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.

No description available.

In recent years there has been growing interest in bio-mineral processing due to its low operating costs and its application in processing lean-grade ores. Bioprocessing is a good alternative to conventional hydrometallurgy process in mineral processing. In recent times microorganisms have been used as surface modifiers in processes such as froth flotation and flocculation. The surface properties of microbes and minerals such as zeta potential and surface hydrophobicity play a major role in determining adhesion of microorganisms to minerals and hence, the efficiency of flocculation and flotation. These properties also depend on solution conditions such as pH and ionic strength. Adhesion of microorganisms to mineral surfaces can alter the surface properties of the minerals. Such surface modification imparting hydrophobicity or hydrophilicity is used in flocculation and flotation of fine particles. In this research work the effect of ionic strength and pH in deteraiining the surface properties and hence adhesion of the bacterium Bacillus polymyxa to minerals such as hematite, quartz and coal has been studied in detail. The effect of the ionic strength and pH on the electrokinetics of the minerals and bacteria and its subsequent effect on adhesion and flocculation were investigated in detail. Contact angle measurements along with the zeta potential results were used to calculate the interaction energies between the mineral and the microorganism to establish a mechanism for the interaction. The following major conclusions can be drawn from this study. Results indicate that increase in the ionic strength significantly changes the zeta potential of hematite and bacteria without varying the isoelectric point. Increase in the ionic strength caused very little change in the zeta potential of quartz and coal. The adhesion of bacterial cells on to the minerals was found to be dependent on pH, ionic strength and conditioning time. Adhesion of bacterial cells was found to be more on hematite and coal when compared to quartz. The adsorption isotherms of Bacillus polymyxa cells with respect to all the three minerals were found to obey Langmuir isotherm. Flocculation studies demonstrated that the settling rate of hematite and coal was enhanced in presence of bacterial cells and electrolyte. However quartz settled much slower under the same conditions indicating that the quartz particles are being dispersed. Thus, selective flocculation of hematite and coal is possible which can be used in separating them from quartz effectively. The different components of total interaction energy arising from Lifshitz-van der Waal forces, acid/base forces and electrostatic forces were calculated using the van Oss approach. Calculation of the components of the acid base free energy showed that coal and hematite were hydrophobic compared to quartz and the bacterium. From total interaction energy calculation based on the extended DLVO theory, hematite and coal were found to have a net negative interaction energy in acidic pH values and hence attractive forces are predominant. Quartz was found to have a net repulsive energy at all the pH values at low ionic strengths but increase in ionic strength the interaction energy become attractive. The AGLW values of quartz was found to be attractive which is probably responsible for bacterial adhesion onto quartz.

Tez (Yüksek Lisans) - Süleyman Demirel Üniversitesi, Fen Bilimleri Enstitüsü, Tekstil Mühendisliği Anabilim Dalı, 2008. / Kaynakça var.

Resumo: A lixiviação bacteriana, ou biolixiviação é um processo biotecnológico que se fundamenta na utilização de microorganismos capazes de solubilizar metais pela oxidação de sulfetos metálicos, sendo nos dias atuais, uma das mais importantes alternativas para a extração de metais, sobretudo do ponto de vista ambiental e econômico. Uma das principais espécies utilizada neste processo é o Acidithiobacillus ferrooxidans, uma bactéria aeróbia, mesofílica e acidofílica, que obtém energia pela oxidação de substratos inorgânicos, basicamente o íon ferroso e compostos reduzidos de enxofre. Todavia, a interação dessa espécie com os sulfetos metálicos é um assunto ainda pouco entendido e de muita controvérsia na literatura. Com intuito de melhor entender estas diferenças, o presente trabalho estudou a oxidação da molibdenita (MoS2) e da covelita (CuS) pelo A. ferrooxidans linhagem LR em algumas condições fisiológicas, destacando-se a fonte energética de crescimento (íon ferroso e S0) e a remoção das substâncias exopoliméricas (EPS) para células crescidas em íon ferroso. A cinética de oxidação destes sulfetos também foi avaliada. Tais estudos foram realizados pela técnica de respirometria celular, que permite avaliar rapidamente a oxidação do substrato a partir de medidas de oxigênio consumido pela bactéria. Em todas as condições testadas a covelita apresentou significativa diferença de oxidação pelo A. ferroxidans LR em comparação com a molibdenita. A análise da cinética de oxidação dos sulfetos demonstrou que a molibdenita apresenta uma cinética que segue Michaelis-Menten, o mesmo não acontecendo para a covelita, provavelmente devido a forma com que estes sulfetos reagem ao ataque químico-bacteriano, fato determinado pelas estruturas eletrônicas dos sulfetos minerais. / Abstract: Bacterial leaching or bioleaching is a biotechnological process that applies microorganisms able to solubilize metals by metallic sulfides oxidation. This process is nowadays one of the most important alternatives for recovering metals, mainly by environmental and economic aspects. One of the most important bacteria employed in this process is Acidithiobacillus ferrooxidans. It is a gram-negative, acidophilic, aerobic and chemoautotrophic bacteria that obtain energy by the oxidation of inorganic substrates like ferrous ion and reduced sulfur compounds, including metal sulfides. Nevertheless, the interaction of this specie with metallic sulfides remains unclear. With the aim to understand these interactions, the present work has studied the covellite (CuS) and molydenite (MoS2) oxidation by A. ferrooxidans strain LR under different physiological conditions such as the source energy for growth (S0 and ferrous ion) and the removal of extracellular polymeric substances (EPS). These studies were performed by respirometric technique tha t allow evaluating very quickly the substrate oxidation by oxygen uptake measures. For all essays realized it was observed that the efficiency of covellite oxidation by A. ferrooxidans LR is much better than molybdenite. On the kinetic oxidation analyses, molybdenite revealed to be according to Michaelis-Menten substrate saturate kinetic. On the other hand, covellite was not in agreement with Michalis-Menten kinetic. This finding is probably associated with the pathway which these minerals sulfide react to chemistry-bacterial attack, what is influenced by electronic structures of mineral sulfides. Regarding essays performed with cells of A. ferrooxidans strain LR grown with different substrates (ferrous ion and sulfur) and to essays which EPS of bacterial cells were removed, the results obtained did not show differences in covellite oxidation. / Orientador: Oswaldo Garcia Júnior / Coorientador: Denise Bevilaqua / Banca: Assis Vicente Benedetti / Banca: Fernanda de Castro Reis / Mestre

No description available.

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.

Although bacterial leaching of sulphidic minerals is a well-known phenomenon, it is only in the last ten years that full-scale bacterial leaching plants have been commissioned for gold processing. In order for bacterial leaching to compete successfully with other pretreatment processes for refractory ores, particularly with established technologies such as roasting and pressure leaching, it needs to be efficient. This requires the optimization of the parameters affecting the leaching reaction and the growth of bacteria. The entire biotreatment process is agitation leaching, carried out in stirred reactors or Pachuca type reactors. The bacterial oxidation is a complex reaction involving gaseous, liquid and solid phases. The interactions are highly complex, and analysis is complicated by the presence of solids in the leaching medium. Inspite of the amount of research that has been performed, kinetic and process models are underdeveloped. Since kinetic data varies widely with the type and source of concentrate, experimental data should be generated before doing the full-scale reactor design. In sizing reactors for a commercial scale process, it would be useful to have a mathematical model that one could use to predict the amount and rate of release of metal, as a function of the various operating parameters of the system. G.R.Halli arsenical gold sulphide concentrate obtained from Hutti Gold Mines Ltd., Karnataka, was chosen for our study, because of its high refractoriness. An indegenous strain of Thiobacillus ferrooxidans was used for biooxidation. The experiments were conducted in a well-agitated stirred tank reactor under controlled conditions. Sparged air was supplemented with carbon-dioxide for optimized growth. In this work, more than 90% gold and 95% silver could be recovered from the sulphidic gold concentrate when bioleaching was used ahead of cyanidation, compared to 40% and 50% by direct cyanidation. A generalized model, which accounts for both direct bacterial attack and indirect chemical leaching, has been proposed for the biooxidation of refractory gold concentrates. The bacterial balance, therefore, accounts for its growth both on solid substrate and in solution, and for the attachment to and detachment from the surface. The overall process is considered to consist of several sub-processes, each of which can be described in terms of a mechanism and related rate expressions. These sub-processes were studied seperately under kinetically controlled conditions. The key parameters appearing in the rate equations were evaluated using the experimental data. Since the refractory concentrate contains pyrite and arsenopyrite as the major leachable entities, leaching studies have been done on pure pyrite and arsenopyrite as test minerals and the key parameters in the rate equations are evaluated using this data. The model so developed is tested with the leaching kinetics of the concentrate. The growth of bacteria is dependent on the availability of the substrate, ferrous iron, and the dependence is modelled by the widely accepted Monod equation. The effect of carbon dioxide supplementation on the bacterial activity was studied and the optimal concentration for growth was found to be l%(v/v). Studies on indirect chemical leaching showed that the rate is sensitive to surface area of concentrate. Indirect rate constant of arsenopyrite was found to be greater than that of pyrite, since pyrite is more nobler than arsenopyrite. Conditions of direct leaching alone was obtained at high pulp density and using substrate adapted bacteria. The rate constant of arsenopyrite was found to be greater than that of pyrite. The parameters obtained were tested with the overall batch leaching data of the concentrate and favourable comparision was obtained. Thus, it has been possible to isolate the various simultaneous sub-processes occurring during the leaching and propose useful models to describe these processes in some detail. The model has been extended successfully to predict the continuous leaching behaviour using the parameters obtained from the batch data. Studies on the effect of residence time and pulp density on steady state behaviour showed that there is a critical residence time and pulp density below which washout conditions occur. The critical residence time at 10% pulp density was found to be 11 hrs. Operation at pulp densities lower than 5% and residence times lower than 72 hrs is not favourable for efficient leaching. Studies on the effect of initial ferric iron concentration showed that there exists an optimum concentration of ferric iron at which the time required to reach steady state is minimum.

Acid mine drainage (AMD) is a worldwide environmental problem associated with the mining wastes, generated from active and inactive mining sites from mineral processing activities. AMD is defined as the drainage that occurs as a result of oxidation of sulphide minerals/wastes/tailings when exposed to air and water in the presence of chemolithotrophs namely the Acidithiobacillus sp. AMD is characterised by low pH and increased acidity due to elevated heavy metals and sulphate concentration. The acid production potential was carried out for sulphide minerals such as pyrite and chalcopyrite and copper tailings sample in the absence and presence of bacteria namely Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans. Acidity was generated in all the cases due to the oxidation of the mineral samples. The oxidation was found to occur at a higher rate in the presence of the bacteria compared to the control samples. Bioremediation experiments were conducted on acid mine water collected from the Ingaldahl Mines, Chitradurga, Karnataka, India, using organic and inorganic substrates. In the experiments with rice husk, complete removal of metal ions from the acid mine water was achieved with an attendant increase in the pH of the acid mine water from 2.3 to 5.5. About 21% of sulphate could be removed using Dsm. nigrificans from acid mine water pretreated with rice husk at pH 5.5 and this was further increased to 40% by the supplementation of organic components. The rice husk filtrate was found to serve as a good growth medium for Dsm. nigrificans. About 96 % of Fe, 75 % of Zn, 92 % of Cu and 41 % of sulphate removal was achieved from the acid mine water of pH 2.4 with a concomitant increase in the pH value by two units after interaction with the tree bark. About 56 % and 71 % of sulphate reduction could be achieved at initial pH values of 4.1 and 5.5 respectively of the acid mine water pretreated with E. tereticornis (Sm) bark, after inoculation with Dsm. nigrificans. The complete removal of Fe2+ and Fe3+, 80% of Zn, 83% of Cu and 62% of sulphate could be removed from acid mine water using fly ash as the substrate with an increase in pH of acid mine water from 2.3 to 7. About 68% of sulphate reduction at pH 6.8 could be achieved in acid mine water pretreated with fly ash in the presence of Dsm. nigrificans. With red mud as the substrate, complete removal of all the metal ions namely Fe2+, Fe3+, Zn, and Cu from acid mine water was achieved with a concomitant increase in the pH from 2.3 to 8. The sulphate reduction was increased to about 51% at pH 7.2 when the acid mine water pretreated with red mud was inoculated with Dsm. nigrificans. The adsorption experiments carried out on the acid mine water using either organic or inorganic substrates indicated that the free energy of adsorption was negative for all the chosen metal ions attesting to favorable interaction. The adsorption isotherms of the metal ions for rice husk exhibited Langmuirian behaviour, while those for the other substrates adhered to both Langmuir and Freundlich relationships. The adsorption process was found to be endothermic in nature for rice husk, fly ash and red mud. On the contrary, the adsorption onto tree bark showed exothermic behaviour. The adsorption kinetics of the metal ions onto the various substrates adhered to the first order Lagergren equation. The metal uptake processes by the organic and inorganic substrates chosen for this study involve ionic, chemical and physical forces of adsorption. The different types of functional groups present on the surface of the substrates such as carboxyl, hydroxyl and carbonyl, as revealed by FTIR spectroscopic studies, partake in metal binding. The metal ions will also be adsorbed by complexing with the negatively charged reaction sites on the substrate surfaces. Furthermore, the complex solution chemistry of the metals as a function of pH has also to be taken into consideration. The mechanism of sulphate reduction by Dsm. nigrificans in the presence of organic carbon can be illustrated as: 2CH2O + SO42- + 2H+  2CO2 + 2H2O + H2S M2 + H2S  MS  + 2H+ where, CH2O represents the organic matter and M represents the metal ion.

Land application of wastes has become increasingly popular, to promote nutrient recycling and environmental protection, with soil functioning as a partial barrier between wastes and groundwater. Dairy shed effluent (DSE), may contain a wide variety of pathogenic micro-organisms, including bacteria (e.g. Salmonella paratyphyi, Escherichia coli. and Campylobacter), protozoa and viruses. Groundwater pathogen contamination resulting from land-applied DSE is drawing more attention with the intensified development of the dairy farm industry in New Zealand. The purpose of this research was to investigate the fate and transport of bacterial indicator-faecal coliform (FC) from land-applied DSE under different irrigation practices via field lysimeter studies, using two water irrigation methods (flood and sprinkler) with contrasting application rates, through the 2005-2006 irrigation season. It was aimed at better understanding, quantifying and modelling of the processes that govern the removal of microbes in intact soil columns, bridging the gap between previous theoretical research and general farm practices, specifically for Templeton soil. This study involved different approaches (leaching experiments, infiltrometer measurements and a dye infiltration study) to understand the processes of transient water flow and bacterial transport; and to extrapolate the relationships between bacterial transport and soil properties (like soil structure, texture), and soil physical status (soil water potential ψ and volumetric water content θ). Factors controlling FC transport are discussed. A contaminant transport model, HYDRUS-1D, was applied to simulate microbial transport through soil on the basis of measured datasets. This study was carried out at Lincoln University’s Centre for Soil and Environmental Quality (CSEQ) lysimeter site. Six lysimeters were employed in two trials. Each trial involved application of DSE, followed by a water irrigation sequence applied in a flux-controlled method. The soil columns were taken from the site of the new Lincoln University Dairy Farm, Lincoln, Canterbury. The soil type is Templeton fine sandy loam (Udic-Ustochrept, coarse loamy, mixed, mesic). Vertical profiles (at four depths) of θ and ψ were measured during leaching experiments. The leaching experiments directly measured concentrations of chemical tracer (Br⁻ or Cl⁻) and FC in drainage. Results showed that bacteria could readily penetrate through 700 mm deep soil columns, when facilitated by water flow. In the first (summer) trial, FC in leachate as high as 1.4×10⁶ cfu 100 mL⁻¹ (similar to the DSE concentration), was detected in one lysimeter that had a higher clay content in the topsoil, immediately after DSE application, and before any water irrigation. This indicates that DSE flowed through preferential flow paths without significant treatment or reduction in concentrations. The highest post-irrigation concentration was 3.4×10³ cfu 100 mL⁻¹ under flood irrigation. Flood irrigation resulted in more bacteria and Br⁻ leaching than spray irrigation. In both trials (summer and autumn) results showed significant differences between irrigation treatments in lysimeters sharing similar drainage class (moderate or moderately rapid). Leaching bacterial concentration was positively correlated with both θ and ψ, and sometimes drainage rate. Greater bacterial leaching was found in the one lysimeter with rapid whole-column effective hydraulic conductivity, Keff, for both flood and spray treatments. Occasionally, the effect of Keff on water movement and bacterial transport overrode the effect of irrigation. The ‘seasonal condition’ of the soil (including variation in initial water content) also influenced bacterial leaching, with less risk of leaching in autumn than in summer. A tension infiltrometer experiment measured hydraulic conductivity of the lysimeters at zero and 40 mm suction. The results showed in most cases a significant correlation between the proportion of bacteria leached and the flow contribution of the macropores. The higher the Ksat, the greater the amount of drainage and bacterial leaching obtained. This research also found that this technique may exclude the activity of some continuous macropores (e.g., cracks) due to the difference of initial wetness which could substantially change the conductivity and result in more serious bacterial leaching in this Templeton soil. A dye infiltration study showed there was great variability in water flow patterns, and most of the flow reaching deeper than 50 cm resulted from macropores, mainly visible cracks. The transient water flow and transport of tracer (Br⁻) and FC were modelled using the HYDRUS-1D software package. The uniform flow van Genuchten model, and the dual-porosity model were used for water flow and the mobile-immobile (MIM) model was used for tracer and FC transport. The hydraulic and solute parameters were optimized during simulation, on the basis of measured datasets from the leaching experiments. There was evidence supporting the presence of macropores, based on the water flow in the post-DSE application stage. The optimised saturated water content (θs) decreased during the post-application process, which could be explained in terms of macropore flow enhanced by irrigation. Moreover, bacterial simulation showed discrepancies in all cases of uniform flow simulations at the very initial stage, indicating that non-equilibrium processes were dominant during those short periods, and suggesting that there were strong dynamic processes involving structure change and subsequently flow paths. It is recommended that management strategies to reduce FC contamination following application of DSE in these soils must aim to decrease preferential flow by adjusting irrigation schemes. Attention needs to be given to a) decreasing irrigation rates at the beginning of each irrigation; b) increasing the number of irrigations, by reducing at the same time the amount of water applied and the irrigation rate at each irrigation; c) applying spray irrigation rather than flood irrigation.