La presente investigación tuvo como objetivo el aislamiento y determinación de las bacterias del género Acidithiobacillus y Leptospirillum presentes en las aguas residuales de las unidades mineras de Recuay – Huaraz. Con este fin se recolectaron 3 muestras del efluente y se llevaron al laboratorio donde fueron sometidas al enriquecimiento en medios de cultivo 9K y TK líquidos, con un pH de 1.5 y se realizó un conteo poblacional cada 24 horas haciendo uso de la cámara de Newbauber. Se obtuvó la mayor concentración bacteriana en el tratamiento 1 con 9.33E+06 cel/ml durante la etapa de enriquecimiento. Por otro lado, para el aislamiento del cultivo se utilizó medio 9K líquido y sólido; el primero fue incubado a temperatura ambiente por 8 días. Transcurrido este tiempo se sembró en placas Petri, por estría y por extensión. En medio sólido 9K con agar-agar se observó crecimiento bacteriano después de 14 a 25 días, en el 50% de placas sembradas por extensión y el 10% por estría. Mientras que en el medio cultivo 9K con agarosa se observó crecimiento después de 7 a 8 días en el 80% y 60% de placas por extensión y estría, respectivamente. Los resultados de la observación microscópica mostraron presencia de bacterias con morfología bacilar, carácter acidófilo por su desarrollo en medios con pH 1.5 y metabolismo quimiolitiotrofo por su crecimiento y mantenimiento durante el tiempo del ensayo a expensas de compuestos inorgánicos presentes en el medio. En el medio sólido con crecimiento bacteriano después de 45 días de incubación se logró diferenciar 3 tipos de colonias que coinciden con el género de Acidithiobacillus . Además se obtuvieron indicios de las biotransformaciones realizadas por las bacterias lixiviadoras, oxidando el sulfato ferroso a sulfato férrico durante la determinación de bioxidación, lo cual se evidenció mediante el cambio de color de verde traslúcido a naranja brillante.The present investigation had as objective the isolation and determination of the species of the genus Acidithiobacillus and Leptospirillum present in the waste water of the mining units of Recuay - Huaraz. To this end, 3 samples of the effluent were collected and taken to the laboratory where they were submitted to enrichment in 9K and T & K liquid culture media, with a pH of 1.5 and a population count was done every 24 hours using the Newbauber chamber. The results showed that the best enrichment was carried out with the medium 9k in agitation 150 rpm, extra aeration and heat 40 ° C, as the bacterial growth curve occurred in ascending and sustained form, being at 432 hours where Observed the highest bacterial concentration, 9.33E + 06 cells / ml. On the other hand, liquid and solid 9K medium were used for the isolation of the culture; The first was incubated at room temperature for 8 days. After this time was planted in Petri plates, by stria and by extension. In solid 9K medium with agar-agar bacterial growth was observed after 14 to 25 days, in 50% of plates seeded by extension and 10% by streak. While in the 9K medium agarose culture growth was observed after 7 to 8 days in 80% and 60% plates by extension and stria, respectively. The results of the microscopic observation showed the presence of bacteria with bacillary morphology, acidophilic character due to their development in media with pH 1.5 and chemiolithotroph metabolism due to their growth and maintenance during the time of the test at the expense of inorganic compounds present in the medium. In solid medium with bacterial growth after 45 days of incubation it was possible to differentiate 3 types of colonies. In addition, indications were obtained of the biotransformations performed by the leaching bacteria, oxidizing the ferrous sulfate to ferric sulfate during the determination of biooxidation. Which was evidenced by the change of color from translucent green to bright orange.

Experiments with microbial biofilms in microgravity and simulated microgravity have revealed altered growth kinetics, but geomicrobial biofilms have not yet been studied in low gravity environments. No characterisation of biofilms, geomicrobial or otherwise, have been conducted at hypergravity. This thesis explores factors affecting microbe-mineral interactions under terrestrial conditions, lays the groundwork for a scheduled microgravity experiment, and provides the first data on biofilms grown at hypergravity. As a first step in understanding microbe-mineral interactions in altered gravity environments, experiments were undertaken to identify factors that constrain attachment in a terrestrial environment. The model organism Sphingomonas desiccabilis and basaltic rock from Iceland were selected, and the minerals that make up the basalt were identified and procured in their pure form. The relative significance of physical factors such as hydrophobicity, surface charge, porosity and nutritional value were examined in relationship to the success with which biofilms colonised the mineral surfaces. Growth was measured by the quantity of biofilm biomass after a ifxed time period, using Crystal Violet stain, in order to draw conclusions about the most influential physical conditions on biofilm attachment to a substrate. It was found that mineral attachment is influenced more by porosity and nutritional value than by hydrophobicity or surface charge. To explore how reduced gravity affects biofilm formation and weathering rates, a European Space Agency experiment, BioRock, is underway. Samples of basalt, with monocultures of three different organisms, will be sent to the International Space Station in 2019 for long-term exposure to Martian and micro-gravity. Research testing proof of concepts, material compatibility, and experimental procedure and equipment is described. Confocal laser scanning microscopy (CLSM) was used to image the biofilms, and inductively coupled plasma mass spectroscopy (ICP-MS) experiments were conducted to compare biotic and abiotic elemental release rates from basalt. Both of these methods will be employed for post-flight analysis of BioRock. Preliminary terrestrial ICP-MS experiments indicated that rare Earth elements (REEs) showed the most reliable reflection of leaching patterns overall, as a consequence of their high molecular weight and low volatility during the ashing procedure. To fully understand gravity's effect on microbiological processes it is important to investigate what occurs when its influences are removed, but also to establish what occurs when extra gravitational force is applied. Using simulated hypergravity, achieved through hyper-acceleration on a geotechnical centrifuge, the effects of 10 x g on biofilm development and the leaching of basalt were investigated. As this was the first time that biofilms had been studied under hypergravity, additional substrates were included with the basalt, to enable characterisation of the more general response of biofilms to hypergravity. In contrast to previous experiments conducted on planktonic bacteria, which found decreased population sizes, the biofilms grown at 10 x g showed greater biomass than the 1 x g samples. ICP-MS showed no difference in the average weathering rates, but greater variability in the higher gravity samples. The data collected here advances our understanding of microbial interactions with geologically important substrates, with implications for an ISS microgravity experiment and future human space exploration. It also presents new intelligence on the previously unstudied effects of hypergravity on biofilms and rock weathering.

Orientador: Laura Maria Mariscal Ottoboni / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-25T23:22:56Z (GMT). No. of bitstreams: 1 Rodrigues_VivianeDrumond_D.pdf: 5631358 bytes, checksum: 6072655858120e417b92336ddd538828 (MD5) Previous issue date: 2014 / Resumo: O conhecimento acerca da diversidade microbiana associada a ambientes de mina é limitado, apesar da importância que alguns micro-organismos podem ter no processo de biolixiviação e biorremediação ambiental. Adicionalmente, micro-organismos que vivem em condições inóspitas, como os diferentes ambientes de mina, vêm despertando interesse cada vez maior por possuírem enzimas de interesse industrial. Neste sendido, a análise da biodiversidade funcional e estrutural de micro-organismos presentes em ambientes de mina é de fundamental importância para entender a estrutura e a complexidade das comunidades microbianas em ambientes extremos. Neste trabalho a diversidade microbiana foi analisada em diversos ambientes da mina de cobre do Sossego, localizada em Canaã dos Carajás, sudeste do Pará por abordagens dependentes e independentes de cultivo. A composição taxonômica associada a ambientes da mina do Sossego: taludes (estruturas geotécnicas) e entorno da drenagem dos depósitos de Sossego (T-SO1, T-SO2, ED-SO1, ED-SO2) e Sequeirinho (T-SE1, T-SE2, ED-SE1, ED-SE2) foi avaliada por pirosequenciamento do gene de rRNA 16S. Os resultados indicaram que a comunidade de bactérias de talude é distinta do entorno da drenagem e o conteúdo de matéria orgânica e maior disponibilidade de água foram os principais fatores para as diferenças. Os principais táxons responsáveis pelas diferenças foram Acidobacteria, Chloroflexi, Gammaproteobacteria e Firmicutes. Por meio de técnicas dependentes de cultivo, 64 bactérias heterotróficas foram isoladas a partir das amostras SO5, SO6, SO7 e SO9. Estes isolados foram identificados e avaliados quanto à capacidade de produção de enzimas (hidrolases, monoxigenases, sulfoxidases e betalactamase) e compostos (sideróforos, biossurfactantes e antimicrobianos). Foram identificadas bactérias afiliadas aos seguintes gêneros: Acidovorax, Acinetobacter, Brevundimonas, Cupriavidus, Curtobacterium, Kocuria, Lysinibacillus, Pseudomonas, Roseomonas, Ralstonia, Stenotrophomonas e Bacillus, sendo o último respresentado por 43 isolados. Com relação à triagem funcional, 95% das bactérias foram capazes de produzir sideróforos, 58% biossurfactantes, 69% betalactamases, 50% antimicrobianos, 53% proteases, 75% esterases, 20% monoxigenases e três isolados (SO5.4, SO5.9 e SO6.2) apresentaram oxidação seletiva para sulfetos orgânicos. A partir de amostras de drenagem (SO5, SO6 e SO7) foram obtidos consórcios de micro-organismos oxidantes de ferro. Estes consórcios foram testados com relação à capacidade de biolixiviação da calcopirita e foram mais eficientes para a dissolução do cobre do que Acidithiobacillus ferrooxidans LR. A identificação dos micro-organismos presentes nos consórcios foi realizada por eletroforese em gel de gradiente desnaturante (DGGE) e as bandas mais evidentes foram classificadas em Bacillus sp., Delftia sp., Phenylobacterium sp. e Methylobacterium sp. A comunidade de bactérias na mina de cobre do Sossego foi diversa e complexa. Estes resultados mostram um inventário da microbiota em diferentes ambientes da mina do Sossego e as enzimas e compostos obtidos destas bactérias poderão ser utilizadas em processos e tecnologias que permitam a recuperação de metais, como a biolixiviação e biorremediação ou em outras aplicações industriais / Abstract: The knowledge concerning microbial diversity associated with mine environments is limited, despite the importance that some microorganisms can have on environmental bioremediation and bioleaching process. Additionally, microorganisms that live in inhospitable conditions, such as different mine environments, have attracted growing interest because they could have enzymes with industrial applications. In this way, structural and functional biodiversity analysis in mine environments is an important issue to understand the structure and complexity of the microbial communities in extreme environments. The present work shows a microbial diversity analyses in some cooper mine environments of Sossego Mine localized in Canaã dos Carajás mineral province, Pará state, Brazil. The bacterial taxonomic composition associated with Sossego cooper mine: slopes (geotechnical structures) and surrounding drainage of Sossego and Sequeirinho deposits was evaluated using pyrosequencing of 16S rRNA gene. The results indicated slope bacterial community differs from surrounding drainage and organic matter content and higher water availably were the main factors of these differences. The foremost taxons accountable by those differences were Acidobacteria, Chloroflexi, Gammaproteobacteria and Firmicutes. Sixty four bacteria were isolated using culture-dependent methods from SO5, SO6, SO7 and SO9 samples. These bacteria were identified and evaluated concerning the capability of enzyme production (hydrolase, betalactamase, monooxygenase and sulphoxidases) and compounds (siderophore, biosurfactants and antimicrobials). It was identified bacteria related with the followed genera: Acidovorax, Acinetobacter, Brevundimonas, Cupriavidus, Curtobacterium, Kocuria, Lysinibacillus, Pseudomonas, Roseomonas, Ralstonia, Stenotrophomonas and Bacillus, the last one showed 43 isolates. In relation with functional screening, 95% of bacteria were capable to produce siderophores, 58% to produce biosurfactants, 69% betalactamases, 50% antimicrobials, 53% proteases, 75% sterases, 20% monooxygenases and three strains (SO5.4, SO5.9 and SO6.2) exhibited selective oxidation for organic sulphides. Iron oxidizing microorganism consortia were obtained from drainage samples and were tested according with its ability for bioleaching of chalcopyrite. The consortia obtained from SO5, SO6, and SO7 samples were more efficient than Acidithiobacillus ferrooxidans LR regarding bioleaching of copper from chalcopyrite. The identification of microorganism presented in the consortia was performed using DGGE technique and the more evident bands were classified as Bacillus sp., Delftia sp., Phenylobacterium sp. and Methylobacterium sp. The bacterial community in Sossego cooper mine was diverse and complex. These results showed a microbiota inventory in distinct mine environments and enzymes and compounds obtained from those bacteria could be used in new processes and technologies that allow to recovery metals as bioleaching, bioremediation or others industrial applications / Doutorado / Genetica de Microorganismos / Doutora em Genética e Biologia Molecular