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Genetic engineering approaches to increase microalgae light use efficiency
Full text linkIn the next fifty years world population will reach 9-10 billion of people. This increment will drastically increase energy and food demand. The current global food and energy supply chain is not sustainable and it causes increasing CO2 emissions, exacerbating the greenhouse effect and the climate change, whose effects we are already experiencing. The Climate Change conference hold in Paris in 2015 showed a global consensus on the need to drastically reduce carbon emissions to avoid the environmental disaster, which is leading to many negative effects like desertification, animal and plants species extinction and ocean acidification.
In order to face this challenge different strategies are under study and development. Microalgae are emerging as an interesting possibility for the production of energy and food. Being photosynthetic organisms, algae biomass is produced from CO2 fixation, and contains proteins, and lipids exploitable as food or fuel.
Despite their potential, microalgae production on a large scale is still not competitive on the food and energy market. One of the main limitation is that photosynthetic efficiency at the industrial scale is reduced, negatively affecting growth and biomass accumulation. All first attempts of algae large scale cultivation have been pursued cultivating wild type strains (WT) that evolved in an environment extremely different and thus they are not adapted to have a maximal productivity in the industrial system. As done with crops domestication, there is the need to modify genetically strains in order to adapt them to grow in industrial systems and increase productivity. There are different algal species that are emerging as promising candidates for food and fuel production and among them the largest part of this work is focused on Nannochloropsis gaditana, a marine microalga able to accumulate lipids and molecules like β-carotene interesting for nutraceutical purposes.
After a general introduction, chapter 2 described the generation of a collection of random mutants of N.gaditana exploiting two different mutagenic approaches, one based on the use of the mutagen compound Ethyl Methane Sulfonate (EMS) and the other one based on random insertion of a resistance cassette to zeocin. The collection was screened for strains altered in the photosynthetic apparatus using a multiple steps in vivo fluorescence analysis. In Appendix 1 I collaborated in evaluation of the productivity for one of these strains (E2) in a fed batch system mimicking an industrial culture. E2 was selected for a reduction in the chlorophyll content and also in the PSII antenna size. One of the limitations in algae mass cultures is that light penetration negatively affects growth and thus a strain with a reduction in the light absorption capacity could have a productivity advantage. This work indeed showed that the strain E2 had increased productivity with respect to WT, but also highlighted the seminal influence of the cultivation parameters on strains performances.
Most of the PhD work was then invested on the characterization of two promising insertional strains. The first strain presented in chapter 3 is I29. This is a low chlorophyll (Chl) content mutant with no difference in the antenna size of PSII. In flask culture I29 showed a reduction of 20% in the Chl content respect to the WT, which leaded also to a higher electron transport rate (ETR). The site of insertion of the resistance cassette was identified but there was no apparent effect on transcription level of closest genes. The full genome of the strain was thus re-sequenced revealing a point mutation in a key gene involved in the chlorophyll biosynthetic pathway, a highly likely candidate to justify the phenotype. I29 in lab scale PBR was also assessed evidencing an increase in productivity of 14% with respect to the WT.
An analogous work was done in chapter 4 for the characterization of another interesting mutant from the collection isolated in chapter 2, the mutant I48. This mutant has a severe reduction in the non-photochemical quenching (NPQ) activation. The site of insertion was not identifiable because of the presence of tandem insertions and again the strain genome was re-sequenced highlighting the presence of point mutations due to electroporation. Among these we found an interesting one in the gene codifying for the protein LHCX1. This is a stress-related antenna protein which in other species like Phaeodactylum tricornutum has already been recognized as a key component of the fast NPQ response. The loss of the protein accumulation was confirmed by Western Blot. Strain response to different illumination conditions was also evaluated highlighting the ability of the mutant to grow also in high light condition. The last part of the characterization was devoted to productivity evaluation since a depletion of NPQ could be advantageous in an industrial condition of a dense culture, where only the external layer are exposed to intense light. A low NPQ phenotype can avoid undesired energy dissipation in the internal layers. We observed a 24% higher productivity for I48 respect to the WT but, as discussed in Appendix 1 this was dependent from growing conditions.
In diatoms NPQ has been associated not only to the high light response but also to the fluctuating light (FL) response, which is a condition easily experienced in outdoor cultivation system. We take advantage from the isolation of this mutant with a severe reduction in NPQ to verify what can be the impact of NPQ in N.gaditana response. In chapter 5 we set up an experiment in which we exposed the cultures to 150 μmol of photons m-2s-1 but in the control sample the light intensity was constant, in FLs it was the result of a combination of a low light exposition with high light flashes given with different frequencies and duration. We found that those FL treatments caused a severe growth reduction in WT but I48 showed no increased sensitivity. The growth reduction was proportional to the flash frequency and stronger with higher flash frequency. An analysis of photosynthetic apparatus functionality evidenced that in FL PSII activity is not affected while PSI is largely inactivated. This suggests that alternatives electron transport around PSI, that are active in avoiding its over-excitation, are not as efficient in N.gaditana as in other organisms.
These first 5 chapters highlight the potential of microalgae engineering to find new strains more productive respect to the WT. FL experiment not only adds new knowledge about NPQ role in N.gaditana, but taking in count that FL is a condition easily found if we want to exploits sunlight in industrial system, it can be the starting point to find new gene targets to improve productivity. This work started with an insertional approach that in principle should facilitate identification of the genes responsible of the phenotype. In this case this advantage is impaired by the frequent presence of tandem insertions and accumulation of point mutations during transformation. Alternative strategies are thus more suitable and will be pursued in the future.
In chapter 6 we tested the chemical mutagenesis and the screening method set up for N.gaditana with Scenedesmus obliquus to evaluate if the approach could be generally applied to any species of interest. Indeed at least a strain with interesting properties was isolated (SOB17). In this work we also evaluated productivity in a flat panel in continuous mode. Indeed the geometry of a flat panel leads to a better light distribution and homogeneity respect to other system like the bottles. Moreover the continuous mode provides a stable culture continuously producing. This culture showed remarkable stability for more than 30 weeks and SOB17 showed a higher productivity in one of the conditions tested but, as observed for Nannochloropsis gaditana strains also in this case operational conditions were shown to have a fundamental influence on strains performances.
In addition to the Appendix 1 described above, other two appendix sections are also included in the thesis. They are the results of two different collaboration in which some of the experimental techniques exploited in this work were applied with different aims.
In Appendix II, there is the published work “Genetic Engineering of algae photosynthetic productivity using mathematical models” that describes the development of a mathematical model to evaluate the key factors influencing algae biomass productivity in PBR. The model was shown to be able to predict the effect of genetic modifications on algae performances in an industrial context, thus providing a valuable tool to identify the genotypes with the best advantages for productivity.
In Appendix III is reported the collaboration with Prof. Angela Falciatore (Diatom Functional Genomics team, at the UPMC-Paris). A screening procedure similar to those one set up for N.gaditana was adapted to search photosynthetic alterations in a collection of Phaeodactylum tricornutum transgenic lines genetically modified by the RNA interference approach, in order to modulate the expression of transcription factors (TF). Thank to this screening procedure we found some promising correlation between the genetic modulation of a class of TFs and the photosynthetic phenotype of mutants strains isolated. Now this correlation phenotype-TF expression level is under validation by confirming the screening phenotype and evaluating the transcripts level
Photosynthesis regulation in response to fluctuating light in the secondary endosymbiont alga Nannochloropsis gaditana
Full text linkIn nature, photosynthetic organisms are exposed to highly dynamic environmental conditions where the excitation energy and electron flow in the photosynthetic apparatus need to be continuously modulated. Fluctuations in incident light are particularly challenging since they drive oversaturation of photosynthesis, with consequent oxidative stress and photoinhibition. Plants and algae have evolved several mechanisms to modulate their photosynthetic machinery to cope with light dynamics, such as thermal dissipation of excited chlorophyll states (Non-Photochemical Quenching, NPQ) and regulation of electron transport. The regulatory mechanisms involved in the response to light dynamics have adapted during evolution and exploring biodiversity is a valuable strategy for expanding our understanding of their biological roles. In this work, we investigated the response to fluctuating light in Nannochloropsis gaditana, a eukaryotic microalga of the phylum Heterokonta originating from a secondary endosymbiotic event. N. gaditana is negatively affected by light fluctuations, leading to large reductions in growth and photosynthetic electron transport. Exposure to light fluctuations specifically damages photosystem I, likely because of ineffective regulation of electron transport in this species. The role of Non-Photochemical Quenching, also assessed using a mutant strain specifically depleted of this response, was instead found to be minor, especially in responding to the fastest light fluctuations
Acclimation of photosynthesis and lipids biosynthesis to prolonged nitrogen and phosphorus limitation in Nannochloropsis gaditana
No full textMicroalgae represent a potential sustainable source of molecules and materials and the improvement of the knowledge of their metabolic regulation is essential to maximize their potential. Nutrients deprivation stimulates the accumulation of reserve lipids, triacylglycerols but also inhibits photosynthesis with a negative impact on biomass production and therefore overall productivity. In this work, the seawater microalga Nannochloropsis gaditana was cultivated long term in a semi-continuous system where the concentrations of nitrogen or phosphorus were limiting but still sufficient to sustain growth indefinitely, to highlight the response to a long-term nutrient limitation and distinguishing it from the one to acute short-term stress. N. gaditana cells can acclimate to chronic nutrients limitation maintaining photosynthetic activity while also accumulating lipids. Both nitrogen and phosphorus limitation induced an increase of triacylglycerols content, although not by the induction of the synthesis of fatty acids but rather by modulating the fluxes of reduced carbon molecules toward lipid biosynthesis. Photosynthetic activity was maintained under P limitation while this was strongly affected by nitrogen depletion, where proteins of photosynthetic apparatus were largely reduced in content but still maintained their functionality and were able to achieve half of the biomass productivity with 30% of the nitrogen supply
The potential of quantitative models to improve microalgae photosynthetic efficiency
Full text linkThe massive increase in carbon dioxide concentration in the atmosphere driven by human activities is causing huge negative consequences and new sustainable sources of energy, food and materials are highly needed. Algae are unicellular photosynthetic microorganisms that can provide a highly strategic contribution to this challenge as alternative source of biomass to complement crops cultivation. Algae industrial cultures are commonly limited by light availability, and biomass accumulation is strongly dependent on their photon-to-biomass conversion efficiency. Investigation of algae photosynthetic metabolism is thus strategic for the generation of more efficient strains with higher productivity
A Mathematical model to guide Genetic Engineering of Photosynthetic Metabolism
Full text linkThe optimization of algae biomass productivity in industrial cultivation systems requires genetic improvement of wild type strains isolated from nature. One of the main factors affecting algae productivity is their efficiency in converting light into chemical energy and this has been a major target of recent genetic efforts. However, photosynthetic productivity in algae cultures depends on many environmental parameters, making the identification of advantageous genotypes complex and the achievement of concrete improvements slow. In this work, we developed a mathematical model to describe the key factors influencing algae photosynthetic productivity in a photobioreactor, using experimental measurements for the WT strain of Nannochloropsis gaditana. The model was then exploited to predict the effect of potential genetic modifications on algae performances in an industrial context, showing the ability to predict the productivity of mutants with specific photosynthetic phenotypes. These results show that a quantitative model can be exploited to identify the genetic modifications with the highest impact on productivity taking into full account the complex influence of environmental conditions, efficiently guiding engineering efforts
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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