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Estudo da tolerância ao etanol de estirpes da cionobactéria Synechocystis sp. produtoras de bioetanol, utilizando a citometria de fluxo
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O objetivo deste trabalho consistiu no estudo da tolerância ao etanol de duas estirpes de Synechocystis PCC6803, a estirpe selvagem e uma estirpe recombinante (UL030) produtora de etanol. Os cultivos foram realizados na gama de 0 - 40g/L de etanol, a 30 C, sob uma intensidade luminosa de 24 μE/(m2s), e caracterizados em termos de concentração de biomassa, integridade da membrana e atividade metabólica. Os dois últimos parâmetros foram monitorizados em tempo real, por citometria de fluxo, com o intuito de se avaliar o efeito do álcool no estado fisiológico das células. Para concentrações de etanol até 10 g/L, os parâmetros avaliados foram pouco afetados, revelando um efeito de toxicidade negligenciável para ambas as estirpes. Contudo, para concentrações superiores, a estirpe selvagem revelou ser mais afetada do que a UL030 indicando que esta última é mais resistente ao álcool. Nomeadamente, na presença de 15 g/L de etanol, a estirpe selvagem revelou um decréscimo substancial na quantidade de biomassa gerada, na taxa de duplicação, e na percentagem de células com atividade metabólica assim como um aumento da percentagem de células com membrana permeabilizada. Para a UL030, o efeito do etanol só se mostrou com efeito tóxico relevante quanto superior a 20 g/L, afetando essencialmente a produção de biomassa e a atividade metabólica. Na presença de concentrações de etanol de 30 e 40 g/L não foi detetado crescimento para ambas as estirpes até às 400 h de ensaio. Contudo, no caso dos ensaios com 30 g/L, as células mantiveram a sua viabilidade e, quando a evaporação do etanol reduziu a concentração de tóxico para 15 g/L (500 h de incubação), observou-se uma recuperação de funções em termos de duplicação celular e aumento da percentagem de células com membrana intacta e atividade metabólica. Em geral, a atividade metabólica e a integridade membranar foram progressivamente afetadas com o aumento da concentração de etanol, sendo este efeito mais pronunciado sobre os sistemas metabólicos.
The aim of the present work was the study of the ethanol tolerance of two strains of Synechocystis PCC6803, the wild-type and the ethanol producing recombinant UL030. The experiments were carried out in the presence of ethanol concentrations within the range of 0 - 40 g/L, at 30 ºC and 24 μE/(m2s) of illuminance. Cyanobacteria cultures were at-line monitored in terms of biomass concentration, membrane integrity and metabolic activity. The latter parameters were evaluated by flow-cytometry in order to assess the ethanol effect on cell physiology. At concentrations of ethanol up to 10 g/L, cell growth and metabolic activity were only slightly affected, revealing a negligible toxicity effect for both strains. However, at higher levels, the wild-type was significantly more affected than UL030 which showed to be more resistant to the toxic. In the presence of 15 g/L of ethanol, the specific growth rate of the wild-type strain cultures, as well as the maximal biomass concentration attained, was substantially reduced. It was also observed a decrease in the proportion of cells with metabolic activity and an increase in the percentage of damaged membrane cells. The effect of ethanol on the UL030 cells revealed only to be negative for concentrations higher than 20 g/L of toxic. Finally, in the presence of 30 and 40 g/L of ethanol growth was completely inhibited. However, cells at 30 g/L, did not die and so, when the ethanol concentration in the vessel reached a level below 15 g/L (~500 h incubation time) a significant recovery of the vital functions was detected (duplication capability and high proportion of cells with intact membrane and with metabolic activity). In general, metabolic activity and membrane integrity were progressively more affected by the ethanol concentration increase, having this effect been more pronounced on the metabolic systems.
The aim of the present work was the study of the ethanol tolerance of two strains of Synechocystis PCC6803, the wild-type and the ethanol producing recombinant UL030. The experiments were carried out in the presence of ethanol concentrations within the range of 0 - 40 g/L, at 30 ºC and 24 μE/(m2s) of illuminance. Cyanobacteria cultures were at-line monitored in terms of biomass concentration, membrane integrity and metabolic activity. The latter parameters were evaluated by flow-cytometry in order to assess the ethanol effect on cell physiology. At concentrations of ethanol up to 10 g/L, cell growth and metabolic activity were only slightly affected, revealing a negligible toxicity effect for both strains. However, at higher levels, the wild-type was significantly more affected than UL030 which showed to be more resistant to the toxic. In the presence of 15 g/L of ethanol, the specific growth rate of the wild-type strain cultures, as well as the maximal biomass concentration attained, was substantially reduced. It was also observed a decrease in the proportion of cells with metabolic activity and an increase in the percentage of damaged membrane cells. The effect of ethanol on the UL030 cells revealed only to be negative for concentrations higher than 20 g/L of toxic. Finally, in the presence of 30 and 40 g/L of ethanol growth was completely inhibited. However, cells at 30 g/L, did not die and so, when the ethanol concentration in the vessel reached a level below 15 g/L (~500 h incubation time) a significant recovery of the vital functions was detected (duplication capability and high proportion of cells with intact membrane and with metabolic activity). In general, metabolic activity and membrane integrity were progressively more affected by the ethanol concentration increase, having this effect been more pronounced on the metabolic systems.
Descrição
Tese de mestrado, Microbiologia Aplicada, Universidade de Lisboa, Faculdade de Ciências, 2017
Palavras-chave
Etanol Synechocystis Citometria de fluxo Membrana celular Atividade metabólica Teses de mestrado - 2017