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Caracterização Da Diversidade De Eucariotas Fototróficos Provenientes De Águas Ácidas De Mina

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Caracterização Da Diversidade De Eucariotas Fototróficos Provenientes De Águas Ácidas De Mina Universidade do Algarve Faculdade de Ciências e Tecnologia Caracterização da Diversidade de Eucariotas Fototróficos Provenientes de Águas Ácidas de Mina Telma Cristina Teixeira Valente Mestrado em Biologia Molecular e Microbiana Faro 2012 Universidade do Algarve Faculdade de Ciências e Tecnologia Telma Cristina Teixeira Valente (Licenciada em Bioquímica) Mestrado em Biologia Molecular e Microbiana Orientada por: Prof.ª Doutora Margarida P. Reis Prof.ª Doutora Filomena Fonseca CIMA – Laboratório de Ecologia Molecular e Microbiana Faro 2012 After a while you learn the subtle difference between holding a hand and chaining a soul and you learn that love doesn't mean leaning and company doesn't always mean security. And you begin to learn that kisses aren't contracts and presents aren't promises and you begin to accept your defeats with your head up and your eyes ahead with the grace of woman, not the grief of a child and you learn to build all your roads on today because tomorrow's ground is too uncertain for plans and futures have a way of falling down in mid-flight. After a while you learn that even sunshine burns if you get too much so you plant your own garden and decorate your own soul instead of waiting for someone to bring you flowers. And you learn that you really can endure you really are strong you really do have worth and you learn and you learn with every goodbye, you learn... Veronica Shoffstall 1971 Este trabalho é da exclusiva responsabilidade de: ____________________________________ Telma Valente Agradecimentos A realização deste trabalho não teria acontecido sem a boa influência de algumas pessoas que foram fundamentais. Primeiro tenho que agradecer à pessoa que motivou o ingresso no Mestrado, mas que foi a que mais vezes ficou privada da minha companhia, a minha filha. Sem ela nada disto teria o mesmo sentido ou o mesmo sabor… Em igualdade de direitos, e com um “muito obrigado” muito especial, agradeço aos meus pais que sempre tiveram ao meu lado, e que, à sua maneira, me foram transmitindo confiança e motivação para seguir em frente. Agradeço à Professora Doutora Margarida Reis que me “deu o bichinho” das microalgas e me orientou. Agradeço à Professora Doutora Filomena Fonseca pela orientação que me proporcionou e pela infinita paciência que teve para as “perguntas que não lembram a ninguém”. Não poderia faltar o agradecimento à Filipa Esteves, que me orientou nas “lides” da biologia molecular e que, também, tem uma paciência muito grande. À Maria Rodrigues, que soube empurrar e “dar nas orelhas” quando foi preciso, que soube incentivar quando a motivação não era muita, mas principalmente quando partilhou comigo os bons momentos aqui vividos. À Célia Domingos que teve sempre uma palavra de incentivo, mesmo quando não sabia que ela era necessária. Às Colega do laboratório 1.73, Ana Flor e Sandra Mesquita, do laboratório 2.8, Cátia Guerra e do gabinete 1.72, Cristina Costa e Sandra Caetano, que sempre se mostraram disponíveis e tiveram sempre uma palavra de incentivo. A alguém especial que soube transmitir, apesar de longe, confiança e que em alguns momentos foi muito importante. Agradeço a todos os que de uma forma mais ou menos presente contribuíram para o sucesso deste trabalho. Estou grata por ter trabalhado com pessoas que me transmitiram um pouco do conhecimento que possuem. A todos, Muito Obrigada! Resumo O presente trabalho teve por objetivos principais caracterizar a diversidade de organismos fototróficos acidófilos ou acidotolerantes presentes nas águas ácidas provenientes da drenagem ácida de mina, na Área Mineira Abandonada de S. Domingos (Mértola, Portugal); produzir culturas unialgais de microalgas acidotolerantes e/ou acidófilas; e verificar o potencial biotecnológico de uma estirpe isolada com base na sua caracterização fisiológica. Procedeu-se à recolha de amostras que foram identificadas morfologicamente, recorrendo a microscopia, e molecularmente, recorrendo a técnicas de extração de DNA, de amplificação por PCR, de clonagem e de sequenciação, com base em marcadores como sequências conservadas dos 18S rDNA e 23S rDNA. Identificou-se quatro géneros de organismos fototróficos acidófilos, nomeadamente, Euglena , Coccomyxa , Chlamydomonas e Klebsormidium , bem como diatomáceas (Bacillariopheceae). Além destes eucariotas, também foi possível identificar, molecularmente, dois géneros de bactérias, Acidiphilium e Variovorax . Isolou-se duas microalgas dos géneros Coccomyxa e Chlamydomonas , em culturas unialgais, mas não axénicas, utilizando o meio MK a pH 2.5 e recorrendo a técnicas como o riscado à exaustão. Com o isolado de Coccomyxa sp. foi possível fazer estudos fisiológicos, de modo a determinar os potenciais biotecnológicos desta microalga, utilizando como alvos a acumulação de carotenóides e a produção malondialdeído (MDA). Verificou-se a taxa de crescimento desta microalga em dois níveis de pH diferentes (2.5 e 6.5/7.0), duas fontes de azoto (ureia - MKU e nitrato - MK) e em duas intensidades luminosas diferentes. A combinação entre o pH 6.5, a intensidade luminosa mais elevada e a fonte de azoto sob a forma de nitrato proporcionou a taxa de crescimento média mais elevada. A acumulação de carotenoides foi maior na cultura em meio MK a pH 6.5 com a intensidade luminosa elevada. Mediu-se MDA como parâmetro indicador da indução de stress oxidativo em duas concentrações de cobre (0.4 e 0.8 mM). Palavras-chave: AMD; microalgas; extremófilos; isolamento; cultivo; interesse biotecnológico; carotenoides; stress oxidativo. Caracterização da Diversidade de Eucariotas Fototróficos Provenientes de Águas Ácidas de Mina i Abstract The objective of this work was to characterize the main diversity of acidophilic and acid tolerant phototrophic organisms present in acidic waters produced by acid mine drainage at the Abandoned Mining Site of S. Domingos (Mértola, Portugal); to produce unialgal cultures of acidophilic / acid tolerant microalgae and to assess their biotechnological potential based on physiological characters. Collected samples were morphologically identified through microscopic observations and molecularly, using DNA extraction techniques, PCR amplification, cloning and sequencing, with 18S rDNA and 23S rDNA genes as markers. Four genera of phototrophic acidophilic organisms were identified, namely Euglena , Coccomyxa , Chlamydomonas , Klebsormidium as well as diatoms (Bacillariopheceae). In addition to these eukaryota, it was possible to molecularly identify two genera of bacteria, Acidiphilium and Variovorax . Two microalgae from the genera Coccomyxa and Chlamydomonas were isolated in unialgal but non axenic cultures, using the MK medium pH 2.5 and techniques such as plate streaking for purification . The Coccomyxa sp. isolate was submitted to physiological studies in order to estimate the biotechnology potential, by targeting carotenoid accumulation and malondialdehyde (MDA) production. Growth rates at two different pH levels (2.5 and 6.5/7.0), two sources of nitrogen (urea - MKU and nitrate - MK) and two different light intensities were determined. The combination of pH 6.5, with the highest light intensity and nitrate as nitrogen source provided the highest median growth rate. Higher carotenoid accumulation was detected in MK medium cultures at pH 6.5 with the high radiation intensity. MDA was measured as indicator parameter for oxidative stress induction, for two concentrations of copper (0.4 and 0.8 mM). Keywords: AMD, microalgae, extremophiles, isolation, cultivation, biotechnological interest, carotenoids, oxidative stress. Caracterização da Diversidade de Eucariotas Fototróficos Provenientes de Águas Ácidas de Mina ii Abreviaturas A – Absorvência AMA – Área Mineira Abandonada AMD – Acid Mine Drainage ARD – Acid Rock Drainage Aps – Ammonium Persulfate BHT – Butylated Hydroxytoluene (hidroxitolueno butilato) BLAST – Basic Local Alignment Search Tool bp – base pair(s) (par(es) de bases) C – Concentração CCAP – Culture Collection of Algae and Protozoa (Coleção de Culturas de Algas e Protozoários) CCMAR – Centro de Ciências do Mar Cu 2+ - Ião Cobre (II) DGGE – Denaturing Gradient Gel Electrophoresis (Electroforese em Gel com Gradiente Desnaturante) DMSO – Dimetil sulfoxide DNA – Deoxyribonucleic Acid (Ácido Desoxirribonucleico) DO – Densidade Ótica EDTA – Ethylenediamine tetraacetic acid EPS – Extracellular Polymeric Substances (substâncias poliméricas extracelulares) Fe 2S – Sulfureto de ferro (pirite) HL – High Light H2S – Ácido Sulfídrico IPB – Iberian Pyrite Belt (Faixa Piritosa Ibérica) IPTG – Isopropyl-beta-D-Thiogalactopyranoside LB Agar – Lysogeny Bertani Agar LL – Low Light MDA – Malondialdeido MK – K9 Modificado (meio de cultura) MKU – K9 Modificado com Ureia (meio de cultura) Caracterização da Diversidade de Eucariotas Fototróficos Provenientes de Águas Ácidas de Mina iii Abreviaturas Mt – Megatoneladas (10 6 toneladas) NCBI - National Center for Biotechnology Information PCR – Polymerase Chain Reaction pf – Peso fresco RNA – Ribonucleic Acid (Ácido Ribonucleico) ROS – Reactive Oxygen Species (Espécies Reativas de Oxigénio) rpm – rotações por minuto RuBisCO – Ribulose-Bisfosfato Carboxilase Oxigenase SSCP – Single Strand Conformation Polymorphism t – Tempo TAE – Tris-Acetate-EDTA TBA – Thiobarbituric Acid (Ácido Tiobarbitúrico) TBE – Tris-Borate-EDTA TCA – Trichloroacetic Acid (Ácido Tricloroacético) TGGE – Temperature Gradient Gel Electrophoresis (Eletroforese Desnaturante por Gradiente de Temperatura) UV – Ultravioleta X-gal – 5-Bromo-4-Chloro-Indolyl-β-D-Galactopyranoside
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