Nowadays, the environment is becoming increasingly deeply affected by high levels of pollution, chemical processes being one of the main sources of pollution. Since synthetic biology only uses renewable resources, it seems to be a promising way to produce all kinds of molecules. However, for an interesting application of synthetic biology, a large amount of sugar would be necessary to feed all the microorganisms used to produce a large range of chemicals such as biofuels and drugs. In fact, more than 145 million tons of sugar are produced every year in about 120 countries [1] to fulfill the various needs in food or biofuel industries for example. Around 60% to 70% of the sugar currently produced comes from sugar canes with the remainder coming from sugar beets[1]. Like all massive monocultures, massive production of sugar has dramatic impacts on the environment (deforestation, loss of biodiversity, soil and water pollution)[1][2][3]. How much would this need of sugar increase if all the cars used biofuels? Would there be enough lands for culture? What will be the impact on the environment?
To meet the considerably growing needs of sugar for synthetic biology while being respectful for the environment, we needed to find sustainable alternatives to the use of arable lands for sugar production. Given the important marine surface on Earth, producing sugar in a marine environment seemed to be a relevant solution. This could bring back synthetic biology to its contribution to sustainable development. To produce sugar in a marine environment we chose a green microalgae, Chlamydomonas reinhardtii, aka, Claude.
Through our SugaRevolution we want to produce sustainable sugar without harming our
soil and competing with food crop! We want to engineer a strain of the green microalgae Chlamydomonas reinhardtii, adapted to live in sea water, and able to produce sugar in large amounts. Microalgae‘s photosynthesis uses sunlight and atmospheric CO2 to accumulate sugar in its stored form: starch. By using a photobioreactor for the containment of the algae in the oceans, sugar production would not compete with arable lands.
Impact of Monocultures on the Environment
For purposes of clarity, some important impacts that monocultures have on the environment are listed hereafter:
Soil erosion due to irrigation, wind and harvest[1][3][4]: Where irrigation application is inefficient or rainfall is high, water withdrawal is generally coupled with the loss of valuable soil from the farm.
Soil salinization and acidification[1][5]: Salinization of soils typically results from over irrigation or inadequate drainage. Acidification is largely due to the use of inorganic nitrogenous fertilizers such as urea and ammonium sulfate. Under high rainfall conditions nitrate leaching occurs, which also promotes acidification.
Water use[1][6]: Sugar cane ranks among a group of crops noted for their significant water consumption (along with rice and cotton). High water withdrawal is generally coupled with the run-off of polluted irrigation water containing sediment, pesticides and nutrients.
Use of chemicals[1]: The use of herbicides in sugar beet cultures is among the highest as compared to other crops. Inorganic fertilizers typically supply nitrogen, phosphorus and/or potassium in the mineral form. Environmental impacts generally arise because the nutrients in the fertilizers are not entirely taken up by the crop but move into the environment. The overuse of fertilizers on cane or beet crops is typical of farming in general.
Discharge of mill effluents[1]: In some countries with weak environmental laws, a tremendous amount of matter is released straight into the streams. Cane mill effluents tend to include heavy metals, oil, grease and cleaning agents.
A sugar factory that mills 1,250 tons of sugarcane daily uses 151,000 liters of water per hour and releases 30,000 to 75,000 liters of liquid, solid and gaseous waste.
Production of Sugar in marine environments by our modified Chlamydomonas reinhardtii
Why an algae?
As a photoautotrophic organism, Chlamydomonas reinhardtii has the ability to fix carbon through carbon dioxide uptake and a source of light. Its metabolism can also be autotrophic, heterotrophic or mixotrophic. All of these characteristics explain why it is an interesting chassis in comparison to other classical model (bacteria or yeasts) which always need an input of energy such as glucose. This microalgae is well-known as a model organism in the research field on photosynthesis and its genome has been characterized[7]. Moreover, growing range of tools in molecular biology have been developed for use in Chlamydomonas, for example a brand new modular cloning tool was created and published in 2018, the MoClo toolkit set by Pierre Crozet et al. This tool offers a new field of possibilities for the use of Chlamydomonas as it is a very interesting microorganism whose biosynthesis and metabolic pathways can be exploited in synthetic biology. Indeed it was established that this microalgae could be used in the future to produce high value compounds for biotechnology industries[8].
How to make this algae survive in marine environments?
To make use of the marine environment during the sugar production, we needed to have a microalgae that can survive in salty water. So we contacted Josianne Lachapelle who worked with Chlamydomonas reinhardtii and adapted some strains to live in high salt concentration medium[9]. And she kindly accepted to provide us with some of these strains that we could use them to make sugar without competing with arable land.
Which relevant sugar to produce in this algae?
The sugar we chose to produce in this microalgae is the trehalose C12H22O11. It is a disaccharide of glucose, that can be metabolized by Escherichia coli and Saccharomyces cerevisiae. While it is also present in Chlamydomonas, it is, however, mainly involved in stress response to H202 as an antioxidant and not as a source of energy. Without a self-production consumption, Chlamydomonas and trehalose are becoming an interesting combination for energy production. As a result, this source of energy would potentially be fully extracted and used by chassis such as E. coli or S. cerevisiae for the production of molecules of interest by synthetic biology.
Our discussions with professionals and scientists who work with Chlamydomonas or other microalgae, on directed evolution or on regulations played a major role in the further perspective of our project.
One main conclusion we got from our meetings was that algae did not bring an added value for the production of sugar or biofuels because of its high production costs. Indeed, the cost of production will highly increase because of the energy to bring to the photobioreactor, the extraction and the collection of sugar.
Thus, the amount of sugar expected by the use of marine surfaces could be sufficient to meet the needs of synthetic biology development but would not enable a profitable production
Yield improvement
In order to improve the yield of trehalose production, we thought about directed evolution. It aims to identify beneficial mutations on a gene or on a biosynthetic pathway. It is a major tool for synthetic biology in eukaryotes. This relies on in vitro generation of diversity. However, since C. reinhardtii can not replicate plasmids, this tool would be limited in this microalgae. Also, C. reinhardtii has a significantly lower speed of replication than the classical chassis of SynBio (Escherichia coli, Saccharomyces cerevisiae). These particularities make the in vitro generation of a large-sized library of mutants by the classical "error-prone PCR" used in directed evolution, more complicated.
This is why we have been interested in the team BOKU-Vienna 2017 project . To make in vivo evolution in yeast and bacteria, they proposed another solution, a model of retrotransposon. As there is no tool to make directed evolution in Chlamydomonas yet, we worked on adapting this strategy to this organism.
Since directed evolution is an important part in the development of a powerful synthetic biology chassis, we met professionals to get expertise on the subject.
The main returns we had were that it was by itself an expensive and complex subject to work on and develop. As the needs of such chassis in the industrial environment for sustainable development were clearly explained by the experts, it seemed more relevant to focus on our directed evolution tool instead of the sugar production itself.
GMOs, environment and legislation
Finally, an important part of our discussions focused on the legislation about GMOs in offshore platforms. The current legislation compels us to use a confined environment. Very interesting alternative systems for the photobioreactor design such as biocontainment measures were discussed with all the experts we met.
From the low added value of the genetically modified microalgae for the production of sugar, we knew how to use the compiled knowledge for a better valuation of our project through our retrotransposon. Indeed, our meetings with professionals brought out the interest of our retrotransposon development for fundamental research and for production of various molecules of interest in biotechnology.
In this case, the engineered microalgae would be an added value to improve the quality and quantity of molecules through a sustainable way of production at industrial scales. Therefore, it would not undergo the same regulations as GMOs used in offshore platforms
What will you do if our Microalgae release in the environnement?
Further details about our strategy is available on the Safety page
"Talents 2024" is a championship that rewards the most innovative projects, at the service of society. These projects must be in adequacy with the values of Olympism (excellence, friendship, respect) but they must also have the objectives of the Olympic agenda 2020 (sobriety, sustainable development, non-discrimination). 24 selected projects are chosen on the basis of their originality, their impact on the metropolitan territory and the populations as well as their potential for realization, development and exemplarity. Projects can be individual or collective.
We were happy to know that we have been selected among 24 projects. The city of Paris sees our project as a technological innovation with an impact on our future society. In October, we started to be accompanied by professionals, "Makesense" a start-up incubator, to clarify and develop our idea before presenting it in December to a jury of personalities from the world of sports and economy as well as representatives of the community. The jury will reward 3 projects which will share a financial endowment of 50 000 €.
References
[1]Sugar and the environment. WWF, 2018. http://d2ouvy59p0dg6k.cloudfront.net/downloads/sugarandtheenvironment_fidq.pdf
[2] Dom Phillips. Brazil senate considers lifting ban on sugarcane production in Amazon. The Guardian, March 27th, 2018. https://www.theguardian.com/world/2018/mar/26/brazil-senate-considers-lifting-ban-on-sugarcane-production-in-amazon
[3] CS Dominy. Long-term effects of sugarcane production on soil quality in the south coast and the midlands areas of Kwazulu-Natal, 2001. https://pdfs.semanticscholar.org/bf2a/6f249aaeec5921663613070452cc6c91b7bb.pdf
[4] Koç Mehmet Tuğrul. Determination of soil loss by sugar beet harvesting. Soil and Tillage Research, March 3rd, 2012. https://www.sciencedirect.com/science/article/pii/S0167198712000773?via%3Dihub
[5] L. R. Ng Cheong , K. F. Ng Kee Kwong & C. C. Du Preez, Effects of sugar cane (Saccharum hybrid sp.) cropping on soil acidity and exchangeable base status in Mauritius, South African Journal of Plant and Soil, 2009
[6]L. R. Ng Cheong , K. F. Ng Kee Kwong & C. C. Du Preez, Effects of sugar cane (Saccharum hybrid sp.) cropping on soil acidity and exchangeable base status in Mauritius, South African Journal of Plant and Soil, 2009
[7]Merchant, S.S., Prochnik, S.E., Vallon, O., Harris, E.H., Karpowicz, S.J., Witman, G.B., Terry, A., Salamov, A., Fritz-Laylin, L.K., Maréchal-Drouard, L., et al. (2007). The Chlamydomonas genome reveals the evolution of key animal and plant functions. Science 318, 245–250. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2875087/pdf/nihms166705.pdf
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[8]Mark A Scaife, Ginnie TDT Nguyen, Juan Rico, Devinn Lambert, †Katherine E Helliwell, and Alison G Smith, Establishing Chlamydomonas reinhardtii as an industrial biotechnology host, The Plant Journal, May 2015. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4515103/pdf/tpj0082-0532.pdf
[9]J. Lachapelle, G. Bell and N. Colegrave, Experimental adaptation to marine conditions by a freshwater algae. Evolution international journal of organic evolution, August 24th, 2015. https://onlinelibrary.wiley.com/doi/pdf/10.1111/evo.12760
At every step of our project, we have informed the public about it and discussed with the professionals, through recurrent meetings with our supervisors, various interviews, an online survey, and a conference. We have collected their opinions and advice to take all of these data into account to improve the relevance and the safety of our project.
We gradually changed the focus of our project according to the interviews performed and went from producing sugar to building a tool to improve an uncommon chassis in synthetic biology . Our meetings have also given us current juridical knowledge and public opinions about GMOs. Concerning safety, very interesting alternative systems for the photobioreactor design such as bio-containment measures were brought out with all the experts that we met. All of our meetings were very useful sources of information for the definition of a relevant project and for the improvement of our photobioreactor safety.
Throughout the year, we were constantly communicating about our project, the iGEM competition and synthetic biology. We have reached a large public through different actions. Indeed, we have organized a professional conference about synthetic biology in collaboration with two other French iGEM teams. To introduce the subject to the next generations, we held an animation for 3 days at the national science fair where we spoke about synthetic biology and C. reinhardtii for the large public including children. We also gave a lecture to high school students and sold crepes on our university campus in order to present iGEM to our university students and non-biologist professors. To reach people around the world we used social media through which we communicated about our project, our experiments and the iGEM competition. Finally, as we deeply give value to understanding the public view and fears about synthetic biology, we created a survey that we shared to the largest public as a tool that helped us address this issue
