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Biotechnological optimization of microalgae for the sustainable production of biocommodities
Full text linkThe current global economy development trends strongly rely on fossil fuels exploitation, which are responsible for a net greenhouse gases (e.g. CO2) release in the atmosphere. In 2014, the Intergovernmental Panel on Climate Change (IPCC) stated that this net atmospheric CO2 increase is anthropogenic, and it will lead to the rising of the global temperature of > 2 °C, before the end of this century. The latter will strongly contribute to change the behavior of climate and oceans (e.g. leading to their acidification and oxygenation) in a permanent way, with a consequent magnification of the demographic pressures on food and water security, as well as on several ecosystems functional bio-diversity.
To avoid this apocalyptic scenario, the development of renewable and clean energy sources to sustain a consistent part of the global economy is an unavoidable challenge for our society. A plant biomass-based economy could meet this need, but several studies predicted a food prices inflation and a concrete carbon debt, as consequence of this scenario. On the contrary, the exploitation of microalgae biomass could indeed avoid these issues and bring a positive effect on atmospheric CO2 levels, leading to its sequestration and fixation in organic carbon. Currently, microalgae are indeed the best CO2 sequestering organisms, thanks to higher photosynthetic rates with respect to plants. Their ability to grow on marginal lands and use wastewaters opens the doors for their application as environment-impact mitigating agents of current industrial processes. Their exploitation could be indeed the key for the development of an integrate process in which the biomass would be used to convert the majority of the current economy toward environmental-friendly processes.
Despite this promising scenario, mainly wild type microalgae species are currently available for these purposes. Their evolution in a natural environment, different from the artificial one exploited during their intensive industrial cultivation, strongly impairs their theoretical biomass productivities, leading to unsatisfactory values. The development of an algae-based economy indeed depends on the efficient conversion of light energy into biomass and the optimization of metabolic pathways to maximize the synthesis of the products of interest. Still far from the development of an economically competitive and energetically sustainable microalgae industrial cultivation, these organisms therefore need to undergo a biotechnological optimization to achieve the competitiveness threshold.
Although there isn’t an ideal species that could serve to meet all human needs, we focused on the seawater species Nannochloropsis gaditana, which is a promising candidate for both basic biological and applied investigations. The following PhD thesis was conceived to provide a molecular insight on N. gaditana photosynthetic efficiency and metabolic regulation, to provide the molecular targets for its biotechnological improvement. The prerequisite of this experimental work was the optimization of the currently available molecular toolkit for N. gaditana genetic manipulation and also the available molecular information was therefore significantly improved.
In chapter II random mutagenesis approaches were performed in order to isolate mutant strains with photosynthetic phenotypes, likely more suitable for intensive growth conditions. Selected strains indeed showed an improved photosynthesis in limiting growth conditions, also serving as biological tools to improve the available information on the underlying molecular elements controlling light-use efficiency in this organism. When tested in lab-scale growth conditions, the strain E2, selected for a reduction in its Chl content, indeed showed an improved biomass productivity, therefore representing a proof of concept for the developed biotechnological approaches, to able to improve the photosynthetic performances of this organism in the artificial environment of a photobioreactor. Among the isolated mutant strains, in chapter III two major photosynthetic phenotypes, the reduction in the Chl content (strain E2) and the inability to activate NPQ (strain I48), were chosen as selection criteria to improve light penetration and energy conversion mechanisms, respectively, in an intensive culture. Both indeed showed an enhanced biomass productivity in industrial-simulating cultures, proving the theoretical advantage underneath their exploitation. However, when the growth conditions changed, leading to altering the light availability, the selected mutants altered their behavior. They weren’t able of performing better than the WT in all the tested conditions. This would explain why the data published to date for the same mutants in other species often provided contrasting results. We concluded that photosynthetic mutants can modulate their phenotype in relation to the growth conditions and some of the latter could indeed highlight their drawbacks rather than their benefits, therefore the genetic engineering efforts have to be tailored properly to the growth conditions used.
The forward genetics strategy here developed could open the doors toward the identification of the molecular basis regulating photosynthesis in this promising species. In chapter IV mutant strain I48 was further investigate for the identification of the genetic basis of its phenotype. Thanks to the whole genome re-sequencing we identified a splicing variant in the 5’-donor splicing site of the 4th intron of the gene Naga_100173g12, which encodes for the LHCX1 protein. This mutation caused the retention of the intron sequence, leading to a truncated protein product, which is likely degraded. The absence of the LHCX1 protein strongly correlates with inability to activate NPQ, since this proteins clade is well known to be involved in the activation of this mechanism. However, the future complementation of the phenotype will serve to validate this conclusion. Moreover, the LHCX1 protein was found co-localized with the PSI in N. gaditana, therefore strain I48 could also serve as an optimal tool to investigate further its biological role.
To develop an efficient biotechnological optimization strategy, the information on the metabolism regulation of N. gaditana has to be highly enriched. Understanding the metabolic fluxes direction could lead to specifically affect those involved in a specific product accumulation, without affecting other pathways, leading to a possible negative impact on growth. In chapter V an integrated analysis of genome-wide, biochemical and physiological approaches helped us in deciphering the metabolic remodeling of N. gaditana that switches its metabolism toward a greater lipid production in excess light conditions. The latter indeed induced the accumulation of DAGs and TAGs, together with the up-regulation of genes involved in their biosynthesis. We saw the induction of cytosolic fatty acids synthase (FAS1) genes and the down-regulation of those of the chloroplast (FAS2). Lipid accumulation is accompanied by the regulation of triose phosphate/inorganic phosphate transport across the chloroplast membranes, tuning the carbon metabolic allocation between cell compartments and favoring the cytoplasm and endoplasmic reticulum at the expense of the chloroplast. This highlighted the flexibility of N. gaditana metabolism to respond to environmental needs.
In chapter VI the information gained from the latter work was exploited to test the potentiality of this prosing species also as protein expression platform. We built up a modular system for protein overexpression in which the regulatory sequences were chosen among those which revealed to induce a high level of transcription or to be highly regulated by light availability. N. gaditana revealed to be a very promising host for protein expression, given the higher luciferase activity monitored with respect to the reference species for these applications, C. reinhardtii. A method to test the efficacy of several regulatory sequences in driving proteins expression was developed, as well as several expression vectors, which are ready to be tested.
The investigation of the N. gaditana metabolism regulation in chapter V, showed a fine tuning of its photosynthetic apparatus components, in different light conditions. Focusing on LHC proteins we identified a new LHCX protein in this species, called LHCX3 (GENE ID: Naga_101036g3), whose gene coding sequence wasn’t annotated correctly. In chapter VII the correct coding sequence of this gene was further investigated and experimentally validated with molecular techniques. The LHCX3 protein revealed to be fused with an N-terminal fasciclin I-like domain and a sequence analysis together with a preliminary evolution study was performed to infer the biological role of this association.
Since algae metabolism entirely relies on light availability, the importance of investigating the light intensity effect on growth is seminal for their industrial application. In chapter VIII we developed a micro-scale platform, that we called micro-photobioreactor, to easy investigate the impact of light intensity on N. gaditana growth. We were able to test the effect of different light regimes, simultaneously, also on the photosynthetic performances in an integrate system which could be merged with nutrients availability studies, speeding up the N. gaditana characterization process.
Three appendix sections are also included in the thesis in which some of the experimental techniques exploited in this work were applied to different organisms toward the common target of investigating light-use efficiency and the molecular elements involved in its regulation.
In appendix I, the development of a mathematical prediction model for growth and fluorescence data of the species Nannochloropsis salina was described. The work was carried out in collaboration with Prof. Fabrizio Bezzo of the industrial engineering department of the University of Padova.
The development of behavior prediction models representing the phenomena affecting algae growth, could be very helpful in designing and optimizing the production systems at industrial level.
The developed model well represented N. salina growth over a wide range of light intensities, and could be further implemented to describe also the influence on growth of other parameters, such as nutrients availability and mixing.
In appendix II, the monitoring of the in vivo chlorophyll fluorescence was exploited to study the photosynthetic features of rice plants exposed to salt stress conditions. The presented results are part of a wider project (in collaboration with Prof. Fiorella Lo Schiavo from the biology department of the University of Padova), aiming to depict the physiological, biochemical and molecular remodeling, undergoing in one of the major food crop in the world, in response to salt stress conditions.
Depicting the impact of environmental stresses on photosynthesis is seminal to control biomass productivity since plants metabolism strongly relies on the former for growth. We showed the activation of the NPQ mechanism in salt tolerant plants, highlighting the importance of photosynthetic features monitoring to predict plants performances, directly on the field.
In appendix III, Chlamydomonas reinhardtii 13C – 15N labeled thylakoids were isolated from the cw15 and npq2 mutant strain in order to study their structure and dynamics in term of protein and lipid components in situ, by applying the solid-state NMR technique, in collaboration with Prof. Anjali Pandit group from the Leiden Institute of Chemistry. These analyses will serve to investigate the photosynthetic membranes remodeling that undergoes from an active (cw15 strain) to a photo-protective state (npq 2 mutant strain), during the switch toward excess light conditions, with the final aim to understand the biochemical processes regulating this event
Understanding regulation in complex environments: a route to enhance photosynthetic light-reactions in microalgae photobioreactors
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Economic feasibility and long-term sustainability criteria on the path to enable a transition from fossil fuels to biofuels.
Full text linkCurrently the production of liquid biofuels relies on plant biomass, which in turn depends on the photosynthetic conversion of light and CO2 into chemical energy. As a consequence, the process is renewable on a far shorter time-scale than its fossil counterpart, thus rendering a potential to reduce the environmental impact of the transportation sector. However, the global economy is not intensively pursuing this route, as current generation biofuel production does not meet two key criteria: (1) economic feasibility and (2) long-term sustainability. Herein, we argue that microalgal systems are valuable alternatives to consider, although it is currently technologically immature and therefore not possible to reach criterion 1, nor evaluate criterion 2. In this review we discuss the major limiting factors for this technology and highlight how further research efforts could be deployed to concretize an industrial reality
Potential of microalgae biomass for the sustainable production of bio-commodities
Full text linkHuman activities are causing major negative environmental impacts, and the development of sustainable processes for production of commodities is a major urgency. Plant biomass represents a valuable alternative to produce energy and materials, but exploiting present crops for commodities production would however require massive resources (i.e. land, water and nutrients), raising serious sustainability concerns. In addition to efforts to improve plant, land and resource use efficiency, it is thus fundamental to look for alternative sources of biomass to complement crops. Microalgae are unicellular photosynthetic organisms that show a huge, yet untapped, potential in this context.
Microalgae metabolism is powered by photosynthesis and thus uses sunlight, a renewable energy source, and the exploitation of microalgae-based products has the potential to provide a beneficial environmental impact. These microorganisms have the ability to synthesize a wide spectrum of bioactive compounds, with many different potential applications (e.g. nutraceutics/pharmaceutics and biofuels). Several, still unresolved, challenges are however present such as the lack of cost-effective cultivation platforms and biomass-harvesting technologies. Moreover, the natural metabolic plasticity of microalgae is not optimized for a production at scale, and low biomass productivity and product yields affect competitiveness. Tuning microalgae metabolism to maximize productivity thus represents an unavoidable challenge to reach the theoretical potential of such organisms
Novel micro-photobioreactor design and monitoring method for assessing microalgae response to light intensity
No full texticroalgae represent a promising feedstock for the sustainable production of biofuels and many other bio-commodities. However, the optimization of their productivity by assessing the impact that several environmental factors have on microalgae growth still requires intense investigation, as well as numerous and time consuming experiments. In this scenario, microscale technologies are emerging as a valuable tool to improve data production and accordingly to speed up the optimization process, while maintaining a high experimental reliability. Using these approaches for microalgae present additional challenges because of the necessity of dealing with light as a source of energy to support cell metabolism. Currently developed microscale platforms are complex, often requiring a deep experience in the microfluidic field to monitor the device functionality. To overcome this drawback while keeping microscale benefits, here we describe the use of a simple and flexible micro-photobioreactor (micro-PBR) coupled with a quick and reliable growth evaluation method. The system is demonstrated to sustain optimal growth of Nannochloropsis gaditana cells, allowing to assess the impact of different light intensities and to monitor the cells photosynthetic functionality by measuring chlorophyll (Chl) fluorescence in vivo, while providing for a significant time reduction when compared to traditional experimental approaches
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
Knowledge of regulation of photosynthesis in outdoor microalgae cultures is essential for the optimization of biomass productivity
Full text linkMicroalgae represent a sustainable source of biomass that can be exploited for pharmaceutical, nutraceutical, cosmetic applications, as well as for food, feed, chemicals, and energy. To make microalgae applications economically competitive and maximize their positive environmental impact, it is however necessary to optimize productivity when cultivated at a large scale. Independently from the final product, this objective requires the optimization of biomass productivity and thus of microalgae ability to exploit light for CO2 fixation. Light is a highly variable environmental parameter, continuously changing depending on seasons, time of the day, and weather conditions. In microalgae large scale cultures, cell self-shading causes inhomogeneity in light distribution and, because of mixing, cells move between different parts of the culture, experiencing abrupt changes in light exposure. Microalgae evolved multiple regulatory mechanisms to deal with dynamic light conditions that, however, are not adapted to respond to the complex mixture of natural and artificial fluctuations found in large-scale cultures, which can thus drive to oversaturation of the photosynthetic machinery, leading to consequent oxidative stress. In this work, the present knowledge on the regulation of photosynthesis and its implications for the maximization of microalgae biomass productivity are discussed. Fast mechanisms of regulations, such as Non-Photochemical-Quenching and cyclic electron flow, are seminal to respond to sudden fluctuations of light intensity. However, they are less effective especially in the 1–100 s time range, where light fluctuations were shown to have the strongest negative impact on biomass productivity. On the longer term, microalgae modulate the composition and activity of the photosynthetic apparatus to environmental conditions, an acclimation response activated also in cultures outdoors. While regulation of photosynthesis has been investigated mainly in controlled lab-scale conditions so far, these mechanisms are highly impactful also in cultures outdoors, suggesting that the integration of detailed knowledge from microalgae large-scale cultivation is essential to drive more effective efforts to optimize biomass productivity
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