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Apresentação Do Powerpoint UM VIRAR DE PÁGINA NA HISTÓRIA DAS ÁGUAS MINERAIS NATURAIS... UM NOVO CAPÍTULO SE ESCREVE: O CÓDIGO HIDROBIÓMICO DAS ÁGUAS. Paula Sá Pereira 1º estudo científico com significado histórico - 1726 pelo doutor Francisco da Fonseca Henriques - “Aquilégio Medicinal” - levantamento das “caldas com virtudes medicinais, dignas de “particular memória”.. Francisco Tavares (1750-1812), professor da Faculdade de Medicina da Universidade de Coimbra, responsável pela primeira Farmacopeia oficial portuguesa, mas também pelo estudo dedicado à Hidrologia Médica. Em 1791 publica a obra “Advertências sobre os abusos, e legítimo uso das águas minerais das Caldas da Rainha” Historia das águas minerais naturais António Herculano de Carvalho Charles Lepierre (1899 – 1986) (1867-1945) …“No momento da infiltração no subsolo, tem início um processo lento e complexo de filtração natural que se encarregará de eliminar os microorganismos e as substâncias em suspensão”… (cit. Livro Branco, 2017) Microbiologia da água ≈ Qualidade da água …….microbismo normal Portaria 1220/2000 de 29 de Dezembro ? Decreto-Lei n.º 156/98 Artigo 4.º Características microbiológicas 1 - À saída da captação, o teor total de microrganismos susceptíveis de se desenvolverem nas águas minerais naturais deve corresponder ao seu microbismo normal e revelar uma protecção eficaz da captação contra qualquer contaminação. 3 - Na captação os teores totais em microrganismos não devem ultrapassar, respectivamente, 20 por mililitro a 20ºC-22ºC às setenta e duas horas e 5 por mililitro a 37ºC às vinte e quatro horas. 5 - Quer na captação quer na comercialização, as águas minerais naturais devem apresentar-se isentas de: a) Parasitas e microrganismos patogénicos; b) Escherichia coli e outros coliformes e de estreptococos fecais, em 250 ml de amostra analisada; c) Anaeróbios esporolados sulfito-redutores, em 50 ml de amostra examinada; d) Pseudomonas aeruginosa, em 250 ml de amostra examinada. Estudo do Microbismo Natural das Águas Minerais Naturais Contrato Nº 2017/ SGME/0118 Paula Sá Pereira Coordenadora Científica Objetivos gerais do estudo 2017 a 2019 (26 meses) 80 AMN - 1 amostragem no inicio do ano hidrológico e 1 amostragem no fim do ano hidrológico, 2 anos, para validação dos resultados Estudo edafo-climático da envolvente associada a cada captação – transferência dos dados para Sistema de Informação Geográfica – Delimitação dos perímetros de proteção das captações – Zonas de risco Caracterização do microbioma associado a cada água mineral natural em estudo Estudo do metatranscriptoma de algumas águas minerais naturais • ISO 9308-1:2014 e Amd 2016 para a análise das bactérias coliformes e Escherichia coli; • ISO 7899-2:2000 para a análise de Enterococos; • ISO 16266:2006 para a análise de Pseudomonas aeruginosa; • NP EN 26461-2:1994 (versão portuguesa da norma ISO 6461-2:1986) para a análise de anaeróbios esporulados sulfito-redutores) Verificação de todos os isolados por métodos de biologia molecular Foi avaliado o de cada água mineral natural, cultiváveis em laboratório de acordo com os métodos de referência, e em condições experimentais. • ISO 6222:1999 - contagem do número de colónias de microrganismos viáveis – número de colónias a 22 e a 37°C. Em todas as condições, e em todas as amostras conforme, nas 4 fases de colheita, que cumpriram a legislação, foi feito o isolamento, caracterização microbiológica e identificação de microrganismos cultiváveis em laboratório. A validação final foi efectuada por sequenciação de Sanger Sequenciação de Nova Geração Preparação de bibliotecas Produção de clusters Sequenciação Illumina Caracterização das comunidades bacterianas de águas minerais naturais de acordo com o quimismo Gammaproteobacteria Fusobacteria Clostridia Dehalococcoidetes 33 Deltaproteobacteria Chthonomonadetes 30 Betaproteobacteria Aquificae 27 Alphaproteobacteria 24 Methanomicrobia 21 Actinobacteria 18 Brocadiae 15 Nitrospira 12 Oscillatoriophycideae 9 Bacili 6 Nostocophycideae 3 Epsilonproteobacteria 0 Thermobacula Deinococci Opitutae Anaerolineae Thermodesulfobacteria Flavobacteria Planctomycetia Sphingobacteriia Acidobacteria Thermotogae Ignavibacteria Methylacidiphilae UnclassifiedBacteroidia MollicutesSpirochaetes Gammaproteobacteria Spartobacteria 10 Clostridia Solibacteres 9 Deltaproteobacteria 8 Fimbriimonadetes 7 Betaproteobacteria 6 Caldithrixae 5 Alphaproteobacteria 4 3 Holophagae 2 Actinobacteria 1 0 Thermobacula Bacili Planctomycetia Sphingobacteriia Epsilonproteobacteria Nitrospira Pedosphaerae Spirochaetes Acidobacteria Unclassified Thermotogae Ktedonobacteria Clostridia Synergistia 14 Deltaproteobacteria 13 Synechococcophycideae 12 Betaproteobacteria 11 Nitriliruptoria 10 Alphaproteobacteria 9 8 Brachyspirae 7 Nitrospira 6 5 4 Ignavibacteria 3 Gammaproteobacteria 2 1 0 Holophagae Actinobacteria Thermotogae Bacili Thermoleophilia Sphingobacteriia Brocadiae Thermodesulfobacteria Mollicutes Deinococci Planctomycetia Unclassified Gammaproteobacteria Acidimicrobiia 10 Clostridia Brachyspirae 9 Betaproteobacteria 8 Aquificae 7 Alphaproteobacteria 6 5 Caldithrixae 4 Deltaproteobacteria 3 2 Holophagae Actinobacteria 1 0 Thermobacula Nitrospira Ktedonobacteria Bacili Thermotogae Deinococci Methanomicrobia Spirochaetes Mollicutes Unclassified Epsilonproteobacteria Gammaproteobacteria Chlorobia 3 Clostridia Brocadiae 2 Deltaproteobacteria Ignavibacteria 1 Alphaproteobacteria 0 Deinococci Actinobacteria Planctomycetia Nitrospira Thermotogae Unclassified Bacili Betaproteobacteria Conclusões As Betaproteobacteria são a classe que predomina em todas as famílias químicas de águas minerais naturais estudadas, à exceção das águas cloretadas Nas águas sulfúreas são encontra-se ainda bem representadas as Gammaproteobacteria Nas águas hipossalinas, encontram-se as classes Alphaproteobacteria, Clostridia e Actinobacteria como predominantes Nas águas bicarbonadatas as Alphaproteobacteria são igualmente dominantes, assim como a Clostridia e Nitrospira Nas águas gasocarbónicas encontra-se igualmente em maioria as Gammaproteobacteria e as Deltaproteobacteria Nas águas cloretadas as Alphaproteobacteria são dominantes seguidas das Deltaproteobacteria, Nitrospira, Actinobacteria, Gammaproteobacteria e Clostridia. Diversidade de géneros e espécies de bactérias mais significativas nas águas minerais naturais estudadas (> 1000 Hits) Nº de Aguas n=10 Nº de Aguas n=4 Nº de Aguas n=10 Nº de Aguas n=35 Nº de Aguas n=14 águas Gasocarbónicas >1000 Hits águas Cloretadas >1000 Hits águas Hipossalinas >1000 Hits águas Sulfúreas >1000 Hits águas Bicarbonatadas >1000 Hits (%) (%) (%) (%) (%) Methylobacterium Thermodesulfovibrio Thermodesulfovibrio Rhodoferax 4 40 3 75 Methylobacterium 7 70 13 37,1 7 50 radiotolerans aggregans aggregans Thermodesulfovibrio Rhodoferax antarcticus 4 40 Desulfosarcina ovata 2 50 Oxalobacter vibrioformis 5 50 Thiovirga sulfuroxydans 13 37,1 6 42,9 thiophilus Methyloversatilis Acidovorax temperans 3 30 Methylobacterium 2 50 Azospirillum 4 40 10 28,6 Methyloversatilis universalis 5 35,7 universalis Methylobacterium Gallionella 3 30 Pseudomonas 2 50 4 40 Thiofaba 10 28,6 Azoarcus 3 21,4 mesophilicum Methylobacterium Nitrosovibrio 3 30 Pseudomonas plecoglossicida 2 50 4 40 Sulfuritalea 9 25,7 Chondromyces pediculatus 3 21,4 radiotolerans Thermodesulfovibrio Rhodoferax ferrireducens 3 30 2 50 Moorella glycerini 4 40 Sulfuricurvum kujiense 8 22,9 Dechloromonas 3 21,4 aggregans Thermodesulfovibrio Thermodesulfovibrio 3 30 2 50 Gallionella 3 30 Thiovirga 7 20 Dechloromonas hortensis 3 21,4 aggregans thiophilus Denitratisoma Burkholderia ubonensis 2 20 Acinetobacter oleivorans 1 25 Paucibacter 3 30 6 17,1 Oxalobacter vibrioformis 3 21,4 oestradiolicum Giesbergeria 2 20 Anaerospora 1 25 Pseudomonas 3 30 Sulfurospirillum 6 17,1 Sphingobium amiense 3 21,4 Methyloversatilis Thermodesulfovibrio 2 20 Aquabacterium 1 25 3 30 Thiobacillus 6 17,1 Sphingomonas panni 3 21,4 universalis thiophilus Propionivibrio Thermodesulfatator 2 20 Azoarcus evansii 1 25 Bradyrhizobium 2 20 Thiothrix 6 17,1 3 21,4 dicarboxylicus atlanticus Ectothiorhodospira Schlegelella aquatica 2 20 Azospira restricta 1 25 Burkholderia 2 20 5 14,3 Zoogloea resiniphila 3 21,4 imhoffii Thermodesulfovibrio Sulfuricurvum kujiense 2 20 Azovibrio 1 25 Burkholderia bryophila 2 20 5 14,3 Candidatus Scalindua brodae 2 14,3 thiophilus Candidatus Tammella Sulfurospirillum 2 20 Bifidobacterium bombi 1 25 2 20 Thermus 5 14,3 Chromatium weissei 2 14,3 caduceiae Uliginosibacterium 2 20 Blautia coccoides 1 25 Chondromyces pediculatus 2 20 Thiomonas intermedia 5 14,3 Desulfobulbus elongatus 2 14,3 gangwonense Zoogloea oryzae 2 20 Chlorobaculum limnaeum 1 25 Cupriavidus basilensis 2 20 Acinetobacter johnsonii 4 11,4 Gallionella 2 14,3 Acinetobacter Methylobacterium Bifidobacterium bombi 1 10 Chondromycespediculatus 1 25 Gallionella ferruginea 2 20 4 11,4 2 14,3 tjernbergiae mesophilicum Methylobacterium Brachyspira ibaraki 1 10 Clostridium 1 25 Haliangium 2 20 Desulfomonile tiedjei 4 11,4 2 14,3 radiotolerans CONSOLIDAÇÃO DE HIDROGENOMAS Estabilidade nos índices de diversidade de microrganismos e estrutura das populações nas amostras estudadas – F1, F3, F75, F7 Famílias Químicas % Consolidação Sulfúreas 80 Hipossalinas 0,1 Bicarbonatadas 85,7 Gasocarbónicas 70 Cloretadas 100 Fatores que, putativamente, possam ter influenciado não consolidação dos hidrogenomas, a variação na diversidade de microrganismos e estrutura das populações nas amostras, além de outros são: Condições climatéricas que ocorreram imediatamente antes e durante o período
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