Orientadores: Lucia Regina Durrant, Alexandre Nunes Ponezi

Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos

Resumo: 17a-etinilestradiol (EE2), carbamazepina (CBZ) e ibuprofeno (IBU) são substâncias farmacêuticas muito utilizadas em todo o mundo e vêm sendo frequentemente detectadas em estações de tratamento de efluentes e em águas naturais em vários países, inclusive no Brasil. A grande preocupação da...

Resumo: 17a-etinilestradiol (EE2), carbamazepina (CBZ) e ibuprofeno (IBU) são substâncias farmacêuticas muito utilizadas em todo o mundo e vêm sendo frequentemente detectadas em estações de tratamento de efluentes e em águas naturais em vários países, inclusive no Brasil. A grande preocupação da presença destes fármacos em quantidades residuais na água potável e nos ambientes aquáticos são os potenciais efeitos adversos para a saúde humana e animal. O objetivo principal deste trabalho foi avaliar o potencial de fungos ligninolíticos e bactérias para degradar esses três compostos, individualmente. Linhagens de bactérias e fungos ligninolíticos foram crescidas em meio mineral com os fármacos, na presença ou ausência de glicose. Primeiramente, foi realizada uma seleção com o objetivo de escolher linhagens bacterianas e fúngicas com maior capacidade de degradação dessas drogas, avaliando a necessidade da presença de glicose para que a degradação ocorresse. As linhagens que apresentaram maior capacidade de degradar tais compostos foram selecionadas e, em seguida, foram realizados ensaios com o intuito de se otimizar o período de incubação, visando-se uma maior porcentagem de degradação no menor período de incubação possível. Posteriormente, foram realizadas análises de atividade das enzimas lacase, lignina peroxidase (LiP) e manganês peroxidase (MnP) produzidas pelos fungos selecionados e foi avaliada a participação dessas na degradação dos fármacos. A atuação das enzimas do Citocromo P450 na degradação dos fármacos foi avaliada por meio da adição de piperonil butóxido (PB), o qual inibe esse complexo enzimático. A toxicidade dos fármacos e seus metabólitos para a bactéria Vibrio fischeri também foram avaliadas. A quantificação dos fármacos em todas as amostras foi realizada por meio de cromatografia líquida de alta eficiência. EE2 foi totalmente degradado por todos os fungos avaliados, sem a necessidade de glicose no meio de cultivo; no entanto, nenhuma das bactérias estudadas foi capaz de degradá-lo significativamente. Pleurotus ostreatus (Jacq.) P. Kumm linhagem P1 foi selecionado para os ensaios subsequentes. Após 6 dias, foi encontrada atividade de MnP igual a 5122,11 U.L-1. A lacase teve como atividade 307,69 U.L-1, valor encontrado após 4 dias de incubação. Não foi detectada atividade da enzima LiP em nenhum dos tempos analisados. Apesar da detecção de atividade dessas enzimas, elas não foram capazes de degradar o EE2 na ausência do micélio fúngico. Nos ensaios de toxicidade foi encontrada uma CE50 igual a 76% para o EE2 e os metabólitos não apresentaram toxicidade. Trametes sp. linhagem BNI foi a selecionado para degradar CBZ, sendo a glicose necessária para o processo de biodegradação. Após 28 dias de incubação, houve 42% de degradação de CBZ. A atividade máxima de lacase foi de 1740,17 U.L-1, sendo encontrada após 21 dias de incubação. LiP teve como atividade máxima 663,08 U.L-1, valor encontrado após 14 dias de incubação. Não foi detectada atividade da enzima MnP em nenhum dos tempos analisados. Não houve a degradação de CBZ utilizando apenas o caldo enzimático. A presença de PB inibiu totalmente a degradação de CBZ. CBZ e seus metabólitos não apresentaram toxicidade. Nenhuma das bactérias foi capaz de degradar CBZ. IBU foi totalmente degradado por todos os fungos avaliados sem a necessidade de glicose no meio de cultivo, sendo Trametes sp. linhagem BNI selecionado para os ensaios posteriores. Após 2 dias de incubação, BNI foi capaz de degradar totalmente IBU. Lacase foi a única enzima que teve atividade detectada nesse ensaio, sendo a atividade máxima detectada igual a 478,18 U.L-1, no sexto dia de incubação. Não foi detectada degradação de IBU utilizando apenas o caldo enzimático e a presença de PB no meio não inibiu a degradação deste fármaco. Nos ensaios de toxicidade foi encontrada uma CE50 igual a 86% para o IBU e os metabólitos não apresentaram toxicidade. Staphylococcus arlettae e Bacillus megaterium foram capazes de degradar significativamente IBU na presença de glicose. B. megaterium foi selecionado para os ensaios subsequentes. Após 3 dias, essa linhagem foi capaz de degradar todo IBU disponível no meio. Nos ensaios de toxicidade para os metabólitos do processo de biodegradação por B. megaterium, o IBU apresentou uma CE50 inicial igual a 47% e os metabólitos não apresentaram toxicidade. Esses resultados comprovam que fungos ligninolíticos e bactérias são capazes de degradar fármacos encontrados em matrizes ambientais, sendo plausível a utilização destes micro-organismos, ou suas enzimas, em sistemas de tratamento de água e esgoto

Abstract: 17a-ethinylestradiol (EE2), carbamazepine (CBZ) and ibuprofen (IBU) are pharmaceutical drugs used worldwide and have been frequently detected in wastewater treatment plants and in natural waters in several countries, including Brazil. The major concern about the occurrence of these drugs...

Abstract: 17a-ethinylestradiol (EE2), carbamazepine (CBZ) and ibuprofen (IBU) are pharmaceutical drugs used worldwide and have been frequently detected in wastewater treatment plants and in natural waters in several countries, including Brazil. The major concern about the occurrence of these drugs in trace amounts in drinking water and aquatic environments are the potential adverse effects on human and animal health. The main objective of this study was to assess the potential of ligninolytic fungi and bacteria to degrade these 3 compounds individually. Bacteria and ligninolytic fungi strains were grown on mineral medium with these drugs and with or without glucose. A selection was carried out to choose bacterial and fungal strains with capacity to degrade these drugs and if an addition of a carbon source (glucose) was needed for degradation. Strains with greater capacity to degrade these compounds were selected and assays were performed in order to optimize the incubation time to obtain the highest degradation rate in the shortest incubation time. Subsequently, the enzymatic activities of laccase, lignin peroxidase (LiP) and manganese peroxidase (MnP) produced by the selected fungi was assessed. Also, the action of these enzymes in the degradation of the drugs was evaluated. The involvement of cytochrome P450 enzymes in degradation of the pharmaceutical drugs was evaluated by the addition of piperonyl butoxide (PB), which inhibits this enzyme complex. The toxicity of the drugs and metabolites to Vibrio fischeri were also evaluated. The quantification of the drugs was performed by high performance liquid chromatography. EE2 was completely degraded by all fungi without glucose in the medium, however none of the studied bacteria was capable to degrade it significantly. Pleurotus ostreatus (Jacq.) P. Kumm strain P1 was selected for subsequent tests. The maximum enzyme activity produced by P1 was 5122.11 UL-1 for MnP after 6 days and 307.69 UL-1 for lacase after 4 days, while LiP activity was not detected. Although the detection of the enzymes activity, they were not able to degrade EE2 without the fungal mycelia. Toxicity studies showed the half maximal effective concentration (EC50) value equal to 76% to EE2 prior to fungal treatment, after this no toxicity was observed. Trametes sp. strain BNI was selected to degrade CBZ, and glucose was shown to be necessary for the biodegradation process. After 28 days of incubation, 42% of CBZ was degraded. The maximum laccase activity was 1740.17 UL-1, after 21 days of incubation. LiP maximum activity was 663.08 UL-1, found after 14 days of incubation, while MnP activity was not detected. There was no CBZ degradation using only the enzymatic supernatant. The addition of PB completely inhibited the degradation of CBZ. CBZ and its metabolites did not show toxicity. IBU was completely degraded by all fungi without glucose in the medium, and Trametes sp. strain BNI was selected for further analyses. 17a-ethinylestradiol (EE2), carbamazepine (CBZ) and ibuprofen (IBU) are pharmaceutical drugs used worldwide and have been frequently detected in wastewater treatment plants and in natural waters in several countries, including Brazil. The major concern about the occurrence of these drugs in trace amounts in drinking water and aquatic environments are the potential adverse effects on human and animal health. The main objective of this study was to assess the potential of ligninolytic fungi and bacteria to degrade these 3 compounds individually. Bacteria and ligninolytic fungi strains were grown on mineral medium with these drugs and with or without glucose. A selection was carried out to choose bacterial and fungal strains with capacity to degrade these drugs and if an addition of a carbon source (glucose) was needed for degradation. Strains with greater capacity to degrade these compounds were selected and assays were performed in order to optimize the incubation time to obtain the highest degradation rate in the shortest incubation time. Subsequently, the enzymatic activities of laccase, lignin peroxidase (LiP) and manganese peroxidase (MnP) produced by the selected fungi was assessed. Also, the action of these enzymes in the degradation of the drugs was evaluated. The involvement of cytochrome P450 enzymes in degradation of the pharmaceutical drugs was evaluated by the addition of piperonyl butoxide (PB), which inhibits this enzyme complex. The toxicity of the drugs and metabolites to Vibrio fischeri were also evaluated. The quantification of the drugs was performed by high performance liquid chromatography. EE2 was completely degraded by all fungi without glucose in the medium, however none of the studied bacteria was capable to degrade it significantly. Pleurotus ostreatus (Jacq.) P. Kumm strain P1 was selected for subsequent tests. The maximum enzyme activity produced by P1 was 5122.11 UL-1 for MnP after 6 days and 307.69 UL-1 for lacase after 4 days, while LiP activity was not detected. Although the detection of the enzymes activity, they were not able to degrade EE2 without the fungal mycelia. Toxicity studies showed the half maximal effective concentration (EC50) value equal to 76% to EE2 prior to fungal treatment, after this no toxicity was observed. Trametes sp. strain BNI was selected to degrade CBZ, and glucose was shown to be necessary for the biodegradation process. After 28 days of incubation, 42% of CBZ was degraded. The maximum laccase activity was 1740.17 UL-1, after 21 days of incubation. LiP maximum activity was 663.08 UL-1, found after 14 days of incubation, while MnP activity was not detected. There was no CBZ degradation using only the enzymatic supernatant. The addition of PB completely inhibited the degradation of CBZ. CBZ and its metabolites did not show toxicity. IBU was completely degraded by all fungi without glucose in the medium, and Trametes sp. strain BNI was selected for further analyses