Early Stage Research Projects

Mathematical modelling of acclimation processes of the photosynthetic chain.

Anna Matuszynska

How algae acclimate to changing light conditions

In the process of photosynthesis, organisms are able to absorb Sun light and in a series of reactions convert it into sugars. Those highly energetic molecules are then stored in different forms in photosynthetic organisms such as plants or green algae, and can be used for various purposes, for instance as a source of energy in the form of biofuels. The way photosynthetic organisms are tuned to capture solar energy amazes researchers. If we could understand better the processes that are involved in capturing and transferring light energy, we could use this knowledge to improve how fast the green algae will grow and how many energetic molecules they will produce. And ultimately, we could improve industrial exploitation of microalgae.

We know that the availability and quality of light influence the photosynthetic efficiency, therefore my task in the project is to help to understand the level of such influences. I am focusing on how mechanisms developed by algae in order to deal with such changes affect the photosynthetic reaction. When algae are lacking light they try to absorb as much light as possible, on the other hand when light is too strong, they ‘waste’ energy to protect themselves against damage.

Based on our current understanding of the photosynthetic reactions I am developing theoretical models of the process that starts with the absorption of light and ends up with the synthesis of ATP: the trading molecule that is necessary for the production of sugars. I am using experimental data to calibrate my model and validate it and any discrepancies between my predictions and observations are considered as a gap in our theoretical understanding and a space for improvement. I hope to contribute to selecting the optimal light conditions for algae growth and to help to understand to what extent we can stimulate the energy transfer by using different light spectra and intensities.

Modelling Chloroplast Signalling Pathways and Optimisation of Photosynthesis.

Antonella Succurro

Project #1: Modelling Chloroplast Signalling Pathways How does the photosynthetic metabolic network in microalgae adapt upon changes in external light conditions and other stresses during short-time intervals? My research is focused on getting a better understanding of the signaling pathways in plants and microalgae, in particular decoding the key role of kinases and phosphatases enzymes. This project requires close collaboration with our experimental partners in Halle, which provide the fundamental proteomic data in which we search for patterns leading us to developing the model.

Project #2: Modelling Algae-Bacteria Consortia Bacteria and microalgae have co-existed for millions of years, very often establishing symbiotic relationships of various kind. We are particularly interested in mutualistic consortia, where both partners take advantage of the presence of the other species. Following a synthetic ecology approach, we want to improve the large-scale cultivation of diatoms (oil-producing microalgae of high interest in industrial applications) by identifying virtuous algal-bacterial consortia where the bacteria (which need organic carbon sources to grow) provides the algae with expensive micronutrients, like the coenzyme B12. Our experimental partners in Bantry are collecting very interesting data, while we are currently working on extending the Flux Balance Analysis (FBA) method to include the role of cofactors like vitamin B12. A software developed at Boston University (COMETS - Computation of Microbial Ecosystems in Time and Space) used dynamic FBA on a lattice to simulate bacteria consortia. We want to implement our findings in this framework to be able to model the growth of bacteria and algae colonies.

Molecular analysis of key-factors for light use efficiency in plant and algae.

Ioannis Dikaios

Photosynthesis is driven by the energy absorbed by the green pigment chlorophyll. Chlorophyll performs this task efficiently because upon absorption it builds long lasting excited states that store the energy until it can be used by reaction centers for photosynthesis. However, this property can be a weak point in the present-day, oxygen-rich atmosphere because a type of excited states - called triplets - reacts with oxygen and produce ROS (Reactive Oxygen Species) which are poisonous for the cell. Of course, triplet level should be kept very low and this is performed by the action of special proteins called PSBS and LHCSR, respectively in plants and algae. What these proteins do is transform the energy of the triplet states into heat thus making them safe. The productivity of plant and algae depends on the capacity of preventing damage without slowing photosynthesis too much. LHCSR and PSBS accomplish this by detecting the presence of excess energy from the acidification of the chloroplast (lumen) and activating energy dissipation. This makes chloroplasts less acidic and inactivates the proteins allowing for optimal photosynthetic yield. In general, organisms privilege survival vs. growth: Algae do that very well, thus dissipating more energy than needed. Engineering LHCSR and PSBS would allow tuning energy dissipation and maximizing productivity. This is what we want to do in our research: Identify the key component of the molecular switch between photosynthesis and heat dissipation in order to understand how it works and modify it for the best.

Light regulation of photosynthetic electron transfer.

Federica Cariti

All organisms on earth are continually exposed to changes in their environment, such as the atmospheric conditions or nutrient availability. Their survival depends on their ability to react and adapt to new conditions. Photosynthetic organisms are continuously required to acclimate rapidly to changes in the quality and quantity of light. Plants and green algae have developed a sophisticated mechanism to best exploit light in a dynamic environment. The organization of the photosynthetic apparatus, located in the thylakoid membranes of the chloroplast, is rapidly modified in response to different metabolic and light conditions. In particular, the light harvesting complex II (LHCII) is dynamically allocated between PSII and PSI in order to redistribute light energy between the two photosystems. This process is known as state transition. The main objective of the project is to investigate in detail state transitions in Chlamydomonas reinhardtii. The allocation of the LHCII complex is finely regulated by reversible phosphorylation. The specific roles of kinases and phosphatases involved in this process will investigated and the kinetics of state transitions will be studied in detail. Moreover, the data obtained will be used to refine a mathematical model of the regulation of photosynthesis, in collaboration with A. Matuszynska and O. Ebenhöh.

Elucidating the interplay between redox poising and remodelling of photosynthesis.

Serena Flori

Phytoplankton is part of a wide network of microorganisms floating on water, which are essential for the Marine Ecosystem. Like plants, they use solar energy to absorb CO2 and produce oxygen via photosynthesis, and it is believed that phytoplankton contributes up to 50% of the overall O2 productivity and CO2 assimilation capacity on this planet. In a dynamic environment like the Oceans, light is a crucial factor, because it can change at different time and space scales depending on latitude, seasons, coverage of the sky, and water mixing. Therefore, Phytoplanktonic organisms must be able to optimize light absorption, utilization or dissipation, depending on the external conditions. During my PhD I am comparing the capacity of light utilization in two model organisms, a marine diatom Phaeodactylum tricornutum and a green microalgae Chlamydomonas reinhardtii. I would like to understand how light is used in the chloroplast (a specific cell structure responsible for photosynthesis). To achieve this, I am studying the structure of the chloroplast and the function of the proteins contained in this compartment under different environmental conditions, which I can reproduce in the laboratory. My aim is to build a general model of algae acclimation, describing how the environment modified the structure and function of the chloroplast, using structural, biochemical and modelling approaches.

Molecular characterisation of diatom photo-acclimation and photo-protection mechanisms.

Lucilla Taddei

Diatoms are photosynthetic microorganisms living in marine and freshwater environments, that use light energy to synthetize carbon compounds from carbon dioxide and water, with the generation of oxygen. Thus, through their photosynthetic activity, diatoms contribute for about one quarter of the global primary productivity and 40% of O2 on Earth. Diatoms are able to grow and perform optimally in variable environments, and to cope with fast light and nutrients changes in moving waters. When the light absorbed exceeds the photosynthetic capacity, harmful Reactive Oxygen Species (ROS) can be generated. To avoid this, photosynthetic organisms can dissipate this energy as heat. The modulation of this process is fundamental for an efficient balance between survival and growth and a complete comprehension of it is still far from being achieved in these organisms.

It has been recently discovered that LHCX1 is a protein required for efficient light responses and growth, likely providing diatoms with a photosynthetic machinery capable of anticipating sudden changes in the underwater light field and offering a selective growth advantage in turbulent waters. In a dynamic environment like the Oceans, diatoms can also experience nutrient deprivation. Three others LHCXs proteins are present in P. tricornutum and play a role in different moments of a diatom’s life, like the light absence and nutrients deprivation. I would like to understand the role of the different LHCXs proteins in the diatoms responses to these different external conditions.

Identification of novel regulator implicated in diatom growth and photosynthesis through reverse genetic approaches.

My research project is focussed on studying photosynthesis and photo protection in the diatom Phaeodactylum tricornutum. Diatoms are prominent marine micro algae which account for ~20% of oxygen production on the planet. Living in a turbulent environment, such as oceans, diatoms have to cope with ever changing light conditions (both for light intensity and light quality, i.e. colour), which is a very stressful situation for photosynthetic organisms. But diatoms have adapted to this kind of environment very well and are able to deal with stressing light conditions much better than land plants.

Diatoms can be used, in an applied research context, to produce high added value products (pigments, anti-oxidants) or biofuels (biodiesel). The understanding of their physiology and of the environmental conditions that promote or inhibit their growth is therefore of great industrial interest.

My project involves the production of different mutants with a modified photosynthetic apparatus to study diatoms’ genetics and physiology. Photosynthesis is conducted in chloroplasts, small subunits of plant and diatom cells; thanks to Cellectis’ know-how on genomic engineering, I can produce enzymes able to cut and modify DNA, called nucleases. With these enzymes I can introduce targeted mutations in specific genes involved in chloroplast activity in order to be able to better understand how these processes work.

In addition I will try to understand the connection between circadian rhythms and photosynthesis regulation: circadian rhythms are endogenous oscillations of biological processes that in animals control, for instance, sleeping time and appetite. In diatoms little is known about circadian regulation of photosynthesis and we will investigate if anticipation of day-to-night switch and vice-versa can help diatoms to increase their fitness and better regulate the components of their photosynthetic apparatus.

Since diatoms’ physiology is still poorly understood these studies will help to improve our knowledge on the dynamic regulation of their photosynthetic apparatus. A better understanding of diatoms physiology will allow us to choose the best growing conditions and eventually to modify selected genes to increase biomass production and specific metabolite accumulation, such as pigments, anti-oxidants or lipids for biodiesel production. The final aim is in fact to increase of micro algae’s performances in photobioreactors for industrial value products synthesis.

Establishing connections in the photosynthetic phosphoproteome network.

ESR project MLU Halle, Germany

Plants use light at certain wavelengths with just the right amount of energy to produce sugars. Light intensity varies throughout the day, and the leaf must adjust how this energy is distributed among the light collecting systems in order to best use it. The two light gathering protein complexes can each absorb a certain light quality, and the whole system works best when the amount of light that they each absorb is balanced. For this to happen, many types of proteins will work together to control the energy distribution between the light harvesting systems in the leaf. These proteins must also be regulated so that they do their job when required and stop when they are not needed. This is achieved by switching the proteins on or off using two chemical reactions called phosphorylation and de-phosphorylation. This means that a phosphate ion is either attached (phosphorylation) or removed (de-phosphorylation) from a protein by an enzyme that works to give or take away phosphate ions. Depending on the protein, the addition or removal of a phosphate ion will often change the properties of the protein, and this turns it on so that it can do its job, or turns it off so that it stops.

In this project, phosphate-adding enzymes, called kinases, and the phosphate-removing enzymes, called phosphatases are studied. The goal is to elucidate which proteins are turning on or off and under what light conditions this is happening. To this end, the modification of proteins will be analysed by a method called mass spectrometry. This method measures the masses of peptides and uses the fact that phosphate-carrying peptides are heavier than their non-phosphorylated counterparts to distinguish them.This type of analysis will be performed in the absence and the presence of a specific kinase or phosphatase, to identify the targets of these enzymes by finding the differences between these experimental conditions.

Regulation of photosynthetic carbon metabolism.

Martina Zanella

The aim of my PhD project is to investigate the role of light in regulating the process of photosynthesis in plants. As autotrophic organisms, plants are capable of capturing light energy and converting it to chemical energy. They use this energy to transform water and atmospheric carbon dioxide into carbohydrates; a primary energy source both for the plants themselves and for animals and microorganisms that depend on them. The biochemical process by which plants make carbohydrates during photosynthesis is called the Calvin-Benson cycle, named after the scientists who discovered it. This cycle is driven by a set of enzymes – proteins found inside the chloroplast organelles in green leaves.

Being sessile organisms, plants have developed a variety of strategies to cope with the wide range of different environmental conditions that they experience, both diurnally and throughout their life cycle. One of the most obvious fluctuating parameters is the constant change in light intensity connected to the day/night cycle. Numerous regulatory mechanisms have evolved to adapt the speed at which the Calvin-Benson cycle operates to bring it into line with the input of light energy.

My research will focus on evaluating the impact of such light-dependent regulatory mechanisms using the model plant Arabidopsis thaliana. I will create a set of Arabidopsis transgenic plants that instead of their normal, regulated enzymes carry altered versions of the enzymes that are insensitive to light-dependent regulation. It is likely that photosynthesis in these plants will behave quite differently from the originals when exposed to variations in light intensity.

Because Arabidopsis is the number 1 model plant for scientific investigation, many different techniques are already available to help me achieve this goal quickly and thereby gain a much deeper understanding of the light regulatory effects on photosynthesis. This fundamental knowledge will be of great value not only for the scientific community, but also for more applied fields like agronomy and crop science. This is important because agriculture faces a looming challenge in terms of providing food, fibre and fuel for an increasing population, despite diminishing fossil fuel reserves and greater climate instability.

Genome-scale metabolic model of Arabidopsis thaliana and Chlamydomonas reinhardtii.

Kailash Adhikari

Human life is dependent on different plant products and with growing population, demand for food and energy is increasing but resources to meet them remains limited on earth, so there is a need to find the ways to balance this chain. Green plants and algae can synthesize useful nutrients using carbon dioxide, water and sunlight form the environment by a process called photosynthesis and it is possible to engineer their metabolic process to increase the yield and also use them for production of biofuels. This project is focused on use of computational modelling techniques to study and analyze the metabolic behavior of Arabidopsis thaliana - a multicellular flowering plant and Chlamydomonas reinhardtii - an unicellular green algae in response to light and other environmental conditions. In order to under-stand their metabolism and engineer them, genome-scale metabolic models (GSM) of both will be assessed and analyzed. A GSM represents the entire metabolic capabilities of an organism and is built from data extracted typically from annotated genome databases. Linear Programming (LP) will then be used to explore distribution of reaction rates i.e. the effect of each reactions over the metabolic network under variety of assumed environmental conditions. LP is a mathematical method to compute an optimal solution under given parameters such as maximizing biomass production in our case. The experimental part of the project involves examination of biomass composition, signaling pathways etc and will be carried out in collaboration with other partners in the consortium. Both modelling and experimental results will be analyzed to identify potential cause of damage to the plants form environmental stress conditions and formulate strategies to mitigate such effects to obtained desired outputs form their metabolic activity. New hypotheses will be proposed based on these observations about operating characteristics of metabolic networks of Arabidopsis and Chlamydomonas and will be tested in collaboration with experimental and industrial partners.

Metabolic models of Phaeodactylum tricornutum.

Dipali Singh

Energy demand has increased tremendously over last decades and continuous use of fossils fuels to meet this demand has not only depleted the fossil fuels but has also raised concerns regarding global climate change. Therefore, there is need to find alternative source of energy in order to meet the future energy demand and to protect the global environment.

Phaeodactylum tricornutum, a species of diatom, is a single celled organism which can use light energy and CO2 from the environment to produce useful storage products. They contribute up to 40% of organic matter production in the ocean and have the capability to store lipid which makes them an important candidate for exploitation and raises new possibilities to increase algal oil production. However, we need to understand their metabolism, to manipulate their pathways and to optimise quantity and composition of lipid, in order to make them an economically valid alternative source of energy.

The technique used in this project, to understand the metabolism of diatoms, is to construct a genome scale metabolic model (GSM) which describes the reaction networks predicted from enzymes encoded by the genome. This allows us to predict the essential reactions involved with particular pathways and to optimise the pathways for the production of useful products.

The goal of this project is to construct a GSM of Phaeodactylum tricornutum by identifying all the enzymes of metabolism that are encoded in the genome and to refine the model so that it is biologically relevant. The model will be analysed for underlying biological properties and behaviour of system under various conditions such as varying light intensities, energy demand and stress conditions. Based on this analysis, strategies will be developed to identify optimised pathways for lipid production and experiments will be designed to test such strategies.

Investigation of the feasibility of large-scale culture at a pilot plant.

Fiona Moejes

The research project is based at Daithi O’Murchu Marine Research Station in Bantry, Ireland. The project aims to evaluate the efficiency of Phaeodactylum tricornutum strains for biofuel production in large-scale industrial outdoor bioreactors and assess the extent to which the models developed for controlled laboratory conditions are applicable to outdoor, industry-scale bioreactors.

One of the main obstacles encountered when scaling up the cultures is the issue of contamination. Rather than looking at contaminants as a negative phenomenon, the project took a new direction looking at these contaminants as symbionts of Phaeodactylum tricornutum – could there be an advantage to having contaminants in the cultures? More specifically, the project is looking at the symbiotic relationship between the bacteria and diatoms in culture systems. Research has shown that bacteria provide the diatoms with essential nutrients required for optimum growth, including vitamin B12 and iron. These nutrients are expensive, especially when needed for large-scale cultures. The bacteria could provide these nutrients for free, therefore keeping costs down. Understanding the interactions will also allow us to create an artificial ecosystem within the cultures using the organisms we want, and therefore reducing the number of unwanted contaminants.

Making microalgal-derived biofuels economically viable is another aspect of the project. Keeping costs as low as possible during the culturing phase is the key to making biofuels a reality. This includes using cheap nutrient sources such as fertilisers and wastewater. Another interesting aspect is looking at culturing microalgae to produce high-value by-products as well as lipids for biofuel production. This will make the culturing process economically viable as the cost of the production process could be covered by the value of the by-products.

All of these aspects of the scaling up process must be weighed up to find the optimum culturing process for industry-scale culturing, and make the availability of microalgal-derived biofuels as an alternative fuel source a reality.

Development of a biochemically based structured model for algal growth in a photobioreactor.

Brieuc Urbain

There have been significant advances in the understanding of the metabolism of microalgae in the past decades and there is now a huge amount of data available on the subject. There is, however, a gap of knowledge as to how these organisms can adapt so well to their changing environment. Most recent mathematical models try to focus on some of the aspects but do not always include data from both the metabolism and engineering. The project aims to develop and refine a mathematical model capable of predicting the growth of the microalga Chlamydomonas reinhardtii, which can reliably represent the adaptation of metabolism within changing environment in photobioreactors (PBRs). The model will be biochemically structured, meaning it will explicitly express energy-yielding and energy-requiring metabolic processes, which ought to be taken into account in the overall biomass growth process. In order to do so, prior knowledge of the metabolic network and energetics coupling is required, requiring specific metabolic investigations that will consider the microalga as a “cell factory” capable of transforming and storing energy under various forms. The model will be compared to experiments at the population scale in PBRs, where the growth parameters can be controlled: light, temperature, pH, dissolved gases being the most common. Because light is the main energy source for growth under autotrophic conditions, light availability within PBR ought to be fully characterized and analyzed during culturing so that photon uptake rate can be used as model input. Using the PBR as a platform, the model parameters will be assessed experimentally and/or identified in order to make the model as robust as possible. The objective is to develop a tool that would be as deterministic/generic as possible, in order to extend it to other microalgal species (eg: Phaeodactylum tricornutum) in the future.

Optimising diatom growth and yield by mixotrophic growth.

Valeria Villanova

Microalgae are unicellular microorganisms able to use the light as an energy source via photosynthesis like plants. Moreover, microalgae are capable of growing in the dark, via the respiration of a carbon source. Eventually, a few microalgae can combine photosynthesis and respiration for growth, so that both processes are optimised. In my PhD I am studying this process (called mixotrophy) using a model microalgae Phaeodactylum tricornutum and glycerol as carbon source. This carbon source supports the growth of P.tricornutum and leads to the production of high-value products (e.g. lipids), which can be used for bioenergy (e.g. biofuel production). The aim of my project is first to understand the consequences of this molecule on the respiration and photosynthesis in P.tricornutum. In addition, I am also trying to identify other molecules that promote mixotrophy with a comparable effect of glycerol using special microplates that allows the simultaneous screening of ~ 100 possible substrates. This part of the work is done in a research institute in Grenoble. The second part of my thesis will focus on the industrial exploitation of my results for bioenergy applications. This will be done in an industrial biotechnology company (Fermentalg) located in the south west of France (Libourne). Here, I will focus on the optimization of yield and productivity of P.tricornutum, using both light and selected carbon sources, in large scale culture systems (e.g. photobioreactors).

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The Marie Curie Initial Training Network AccliPhot is funded by the European Union under the Seventh Framework Programme (SP3-People) under the grant agreement number PITN-GA-2012-316427