Berlin, Techn. Univ., Diss., 2001. / Computerdatei im Fernzugriff.

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Acidithiobacillus ferrooxidans is a gram-negative bacterium that is able to extract energy from oxidation of Fe²⁺ 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 Fe²⁺ 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 Fe²⁺ at pH 1.8. The Fe²⁺ concentration and pH were varied in the experiments to assess their impacts on growth rate, cell yield (g cells/g Fe²⁺) 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 Fe²⁺) 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 Fe³⁺ and 70 mM citrate at pH 2.2 was explored during the electrochemical reduction of Fe³⁺. It turned out that electrochemical reduction of Fe³+ 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 Fe³⁺. The conductivity was improved by adding 500 mM Mg²⁺ to the medium. Vanadium was used as an alternative redox mediator that has a much better solubility than Fe³⁺ 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 Fe²⁺ concentration and addition of citrate. Further optimization of the growth medium was done by increasing Fe²⁺ 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.

Laboratory experiments were carried out to evaluate the effectiveness of antibacterial substances and antibiotics against Thiobacillus ferrooxidans, the organisms responsible for bacterial mediated acidic mine drainage. Twenty two antibiotics (obtained from Lilly and Co.) and two antibacterial substances were added to: bacterial culture ATCC 19859 grown in 9K medium. Appropriate controls were maintained. Inhibition of iron oxidizing bacteria was recorded in terms of changes in Eh of the medium treated with the compound. Seven antibiotics (A38533A:, A38533B, 197506, 13780, 171541, chloramphenicol and cephalexin) and the two antibacterial substances [N-serve(nitrapyrin) and Dicyandiamide] effectively inhibited the oxidation of Fe²⁻ ions in the medium. The kinetics of Fe²⁻ oxidation with the addition of antibiotics and the antibacterial substances was studied. N-serve [2-chloro-6-(trichloromethyl) pyridine], used as a nitrification inhibitor in agriculture, was highly effective at concentrations greater than 0.1 ml/l. Iron oxidation levels were reduced to levels close to that in uninoculated controls (abiotic oxidation). The use of N-serve to inhibit acid mine drainage (AMD) causing bacteria seems to be both economical and environmentally safe. / Master of Science

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Orientador: Laura Maria Mariscal Ottoboni / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-07-23T19:15:17Z (GMT). No. of bitstreams: 1 Novo_MariaTeresaMarques_D.pdf: 4388960 bytes, checksum: 4e9cc7ee25e2a967d6cd5387992b880d (MD5) Previous issue date: 1998 / Doutorado / Genetica de Microorganismos / Doutor em Ciências Biológicas

Biomining technologies have been developed to use acidophilic microorganisms and the reactions that they catalyze to extract metals from ores in the mining industry. This biological processing through hydrometallurgy is responsible for the production of a significant portion of the world’s copper and gold supplies. Acidithiobacillus ferrooxidans is one of the better-studied and important chemolithotrophic bacterial species that is a part of the natural consortia found in mines across the world. This acidophile is unique in the array of redox reactions it participates in as it is capable of oxidizing both iron and reduced inorganic sulfur species, enabling dissolution of metal from minerals. As the transition to renewable energy continues and the demand for electronic devices grows, more copper and other valuable metals will need to be extracted from increasingly low-grade ores, such as chalcopyrite. Additionally, there has been a growing interest in further developing this biotechnology for the leaching and the recovery of valuable metals from scrap alloys and electronic waste as these feedstock streams can contain rare metals at concentrations above those found in the earth. However, the challenge in deploying biomining to these applications involves understanding the interactions that can potentially inhibit the extraction of these metals. In this dissertation, we expanded the genetic toolbox for A. ferrooxidans by using a transposition technique for the chromosomal integration of exogenous genes. The ability to permanently modify the genome enables engineering of strains that can be used in industry without the need of maintaining selective pressure for plasmid-based expression. Next, we investigated the potential role of A. ferrooxidans in microbially influenced corrosion. We focused on finding conditions that would enable the corrosion of stainless steel, which is resistant to the medium typically used for the growth of the bacterium. Additionally, the further optimization of the corrosive environment and the introduction of genetically engineered cells led to additional corrosion of a higher-grade stainless steel. Then, we explored how altering the bioavailability of sulfur in different formulations could shift the population phenotypes in A. ferrooxidans. We found that a unified description with a few parameters could describe the wide range of behaviors observed in the presence of iron and sulfur. Thus, using this improved understanding of A. ferrooxidans, we are able to engineer phenotypes of interest to generate robust strains that can modulate leaching conditions.