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.

Microbial leaching plays a significant role in the natural weathering of silicate containing ores such as diamond-bearing kimberlite. Harnessing microbial leaching processes to pre-treat mined kimberlite ores has been proposed as a means of improving diamond recovery efficiencies. The biomineralization of kimberlite is rarely studied. Therefore, this study investigated the feasibility of exploiting both chemolithotrophic and heterotrophic leaching processes to accelerate the weathering of kimberlite. Preliminary investigations using mixed chemolithotrophic leaching cultures were performed on four finely ground kimberlite samples (<100μm) sourced from different mines in South Africa and Canada. Mixed chemolithotrophic cultures were grown in shake flasks containing kimberlite and inorganic basal media supplemented either with iron (Fe2+, 15g/l) or elemental sulfur (10g/l) as energy sources. Weathering due to dissolution was monitored by Inductive Coupled Plasma (ICP) analyses of Si, Fe, K, Mg and Ca in the leach solutions at known pH. Structural alterations of kimberlite after specified treatment times were analyzed by X-ray Powder Diffraction (XRD). The results of the preliminary investigation showed that weathering can be accelerated in the presence of microbial leaching agents but the degree of susceptibility and mineralogical transformation varied between different kimberlite types with different mineralogical characteristics. In general, the results showed that the kimberlite sample from Victor Mine was most prone to weathering while the sample from Gahcho Kue was the most resistant. It was therefore deduced that kimberlite with swelling clays as their major mineral component weathered relatively more easily when compared to kimberlite that consisted of serpentine and phlogopite as their major minerals. Gypsum precipitates were also distinguished indicating that a partial alteration in the kimberlite mineralogical structure occurred. Both energy sources positively influenced the dissolution process, with sulfur producing superior results. This was attributed to the generation of sulfuric acid which promotes cation dissolution and mineral weathering. Success in the preliminary investigations led to further experimental testing performed to determine the effect of particle size and varying energy source concentrations on the biotransformation of kimberlite. It was observed that although weathering rates of the larger kimberlite particles (>2mm<5mm) were lower than that of the finer particles, slight changes in their mineralogical structures represented by the XRD analyses were seen. Optimisation studies of energy source concentration concluded that although the highest concentration of elemental sulfur (20% w/w) and ferrous iron (35% w/w) produced the most pronounced changes for each energy source tested, the leaching efficiency at these concentrations were not drastically greater than the leaching efficiency of the lower concentrations, as expected. Following the success of batch culture shake flasks weathering tests, the effect of continuous chemolithotrophic cultures on the biotransformation of larger kimberlite particles (>5mm<6.7mm) was investigated. A continuous plug-flow bioleach column was used to model the behaviour of chemolithotrophic consortia in a dump- or heap leaching system. Two sequential columns were setup, in which the first consisted of kimberlite mixed with sulfur and the second purely kimberlite. Inorganic growth medium was pumped to the first column at a fixed dilution rate of 0.25h-1 and the leachate from the first column dripped into the second. After an 8 week investigation period, the ICP and XRD data showed that weathering did occur. However, the pH results showed that the leaching process is governed by the amount of acid produced by the growth-rate independent chemolithotrophic consortia. Data from pH analyses also showed that the leaching bacteria reached ‘steady state’ conditions from day 45 onwards. The pH also remained higher in the second column than in the first column highlighting the alkaline nature of the kimberlite ores and its ability to act as a buffering agent and resist weathering. This important factor, as well as further optimisation studies in process operating conditions and efficiency, needs to be considered when establishing heap-leaching technology for these kimberlite ores. In the preliminary heterotrophic investigation, Aspergillus niger was used to produce organic metabolites to enhance kimberlite mineralization. The results demonstrated that the organic acid metabolites generated caused partial solubilization of the kimberlite minerals. However, it was deduced that for more significant changes to be observed higher amounts of organic acids need to be produced and maintained. The results obtained in this study also showed that the type of kimberlite presents a different susceptibility to the dissolution process and the presence of the fungal cells may improve the leaching efficiency. The results in this study provided an optimistic base for the use of microbial leaching processes in accelerating the weathering of kimberlite. These findings may also serve to supply data to formulate recommendations for further and future column microbial leach tests as well as validation and simulation purposes. / Thesis (M.Sc.) - University of KwaZulu-Natal, Pietermaritzburg, 2008.

Thesis (MSc (Microbiology))--University of Stellenbosch, 2005. / Plasmid pTC-F14 is a 14.2kb promiscuous, broad-host range IncQ-like mobilizable plasmid isolated from Acidithiobacillus caldus f. At. caldus is a member of a consortium of bacteria (along with Acidithiobacillus ferrooxidans and Leptospirilum ferrooxidans) that is used industrially for decomposing metal sulphide ores and concentrates at temperatures of 40ºC or below which is now a well-established industrial process to recover metals from certain copper, uranium and gold-bearing minerals or mineral concentrates. These biomining microbes are usually obligately acidophilic, autotrophic, usually aerobic iron- or sulphur-oxidizing chemolithotrophic bacteria. Their remarkable physiology allows them to inhabit an ecological niche that is largely inorganic and differs from those environments populated by the more commonly studied non-acidophilic heterotrophic bacteria. At. caldus, is a moderately thermophilic (45 to 50ºC), highly acidophilic (pH1.5 to 2.5) sulphur-oxidizing bacterium, and its role as one of the major players in the industrial decomposition of metal sulphide ores has become evident in recent years. At. caldus f from which pTC-F14 was isolated was found to be one of two dominant organisms in a bacterial consortium undergoing pilot-scale testing for the commercial extraction of nickel from ores.

Thesis (MSc (Microbiology))--University of Stellenbosch, 2009. / Biomining organisms are generally found in metal-rich, inorganic environments such as iron and sulfur containing ores; where they play a vital role in mineralization and decomposition of minerals. They are typically obligatory acidophilic, mesophilic or thermophilic, autotrophic, usually aerobic, iron-or sulfur oxidizing chemolithotrophic bacteria. The most prominent biomining organisms used in bioleaching of metal sulfides are Acidithiobacillus ferrooxidans, At. thiooxidans, At. caldus, Sulfobacillus spp. and Leptospirillum spp. Biomining enables us to utilize low grade ores that would not have been utilized by conventional methods of mining. Research has focused on the backbone features of plasmids isolated from bacteria of biomining environments. The aim of this study is to sequence and analyze an 18 kb region of the 66 kb plasmid pTcM1 isolated from At. caldus MNG, focusing on accessory genes carried by this plasmid. Fifteen putative genes / open reading frames were identified with functions relating to metabolism and transport systems. The genes are located in two divergently located operons. The first operon carries features related to general metabolism activities and consists of a transcriptional regulator (ORF 2), a succinate / fumarate dehydrogenase-like subunit (ORF 3), two ferredoxin genes (ORF 4 and ORF 7), a putative HEAT-like repeat (ORF 6) which is interrupted by an insertion sequence (ORF 5) and a GOGAT-like subunit (ORF 8). The second operon contains an ABC-type nitrate / sulfonate bicarbonate-like gene (ORF 9), a binding protein-dependent inner membrane component-like gene, another ABC sulfonate / nitrate-like gene (ORF 12i and 12ii) which is interrupted by an insertion sequence (ORF 13) and two hypothetical proteins with unknown functions (ORF 14 and ORF 15). Southern hybridization analysis have shown that most of the genes from the two operons are found in other At caldus strains #6, “f”, C-SH12 and BC13 from different geographical locations. Expression of the GOGAT-like subunit and the succinate / fumarate-like subunit was demonstrated in At. caldus MNG showing that these genes are functional and actively transcribed. The transcriptional regulator (ORF 2) has been shown to repress the downstream genes of putative operon 1. The persistence of these genes on plasmids together with the fact that they are being expressed, represents a potential metabolic burden, which begs the question why they have been maintained on the plasmid from geographically separated strains (and perhaps also growing under very different nutrient availability conditions) and therefore what possible role they may play.

Thesis (MSc)--University of Stellenbosch, 2006. / ENGLISH ABSTRACT: In this study the chromosomal arsenic resistance (ars) genes shown to be present in all Acidithiobacillus. caldus isolates were cloned and sequenced from At. caldus #6. Ten open reading frames (ORFs) were identified on a clone conferring arsenic resistance, with three homologs to arsenic genes, arsC (arsenate reductase), arsR (regulator) and arsB (arsenite export). This ars operon is divergent, with the arsRC and arsB genes transcribed in opposite directions. Analysis of the putative amino acid sequences of these arsRC and arsB genes revealed that they are the most closely related to the ars genes of Acidithiobacillus ferrooxidans. These ars genes were functional when transformed into an Escherichia coli ars deletion mutant ACSH50Iq, and conferred increased levels of resistance to arsenate and arsenite. ArsC was required for resistance to arsenate, but not for resistance to arsenite. None of the other ORFs enhanced arsenic resistance in E. coli. A transposon located arsenic resistance system (TnAtcArs) has been described for highly arsenic resistant strains of the moderately thermophilic, sulfur-oxidizing, biomining bacterium At .caldus #6. In the latter study it was shown that TnAtcArs confers higher levels of resistance to arsenate and arsenite than the chromosomal operon. TnAtcArs was conjugated into a weakly ars resistant At. caldus strain (C-SH12) and resulted in greatly increased arsenite resistance. RT-PCR analysis revealed that arsR and arsC are co-transcribed. Despite ORF1 (cadmium inducible-like protein) and ORF5 (putative integrase for prophage CP-933R) not being involved in resistance to arsenic, ORF1 was co-transcribed with arsRC and ORF5 with arsB. Using arsR-lacZ and arsB-lacZ fusions it was shown that the chromosomal ArsR-like regulator of At. caldus acts as a repressor of the arsR and arsB promoter expression. Induction of gene expression took place when either arsenate or arsenite was added. The chromosomal located ArsR was also able to repress TnAtcArs, but the transposon-located ArsR was unable to regulate the chromosomal system. / AFRIKAANSE OPSOMMING: In hierdie studie is die chromosomale arseen weerstandbiedendheidsgene (ars gene), teenwoordig in alle Acidithiobacillus caldus isolate, gekloon en die DNA volgorde daarvan vanaf At. caldus #6 bepaal. Tien oopleesrame (ORFs) is geïdentifiseer op ‘n kloon wat arseen weerstandbiedend is, met drie homoloog aan ars gene, nl. arsC (arsenaat reduktase), arsR (reguleerder) en arsB (membraan-geleë pomp wat arseniet uitpomp). Die ars operon is gerangskik met die arsRC en arsB gene wat in teenoorgestelde rigtings getranskribeer word. Analise van die afgeleide aminosuurvolgorde van dié ars gene het getoon hulle is naverwant aan die ars gene van Acidithiobacillus ferrooxidans. Die ars gene was funksioneel na transformasie na ‘n E. coli ars mutant (ACSH50Iq), en het ‘n hoër vlak van weerstand teen arsenaat en arseniet gebied. ArsC was nodig vir weerstand teen arsenaat, maar nie vir weerstand teen arseniet nie. Geen van die ander ORFs het arseen weerstandbiedendheid in E. coli bevorder nie. Voorheen is ‘n ars operon, geleë op ‘n transposon (TnAtcArs), in ‘n hoogs arseen-weerstandbiedende stam van die middelmatige termofiliese, swawel-oksiderende, bio-ontgunning (“biomining”) bakterie Acidithiobacillus caldus #6 beskryf. In laasgenoemde studie is gevind dat TnAtcArs hoër vlakke van weerstand bied teen arsenaat en arseniet as die chromosomale operon. TnAtcArs is na ‘n lae arseen-weerstandbiedende At. caldus stam (C-SH12) gekonjugeer. Die resultaat was ‘n groot verhoging in arseen weerstandbiedendheid. RT-PCR analise het onthul dat arsR en arsC saam getranskribeer word. Benewens die feit dat ORF1 (kadmium induseerbare protein) en ORF5 (afgeleide integrase vir profaag CP-933R) nie betrokke is in weerstand teen arseniet and arsenaat nie, is ORF1 saam met arsRC getranskribeer en ORF5 saam met arsB. Deur gebruik te maak van die fusie-gene arsR-lacZ en arsB-lacZ is bewys dat die chromosomale ArsR reguleerder van At. caldus as ‘n inhibeerder van die arsR en arsB promoter uitdrukking funksioneer. Indusering van geen uitdrukking vind plaas wanneer arseniet of arsenaat bygevoeg word. Die chromosomaal-geleë ArsR is ook in staat om TnAtcArs te inhibeer, terwyl die transposon geleë ArsR nie daartoe in staat is om die chromosomale ars sisteem te reguleer nie.

Thesis (Ph.D. (Microbiology)) -- University of Limpopo, 2022 / NRF and TIA