In order to better understand alga’s biology and allow to design biotechnological approaches to improve biomass yield, in this PhD thesis we investigated the molecular mechanism involved in the microalgae carbon use efficiency. In the Chapter 1 we studied the model algae C. reinhardtii. In section A Photosystem II assembly were investigated. Indeed, no detailed studies of the assembly factors of PSII have been performed. In this work we focus on a putative assembly factor of the CP43 subunit, called LPA2 (low PSII accumulation 2), previously identified in A. thaliana. A candidate lpa2 gene in C. reinhardtii was identified by homology and its role was studied in vivo thank to a CRISPR-cas9 mutant. The data collected demonstrated that LPA2 protein is involved in both de novo biogenesis and repair of PSII. In the section B the relationship between chloroplast and mitochondrion metabolism was explored studying a mutant of C. reinhardtii knockout for a mitochondrial transcription factor. Previous studies demonstrated that the mutation affect the mitochondrial respiration and resulted in a light-sensitive phenotype. In this work we investigated how a mutation affecting the mitochondrial respiration perturbed light acclimation of the strain. Chapter 2 regards two species of Chlorella genus. In the section A we elucidated the molecular basis of the improved growth and biomass yield in mixotrophic condition, where the cross-talk between chloroplast and mitochondria metabolism is essential for efficient biomass production. C. sorokiniana is able to combine an autotrophic metabolism with the utilization of reduced carbon source (mixotrophic condition). The de novo assembly transcriptome allowed to identify the regulation of several genes involved in control of carbon flux. In section B genetic basis of the highly productive phenotype of C. vulgaris in low light vs. high light condition was examined. Nuclear and organelle genomes were obtained combining short-reads Illumina, long PacBio reads and Bionano optical mapping, allowing to assembly a near-chromosome scale genome of 14 scaffolds and the two complete circular organelle genomes. All the genes encoding for photosynthetic subunit, as well as, genes involved in the key metabolic pathway were identified. In section C the two Chlorella species was compared for their adaptation to high CO2 level. In C. sorokiniana in 3% CO2 were observed several reorganizations of the photosynthetic machinery leading to an improved carbon fixation, while mitochondrial respiration was essentially unaffected. Instead, in C. vulgaris the 3% CO2 induced an improved uptake of reducing power by chloroplast leading to a reduced mitochondrial respiration. Chapter 3 is focused on the marine algae. In the section A was isolated a chemical mutant of N. gaditana with a reduction chlorophyll content per cell combined with increased lipids productivity. The mutant did not show an increased biomass accumulation but induced an increased lipid content, a class of macromolecules with a higher energy content per gram. This is in any case an indication of improved light energy conversion in line with an improved light penetration in the photobioreactor and more homogenous light availability due to the reduced chlorophyll content per cell in the mutant. Moreover, thank to Illumina sequencing, we found putative genes responsible of the observed phenotype. In the section B cells of T. weissflogii were grown together with an artificial cyanine molecular antenna (Cy5) that extends the absorbance range of the photosynthetic apparatus exploiting light energy in the orange spectral region. The dye was incorporate in the algae increasing light dependent growth, oxygen and biomass production. Time-resolved spectroscopy data indicates that a Cy5-chlorophyll a energy transfer mechanism happen, compatible with a FRET process.

Study of molecular mechanisms to increase carbon use efficiency in microalgae

michela cecchin
2020

Abstract

In order to better understand alga’s biology and allow to design biotechnological approaches to improve biomass yield, in this PhD thesis we investigated the molecular mechanism involved in the microalgae carbon use efficiency. In the Chapter 1 we studied the model algae C. reinhardtii. In section A Photosystem II assembly were investigated. Indeed, no detailed studies of the assembly factors of PSII have been performed. In this work we focus on a putative assembly factor of the CP43 subunit, called LPA2 (low PSII accumulation 2), previously identified in A. thaliana. A candidate lpa2 gene in C. reinhardtii was identified by homology and its role was studied in vivo thank to a CRISPR-cas9 mutant. The data collected demonstrated that LPA2 protein is involved in both de novo biogenesis and repair of PSII. In the section B the relationship between chloroplast and mitochondrion metabolism was explored studying a mutant of C. reinhardtii knockout for a mitochondrial transcription factor. Previous studies demonstrated that the mutation affect the mitochondrial respiration and resulted in a light-sensitive phenotype. In this work we investigated how a mutation affecting the mitochondrial respiration perturbed light acclimation of the strain. Chapter 2 regards two species of Chlorella genus. In the section A we elucidated the molecular basis of the improved growth and biomass yield in mixotrophic condition, where the cross-talk between chloroplast and mitochondria metabolism is essential for efficient biomass production. C. sorokiniana is able to combine an autotrophic metabolism with the utilization of reduced carbon source (mixotrophic condition). The de novo assembly transcriptome allowed to identify the regulation of several genes involved in control of carbon flux. In section B genetic basis of the highly productive phenotype of C. vulgaris in low light vs. high light condition was examined. Nuclear and organelle genomes were obtained combining short-reads Illumina, long PacBio reads and Bionano optical mapping, allowing to assembly a near-chromosome scale genome of 14 scaffolds and the two complete circular organelle genomes. All the genes encoding for photosynthetic subunit, as well as, genes involved in the key metabolic pathway were identified. In section C the two Chlorella species was compared for their adaptation to high CO2 level. In C. sorokiniana in 3% CO2 were observed several reorganizations of the photosynthetic machinery leading to an improved carbon fixation, while mitochondrial respiration was essentially unaffected. Instead, in C. vulgaris the 3% CO2 induced an improved uptake of reducing power by chloroplast leading to a reduced mitochondrial respiration. Chapter 3 is focused on the marine algae. In the section A was isolated a chemical mutant of N. gaditana with a reduction chlorophyll content per cell combined with increased lipids productivity. The mutant did not show an increased biomass accumulation but induced an increased lipid content, a class of macromolecules with a higher energy content per gram. This is in any case an indication of improved light energy conversion in line with an improved light penetration in the photobioreactor and more homogenous light availability due to the reduced chlorophyll content per cell in the mutant. Moreover, thank to Illumina sequencing, we found putative genes responsible of the observed phenotype. In the section B cells of T. weissflogii were grown together with an artificial cyanine molecular antenna (Cy5) that extends the absorbance range of the photosynthetic apparatus exploiting light energy in the orange spectral region. The dye was incorporate in the algae increasing light dependent growth, oxygen and biomass production. Time-resolved spectroscopy data indicates that a Cy5-chlorophyll a energy transfer mechanism happen, compatible with a FRET process.
microalgae, photosynthesis, carbon use efficiency, genome, transcriptome
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11562/1018036
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