The application of biotechnologies in the mining sector has intensified over the last 30 years, driven by the increasing demand for metals associated with the rise in energy costs and the awareness for environmentally responsible mining practices. Acidophilic prokaryotes play an important role in biohydrometallurgy, facilitating the solubilisation and recovery of base metals from ores and waste materials. The potential of novel acidophiles of the phylum Firmicutes for applications in biohydrometallurgical processes is examined in this thesis. Eight strains of extremely acidophilic bacteria were studied and shown to belong to the proposed novel genus “Acidibacillus”. These had been isolated previously from several distinct global locations and were shown to be obligately heterotrophic bacteria with potential to carry out tasks critical to biomining such as regenerating ferric iron (by catalysing the dissimilatory oxidation of ferrous iron), generating sulfuric acid (by the oxidation of zero-valent sulfur and tetrathionate; two strains only), and removing potentially inhibitory dissolved organic carbon. These isolates also demonstrated the ability to catalyse the dissimilatory reduction of ferric iron in anaerobic conditions. Results obtained during this study provide the basis for future research to assess their potential roles in microbial consortia applied in the bio-processing of metal ores. A novel obligately anaerobic acidophilic Firmicute (strain I2511) isolated from sediment obtained from an abandoned copper mine, was characterised in terms of its phylogeny and physiology. This isolate formed a separated clade within the Firmicutes, and was considered to represent a novel candidate genus. It also displayed a unique set of physiological traits, distinct from currently validated species of acidophilic Firmicutes. The isolate was an obligate anaerobe that grew via zero-valent sulfur (ZVS) respiration, generating H2S over a wide pH range (1.8 - 5.0), and also catalysed the dissimilatory reduction of ferric iron. Strains of acidophilic sulfatereducing bacteria (aSRB), also Firmicutes, were shown to reduce ZVS at pH as low as 3. These aSRB, together with isolate I2511, populated a novel variant of a low pH sulfidogenic bioreactor. The “hybrid sulfidogenic bioreactor” (HSB) operated using both sulfate and ZVS as electron acceptors, and glycerol as electron donor. The bioreactor successfully remediated and recovered zinc from circum-neutral pH mine-impacted waters with distinct chemical composition collected from two abandoned lead/zinc mines in the U.K. The microbial consortium used in this system proved to be robust, in which the HSB generated H2S consistently under a wide pH range (2 – 7). Experiments demonstrated that H2S could also be generated abiotically in a non-inoculated low pH reactor, by the chemical reaction of ZVS and zero-valent iron to form iron sulfide, and the consequent acid dissolution of the latter. Operational costs and the advantages of biogenic and abiotic generation of H2S for recovery of transition metals from mine waters are discussed.

CoordenaÃÃo de AperfeiÃoamento de NÃvel Superior / O presente estudo teve o objetivo de avaliar o uso de reatores biolÃgicos, sob condiÃÃes anaerÃbias e microaerÃbias, como opÃÃo de biorremediaÃÃo ex situ de Ãguas contaminadas com BTEX. Inicialmente, foi desenvolvido, otimizado e validado um mÃtodo analÃtico para a detecÃÃo e quantificaÃÃo de BTEX em Ãguas e efluentes, o qual consistia em extraÃÃo por headspace seguida de cromatografia gasosa com detecÃÃo por fotoionizaÃÃo. Posteriormente, foram conduzidos experimentos em fluxo contÃnuo em dois reatores anaerÃbios mesofÃlicos (27 ÂC) â um deles operado sob condiÃÃes metanogÃnicas e, em seguida, sob condiÃÃes microaerÃbias, e o outro, apenas sob condiÃÃes sulfetogÃnicas â a fim de verificar a melhor condiÃÃo operacional para a remoÃÃo de BTEX. Os reatores foram alimentados com Ãgua contaminada com BTEX (~3 mgÂL-1 de cada composto) e etanol (co-substrato), e, dependendo da condiÃÃo redox avaliada, investigou-se o efeito de diferentes parÃmetros operacionais, tais como tempo de detenÃÃo hidrÃulica (24, 36 e 48 h), recirculaÃÃo de efluente, concentraÃÃo de co-substrato, relaÃÃo DQO/SO4 2- e microaeraÃÃo, no desempenho de remoÃÃo de BTEX. AlÃm disso, o reator metanogÃnico sob condiÃÃes microaerÃbias foi submetido a simulaÃÃes de choques de carga e de ausÃncia desses compostos, e de falhas operacionais, como desligamento do sistema e desligamento da microaeraÃÃo, para verificaÃÃo de sua robustez. Sob condiÃÃes metanogÃnicas, dependendo do composto, as eficiÃncias de remoÃÃo variaram de 38 a 97%. PorÃm, o aumento da carga aplicada de BTEX, em consequÃncia da reduÃÃo do tempo de detenÃÃo hidrÃulica de 48 para 24 h, parece ter afetado negativamente o processo de remoÃÃo. Ainda sob condiÃÃes metanogÃnicas, tambÃm se verificou o efeito da recirculaÃÃo de efluente na remoÃÃo de BTEX para altas e baixas concentraÃÃes de co-substrato (etanol). Quando altas concentraÃÃes de etanol foram utilizadas, o impacto da recirculaÃÃo de efluente nÃo foi evidente, jà que, provavelmente, a elevada produÃÃo de biogÃs teria sido suficiente para garantir uma transferÃncia de massa efetiva. Sob condiÃÃes sulfetogÃnicas, a adiÃÃo de sulfato em diversas relaÃÃes DQO/SO4 2- nÃo alterou a remoÃÃo de BTEX, sugerindo que as bactÃrias redutoras de sulfato nÃo estariam diretamente relacionadas à ativaÃÃo inicial dos compostos aromÃticos. Sob condiÃÃes microaerÃbias, elevadas eficiÃncias de remoÃÃo de BTEX foram alcanÃadas (> 90%). à provÃvel que a adiÃÃo de baixas concentraÃÃes de oxigÃnio tenha facilitado a ativaÃÃo inicial dos compostos BTEX, a qual à considerada a etapa limitante do processo de degradaÃÃo anaerÃbia, principalmente para o benzeno. Ainda, constatou-se que a presenÃa de altas concentraÃÃes de etanol afetou negativamente a remoÃÃo de BTEX, notadamente para o benzeno, sob as diferentes condiÃÃes redox testadas, jà que à um substrato preferencialmente degradÃvel em relaÃÃo aos compostos aromÃticos. Finalmente, com relaÃÃo à robustez do reator metanogÃnico sob condiÃÃes microaerÃbias, o sistema conseguiu lidar com os choques de carga de BTEX embora choques consecutivos tenham aumentado seu tempo de recuperaÃÃo. O perÃodo de ausÃncia de BTEX parece ter prejudicado a microbiota do reator, pois a qualidade do efluente deteriorou-se consideravelmente apÃs reintroduÃÃo dos compostos. O desligamento da microaeraÃÃo impactou negativamente a remoÃÃo de BTEX, mas o sistema recuperou-se rapidamente apÃs restabelecimento das condiÃÃes microaerÃbias. / The present study aimed to evaluate the use of biological reactors under anaerobic and microaerobic conditions, as an option of ex situ bioremediation of BTEX-contaminated waters. Initially, an analytical method for BTEX detection and quantification in waters and wastewaters, which consisted of headspace extraction followed by gas chromatography with detection by photoionization, was developed, optimized and validated. Subsequently, continuous-flow experiments were conducted in two mesophilic (27 ÂC) anaerobic reactors â one of them operated under methanogenic conditions and, afterwards, under microaerobic conditions, and the other one only under sulfidogenic conditions â a in order to determine the best operational condition for BTEX removal. The reactors were fed with water contaminated with BTEX (~3 mgÂL-1 of each compound) and ethanol (co-substrate), and, depending on the redox condition evaluated, the effect of some operational parameters, such as hydraulic retention time (24, 36 and 48 h), effluent recirculation, co-substrate concentration, DQO/SO4 2- ratio and microaeration, was investigated in BTEX removal performance. Furthermore, the methanogenic reactor under microaerobic conditions was submitted to simulated situations of shock loading and absence of these compounds, and operational failures, such as system and microaeration shutdown to assess its robustness. Under methanogenic conditions, depending on the compound, removal efficiencies ranged from 38 to 97%. However, the increase of applied BTEX load, as a consequence of hydraulic detention time reduction from 48 to 24 h, seems to have adversely affected the removal process. Moreover, under methanogenic conditions, the effluent recirculation effect on BTEX removal was also assessed when high and low co-substrate (ethanol) concentrations were applied. For high ethanol concentrations, the impact of effluent recirculation was not evident since, probably, the high biogas production would have been sufficient to ensure effective mass transfer. Under sulfidogenic conditions, sulfate addition at different DQO/SO4 2- ratios did not change BTEX removal, which suggests sulfate-reducing bacteria would not be directly related to initial activation of aromatic compounds. Under microaerobic conditions, high BTEX removal efficiencies were achieved (> 90%). It is likely the addition of low oxygen concentrations has facilitated the initial activation of BTEX compounds, which is considered the limiting step of the anaerobic degradation process, mainly for benzene. Furthermore, the presence of high ethanol concentrations negatively affected BTEX removal, particularly for benzene, under the different redox conditions tested, since it is a preferentially degradable substrate when compared to the aromatic compounds. Finally, regarding the methanogenic reactor robustness under microaerobic conditions, the system could cope with BTEX load shocks although consecutive shocks have increased its recovery time. The period of BTEX absence seems to have negatively affected the reactor microbiota because the effluent quality deteriorated considerably after compounds reintroduction. The microaeration shutdown also negatively impacted the removal of BTEX, but the system recovered quickly after microaerobic conditions reestablishment.

Neste trabalho foi realizada a caracterização microbiana anaeróbia de reatores anaeróbios diferenciais horizontais e em batelada, operados sob condições sulfetogênicas e mesofílicas (30ºC). Os reatores diferenciais foram preenchidos com diferentes materiais suportes (espuma de poliuretano, carvão vegetal, polietileno reciclado de baixa densidade e cerâmica porosa à base de alumina) visando a seleção do suporte adequado para otimização do processo sulfetogênico, para a relação DQO/sulfato de aproximadamente 0,67. Os reatores diferenciais foram alimentados diariamente com esgoto sintético, contendo aproximadamente 1000 mg/L de DQO e 1500 mg/L de sulfato, durante 28 dias de operação. A caracterização microbiana foi realizada através da técnica de hibridação in situ fluorescente (FISH), microscopia óptica e eletrônica de varredura. Foram realizadas quantificações, em termos de porcentagens, de microrganismos pertencentes ao Domínio Bacteria (EUB338), Domínio Archaea (ARC915) e bactérias redutoras do íon sulfato (BRS) da subdivisão delta de Proteobacteria (SRB385). Nos reatores diferenciais, houve predomínio de bactérias em todos os suportes estudados. Os reatores diferenciais operados com espuma e carvão apresentaram maiores porcentagens de BRS, com valores iguais a 57,6% e 69,7%, respectivamente. A cerâmica foi o material que apresentou melhor equilíbrio de bactérias e arqueas metanogênicas, com 59,6% e 40,9%, respectivamente. Os reatores em batelada foram operados com espuma de poliuretano e carvão vegetal com relação DQO/sulfato de aproximadamente 3. As porcentagens de BRS quantificadas pelo FISH foram iguais a 65,3% e 69,1% para espuma e carvão, respectivamente. Em ambos os reatores o carvão vegetal foi o material mais favorável à sulfetogênese. / This research reports an anaerobic microbial characterization of both, a horizontal differential anaerobic and a batch reactors, operated at sulfidogenic and mesofilic conditions at 30ºC. The differential reactors were filled with four support materials (polyurethane foam, vegetable coal, recycled polyethylene of low density and alumin based porous ceramic) aiming the selection of a more appropriated support for optimization of sulfidogenic processes (ratio COD/sulfate of approximately 0.67). Differential reactors were fed daily with synthetic sewage, containing approximately 1000 mg/L of COD and 1500 mg/L of sulfate concentrations, during 28 days of operation. Microbial characterization was accomplished using fluorescent in situ hybridization (FISH), optic and scanning electronic microscopy. It was realized a quantification, in percentages, of microorganisms belong to Bacteria Domain (EUB338), Archaea Domain (ARC915) and sulfate-reducing bacteria (SRB) of delta subdivision Proteobacteria (SRB385). Differential reactors have shown predominance of bacteria in all the support materials studied. Differential reactors operated with foam and coal presented the greatest percentages of SRB, with values equal to 57.6% and 69.7%, respectively. The ceramic was the material that presented the best equilibrium of bacteria and methanogenic archaea, with 59.6% and 40.9%, respectively. Batch reactors were operated with polyurethane foam and vegetable coal with COD/sulfate ratio of approximately 3. Percentages of SRB quantified by FISH were equals to 65.3% and 69.1% for foam and coal, respectively. In both reactors the vegetable coal have shown to be the most favorable material to sulfidogenesis.

Neste trabalho foi realizada a caracterização microbiana anaeróbia de reatores anaeróbios diferenciais horizontais e em batelada, operados sob condições sulfetogênicas e mesofílicas (30ºC). Os reatores diferenciais foram preenchidos com diferentes materiais suportes (espuma de poliuretano, carvão vegetal, polietileno reciclado de baixa densidade e cerâmica porosa à base de alumina) visando a seleção do suporte adequado para otimização do processo sulfetogênico, para a relação DQO/sulfato de aproximadamente 0,67. Os reatores diferenciais foram alimentados diariamente com esgoto sintético, contendo aproximadamente 1000 mg/L de DQO e 1500 mg/L de sulfato, durante 28 dias de operação. A caracterização microbiana foi realizada através da técnica de hibridação in situ fluorescente (FISH), microscopia óptica e eletrônica de varredura. Foram realizadas quantificações, em termos de porcentagens, de microrganismos pertencentes ao Domínio Bacteria (EUB338), Domínio Archaea (ARC915) e bactérias redutoras do íon sulfato (BRS) da subdivisão delta de Proteobacteria (SRB385). Nos reatores diferenciais, houve predomínio de bactérias em todos os suportes estudados. Os reatores diferenciais operados com espuma e carvão apresentaram maiores porcentagens de BRS, com valores iguais a 57,6% e 69,7%, respectivamente. A cerâmica foi o material que apresentou melhor equilíbrio de bactérias e arqueas metanogênicas, com 59,6% e 40,9%, respectivamente. Os reatores em batelada foram operados com espuma de poliuretano e carvão vegetal com relação DQO/sulfato de aproximadamente 3. As porcentagens de BRS quantificadas pelo FISH foram iguais a 65,3% e 69,1% para espuma e carvão, respectivamente. Em ambos os reatores o carvão vegetal foi o material mais favorável à sulfetogênese. / This research reports an anaerobic microbial characterization of both, a horizontal differential anaerobic and a batch reactors, operated at sulfidogenic and mesofilic conditions at 30ºC. The differential reactors were filled with four support materials (polyurethane foam, vegetable coal, recycled polyethylene of low density and alumin based porous ceramic) aiming the selection of a more appropriated support for optimization of sulfidogenic processes (ratio COD/sulfate of approximately 0.67). Differential reactors were fed daily with synthetic sewage, containing approximately 1000 mg/L of COD and 1500 mg/L of sulfate concentrations, during 28 days of operation. Microbial characterization was accomplished using fluorescent in situ hybridization (FISH), optic and scanning electronic microscopy. It was realized a quantification, in percentages, of microorganisms belong to Bacteria Domain (EUB338), Archaea Domain (ARC915) and sulfate-reducing bacteria (SRB) of delta subdivision Proteobacteria (SRB385). Differential reactors have shown predominance of bacteria in all the support materials studied. Differential reactors operated with foam and coal presented the greatest percentages of SRB, with values equal to 57.6% and 69.7%, respectively. The ceramic was the material that presented the best equilibrium of bacteria and methanogenic archaea, with 59.6% and 40.9%, respectively. Batch reactors were operated with polyurethane foam and vegetable coal with COD/sulfate ratio of approximately 3. Percentages of SRB quantified by FISH were equals to 65.3% and 69.1% for foam and coal, respectively. In both reactors the vegetable coal have shown to be the most favorable material to sulfidogenesis.

The oil production process is an activity that generates significant negative environmental impacts. The production water or produced water (PW) is the wastewater generated during the extraction of oil and stands out due to its high level of salinity and toxicity. Due to the large volume produced and their complex composition, the treatment of this waste presents a major challenge that requires the urgent need for accessible and efficient treatment techniques development and studies according to the economic reality of each region. In order to facilitate a biological treatment of the residue, it was diluted with Synthetic wastewater with a high sulfate concentration to stimulate anaerobic digestion via sulfidogenesis, since Sulphate Reducing Bacteria (SRB) have the characteristic of being resistant to saline environments. Therefore, an anaerobic reactor was used with suspended biomass, operated in sequential batch with cycles of 24 hours in laboratory scale. The reactor was operated with increasing proportions of PW (0%, 2% and 5%), with average chloride concentrations, respectively, 150 mg.L-1, 2.000 mg.L-1 and 4.800 mg.L-1 and COD/SO42- ratios ranging from 0.7 to 2.4. In the stimulation of sulfidogenesis phase the average COD removal efficiency was reduced by 72% (step without sulfate) to 51%, even without adding PW (COD/SO42- = 2.4 and chloride concentration of 150 mg.L- 1). When the system was fed with 2% of PW (COD / SO42- = 0.7 and chloride concentration 2,000 mg.L-1), the COD efficiency remained at 50% and only 15% of this was removed via sulfidogenesis. The proportion of 2% PW was maintained and the ratio COD/SO42- was raised to 1.5 and the average COD removal increased to 55%. By increasing the ratio of Apr to 5% (COD / SO42- = 1.8 and chloride concentration 4.800 mg.L-1) COD removal efficiency was 47%, and 68% of this was removed via sulfidogenesis. These results indicate the viability of using sulfate as an electron acceptor in the treatment of PW, given the occurrence of increase in the reduction of sulphate when the salinity was high, indicating that the effect of salinity stimulated the removal of COD via sulfidogenesis and inhibited via methanogenic. / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / O processo de produção de petróleo é uma atividade geradora de significativos impactos ambientais negativos. A água de produção ou água produzida (APr) é o efluente gerado durante a extração do petróleo e destaca-se por apresentar alto teor de salinidade e toxicidade. Devido ao elevado volume produzido e à sua complexa composição, o tratamento desse resíduo apresenta-se como um grande desafio que requer a urgente necessidade do estudo e do desenvolvimento de técnicas de tratamento eficientes e acessíveis à realidade econômica de cada região. No intuito de viabilizar o tratamento biológico da APr, ela foi diluída em água residuária sintética com elevada concentração de sulfato, visando estimular a digestão anaeróbia via sulfetogênese, visto que as Bactérias Redutoras de Sulfato (BRS) possuem a característica de serem resistentes a ambientes salinos. Para tanto, foi utilizado um reator anaeróbio com biomassa suspensa, operado em bateladas sequenciais com ciclos de 24 horas, em escala laboratorial. O reator foi operado com proporções crescentes de APr (0%, 2% e 5%), apresentando concentrações médias de cloretos de, respectivamente, 150 mg.L-1, 2.000 mg.L-1 e 4.800 mg.L-1 e razões DQO/SO42- variando de 0,7 a 2,4. Na fase de estímulo à sulfetogênese, a eficiência de remoção média de DQO foi reduzida de 72% (fase sem sulfato) para 51%, mesmo sem adição de APr (DQO/SO42- = 2,4 e concentração de cloretos de 150 mg.L-1). Quando o sistema foi alimentado com 2% de APr (DQO/SO42- = 0,7 e concentração de cloretos de 2.000 mg.L-1), a eficiência de DQO manteve-se em 50%, sendo que apenas 15% dessa era removida via sulfetogênese. Foi mantida a proporção de 2% de APr e a razão DQO/SO42- foi elevada para 1,5 e a remoção média de DQO aumentou para 55%. Ao elevar a proporção de APr para 5% (DQO/SO42- = 1,8 e concentração de cloretos de 4.800 mg.L-1), a eficiência média de remoção de DQO foi de 47%, sendo que 68% dessa foi removida via sulfetogênese. Esses resultados indicam a viabilidade do uso do sulfato como aceptor de elétrons no tratamento da APr, tendo em vista a ocorrência do aumento da redução de sulfato quando a salinidade foi elevada, demonstrando que o efeito da salinidade estimulou a remoção de DQO via sulfetogênese e inibiu a via metanogênica.