Engenharia metabólica de Chlamydomonas reinhardtii e otimização ambiental da produção de hidrogénio

Mestrado em Biologia Funcional - Instituto Superior de AgronomiaThe H2 is an alternative fuel, with a high calorific value and its combustion is absent of reaction products that bear greenhouse effects. Hydrogen can be produced by microalgae, which have the ability to reduce free protons into H2 via the action of hydrogenases. However, the Calvin cycle is the main competitor of reducing power used by the hydrogenases, and rubisco is a key enzyme of this cycle. Taking advantage of the availability of Chlamydomonas rubisco mutants we intended to increase the hydrogen productivity by metabolic engineering. To achieve such aim two kinds of culture media, a sulfur-depleted and a repleted, in hypoxia conditions were used, and a biochemical characterization of the different mutants was performed. In this study the photosynthetic activity, the maximum photochemical efficiency, the chlorophyll, protein and starch were measured. In addition, the expression of rubisco, hydrogenase, D1 and Lhcb1 were investigated. Quantification of H2 was performed by gas chromatography. The protein Lhcb1 was more stable than the others proteins studied and D1 was deeply degraded, showing great sensitivity during the assays. It was observed that the sulfur depleted medium was more suitable than the repleted medium for H2 production. However, in complete medium under the conditions studied the H2 production was also attained. The rubisco mutant, Y67A, produced hydrogen in much higher amounts than the others mutants and control, and display more differences in the parameters studied