to the highest observed current output of 260 ± 72 μA observed by the low Mg treatment at 1100Ω. The superior performance of MFCs containing the low treatment of Mg was observed both in single-chamber MFCs and in MFCs where direct anode contact by the bacteria was precluded. Low Mg concentration in MFCs where direct anode contact was precluded not only increased the maximum current generating potential, but significantly decreased the time necessary to utilize the avaiable substrate. A genome-scale, FBA model was used to evaluate the metabolic capabilities of S. oneidensis. The model, originally calibrated for aerobic conditions, was evaluated under anaerobic growth conditions using lactate and fumarate. The model correctly predicted the acetate secretion rate, however, was off by several factors in the prediction of succinate and the biomass growth rate. The difference between experimental and computer simulations was small enough to conclude qualitative validation of the model. FBA model simulations were used to evaluate the parameters of growth rate, electron shuttle production rate, and anaerobic respiration rate. A trade-off between electron shuttle production and biomass accumulation was predicted to be -0.106 gAFDW mM−1 for riboflavin production and -0.111 gAFDW mM−1 for FMN production. Similarly, a decrease in anaerobic respiration rate was predicted for rate increases to flavin compound synthesis at a ratio of -3.185 mM e− mM−1 and -3.103 mM e− mM−1 for riboflavin and FMN, respectively. A comparison between extreme cases of FMN production indicated an increased demand for phosphorous and nitrogen under conditions of high FMN synthesis. Review of central carbon metabolism under these conditions demonstrates that this increased demand is due to the increased flow of material through the pentose phosphate pathway. The model was used to screen single reaction knockouts for potentially useful genetic modifications. Removal of acetate kinase resulted in an increase of the anaerobic respiration rate, however, this mutation has been previously identified as a fatal knockout. The knockout of cytochrome-c oxidase also indicated an increase to the anaerobic respiration rate and has previously been identified as a mutant with superior current generating performance in an MFC as compared to the wildtype strain. Several other potentially beneficial knockouts are identified, including non-intuitive knockouts such as homoserine kinase. The results of this study demonstrate that microbial metabolism significantly influences the performance of MFCs not only though respiration rates, but through the rate of electron shuttle production. Modification of electron shuttle production rates was successful by changing the availability of Mg to S. oneidensis, however, FBA simulations suggest that higher production rates are still possible. The results of this study provide a practical framework for the targeting of S. oneidensis flavin metabolism as a means of enhancing MFC current densities. c ©Copyright by Robert G. Hall August 3, 2011 All Rights Reserved Metabolic Engineering of Shewanella oneidensis MR-1 for Microbial Fuel Cell Application