Engineering and Characterization of Acidithiobacillus ferrooxidans for Biotechnological Applications Xiaozheng Li Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Graduate School of Arts and Sciences COLUMBIA UNIVERSITY 2016 © 2015 Xiaozheng Li All rights reserved ABSTRACT Engineering and Characterization of Acidithiobacillus ferrooxidans for Biotechnology Applications Xiaozheng Li Acidithiobacillus ferrooxidans is a gram-negative bacterium that is able to extract energy from oxidation of Fe 2+ and reduced sulfur compounds and fix carbon dioxide from atmosphere. The facts that A. ferrooxidans thrives in acidic pH (~2), fixes carbon dioxide from the atmosphere and oxidizes Fe 2+ for energy make it a good candidate in many industrial applications such as electrofuels and biomining. Electrofuels is a new type of bioprocess, which aims to store electrical energy, such as solar power, in the form of chemical bonds in the liquid fuels. Unlike traditional biofuels made from agricultural feedstocks, electrofuels bypass the inefficient photosynthesis process and thus have potentially higher photon-to-fuel efficiency than traditional biofuels. This thesis covers the development of a novel bioprocess involving A. ferrooxidans to make electrofuels, i.e. isobutyric acid and heptadecane. There are four major steps: characterization of wild-type cells, engineering of medium for improved electrochemical performance, genetic modification of A. ferrooxidans and optimization of operating conditions to enhance biofuel production. Each is addressed in one of the chapters in this thesis. In addition, applications of A. ferrooxidans in biomining processes will be briefly discussed. An economic analysis of various applications including electrofuels and biomining is also presented. Wild-type A. ferrooxidans were first characterized in both batch and continuous cultures. A modified 9-K medium suggested by American Type Culture Collection (ATCC) was used as a starting point which has 72 mM Fe 2+ at pH 1.8. The Fe 2+ concentration and pH were varied in the experiments to assess their impacts on growth rate, cell yield (g cells/g Fe 2+ ) and maintenance (energy used to keep cell viability). Citrate was added to the growth medium to dissolve precipitates which can be problematic in a continuous operation. It was found out that cells exhibited higher cell yield (g cells/g Fe 2+ ) and lower maintenance with higher pH and addition of citrate. This indicates that cells grow in a more energy-efficient manner at such conditions since cells spend less energy in maintenance and more energy in biomass formation. Next the growth medium containing 72 mM Fe 3+ and 70 mM citrate at pH 2.2 was explored during the electrochemical reduction of Fe 3+ . It turned out that electrochemical reduction of Fe 3+ could not be carried out effectively due to a low electrolyte conductivity and low energy density of the medium. Citrate was also found to negative affect electrochemical performance due to a strong complexation with Fe 3+ . The conductivity was improved by adding 500 mM Mg 2+ to the medium. Vanadium was used as an alternative redox mediator that has a much better solubility than Fe 3+ to improve the energy density. Genetic modification was achieved by introducing genes from two foreign pathways i.e. valine synthesis and fatty acid synthesis into A. ferrooxidans to enable cells to produce either isobutyric acid (IBA) or heptadecane. Transformed cells were characterized based on the findings in wild-type cells. Isobutyric acid production was found to increase with increasing pH and Fe 2+ concentration and addition of citrate. Further optimization of the growth medium was done by increasing Fe 2+ to 288 mM, holding pH at 2.2 and using gluconate as the iron chelator instead of citrate. An economic analysis was performed on the electrofuel process and applications of genetically modified A. ferrooxidans in copper biomining processes. At electricity prices of $0.05/kWh, further improvement in biological efficiency needs to be achieved before the electrofuel process may become economically viable. The use of genetically modified cells in copper biomining process could open new opportunities to co-produce valuable chemicals and copper from the reduced material associated with the copper ores. The chemicals co-produced during copper processing could be sold for additional revenue or used on-site. Table of Contents TABLES………. ...................................................................................................................................... iii FIGURES……… ....................................................................................................................................... v ACKNOWLEDGMENTS ...................................................................................................................... ix CHAPTER 1. INTRODUCTION ...................................................................................................... 1 1.1 BACKGROUND ......................................................................................................................................... 1 1.2 BACTERIA GROWTH .............................................................................................................................. 5 1.3 ELECTROFUELS ....................................................................................................................................... 6 1.4 OXIDATION OF FERROUS IONS .......................................................................................................... 8 1.5 GENETIC MODIFICATION ..................................................................................................................... 9 1.6 COPPER BIOLEACHING ...................................................................................................................... 11 1.7 GOALS .................................................................................................................................................... 12 1.8 TABLES AND FIGURES ....................................................................................................................... 15 CHAPTER 2. CHARACTERIZATION OF WILD-TYPE A. FERROOXIDANS AND GROWTH MEDIUM * ........................................................................................................................... 23 2.1 ABSTRACT ............................................................................................................................................. 23 2.2 INTRODUCTION .................................................................................................................................... 24 2.3 MATERIAL AND METHODS .............................................................................................................. 26 2.4 RESULTS ................................................................................................................................................ 31 2.5 DISCUSSION .......................................................................................................................................... 33 2.6 ACKNOWLEDGEMENT ....................................................................................................................... 40 2.7 TABLES AND FIGURES ....................................................................................................................... 41 2.8 SUPPLEMENTARY INFORMATION ................................................................................................... 50 CHAPTER 3. ENGINEERING ACIDITHIOBACILLUS FERROOXIDANS GROWTH MEDIUM FOR ENHANCED ELECTROCHEMICAL PROCESSING * ............................... 61 3.1 ABSTRACT ............................................................................................................................................. 61 3.2 INTRODUCTION .................................................................................................................................... 61 3.3 MATERIAL AND METHODS .............................................................................................................. 65 3.4 RESULTS ................................................................................................................................................ 67 3.5 DISCUSSION .......................................................................................................................................... 70 3.6 CONCLUSION ........................................................................................................................................ 74 3.7 FIGURES ................................................................................................................................................. 75 CHAPTER 4. ENGINEERING THE IRON-OXIDIZING CHEMOLITHOAUTOTROPH ACIDITHIOBACILLUS FERROOXIDANS FOR BIOCHEMICAL PRODUCTION * ......... 82 4.1 ABSTRACT ............................................................................................................................................. 82 4.2 INTRODUCTION .................................................................................................................................... 82 4.3 MATERIAL AND METHODS .............................................................................................................. 85 4.4 RESULTS AND DISCUSSION .............................................................................................................