Project Description

Why?

OUR MOTIVE, STIMULATING SUSTAINABLE SYSTEMS

Scientific achievements and innovation in all its glory - we humans with our creative and voracious minds have undoubtedly put both ourselves, as well as the biological world around us, in a complex situation in the 21st century. Our greed for knowledge and productivity has led to many social and economic advancements. Unfortunately, our success has often come at the expense of nature; either by overuse of limited natural resources or by means of the pollution and waste we generate in the process. Anthropogenic effects on climate, biological diversity, air quality and environmental pollution are all evident. This leaves us with great challenges to overcome in a very limited amount of time. If we want to overcome these challenges, we need to work together, communicate, coordinate, and most importantly: we need to act now.

Our project is driven by the motivation to use synthetic biology in a sustainable context. Genetic manipulation can seem scary, whereby it is essential to reflect on how and why we apply these tools. If used in the right way at the right time, GMO could potentially provide us with new systems and solutions. For example, replacing fossil fuels with more sustainable alternatives of biofuels or cleaning waste water from toxic compounds which could harm the environment or human health. Science and innovation can arguably be seen as part of the problem, but it could perhaps also be part of the solution. As members of the iGEM Uppsala team of 2025, we want to contribute to that solution, in every way possible.

What?

OUR ORGANISM: CHLAMYDOMONAS REINHARDTII

In this year's project, we decided to work with the unicellular algae Chlamydomonas reinhardtii, a well-known model organism used for basic research on photosynthetic systems, flagellar motility, phototaxis, cell cycle and much more. Members of the genus Chlamydomonas ( >500 known species) are widely spread in freshwater and soil habitats across the globe ^[1], though most of the laboratory strains we use today originate from a common background strain (CC-125) isolated in Massachusetts, USA in 1945 ^[2]. Having a haploid genome, it is relatively easy to make observable knock-out mutations in these algae and due to their light perception, the cells in a culture can be synchronized to a circadian rhythm, making them more easily comparable ^[3].

However, Chlamydomonas species do not only have applications in basic science, they are also great tools for applied biotechnology. These organisms are photoautotrophic, meaning they get their energy from sunlight and their carbon from inorganic CO₂ in the atmosphere. Hence, they are very cheap and easy to cultivate. ^[4] Furthermore, they do not create any toxic compounds when grown in bulk, like those you might get from their bacterial counterparts: cyanobacteria ^[5]. In fact, C. reinhardtii biomass has been approved for consumption by both humans and animals by the American Food & Drug Administration (FDA) ^[6]. So, perhaps we will soon be having algae-cereal for breakfast!

The list of biotechnological applications of Chlamydomonas is long and covers many different fields of research. One of the most commonly occurring themes is phycoremediation, i.e. using algae for wastewater treatment. Chlamydomonas species have previously been shown to reduce the amount of phosphorous and nitrogen in industrial wastewater ^[7][8] and piggery water ^[9] by using the excess nutrients to continuously generate more algal biomass. This biomass could subsequently be used for other purposes. For example, Chlamydomonas has proven capable of acting as a biofertilizer ^[10][11], providing a more sustainable alternative to highly energy-demanding urea production. Another intriguing use of this microalgal biomass is for production of various types of biofuels. In 2015 Chen et al. showed that wet biomass of Chlamydomonas sp. JSC4 was capable of generating biodiesel at a conversion rate of >97% ^[12] and in 2020, Qu et al. demonstrated empirical evidence of efficient bioethanol production from Chlamydomonas sp. QWY37 grown on swine wastewater^[13]. C. reinhardtii is also being evaluated as a potential candidate for green hydrogen production^[14].

One of the many advantages with C. reinhardtii is of course that it is photosynthetic, which not only means that it does not require any organic carbon source nor chemical energy, it also means that these cells will retain CO₂ and fixate this carbon in their biomass. So not only can we look at Chlamydomonas as potential providers of sustainable biomass, they can also be considered as carbon sequestrators! In fact, a study by Choi et al. in 2021 developed a transgenic C. reinhardtii strain, which by the overexpression of a transmembrane proton pump could survive and grow at a CO₂ concentration of 20%, effectively making them capable of growing on coal-derived flue gas and putting Chlamydomonas on the map as a potential candidate for energy-efficient carbon capture systems.^[15]

How?

OUR PROJECT: SELF-FLOCCULATING MICROALGAE

If microalgae are so great, how come we aren't seeing more of them?

We have already seen many practical and promising trials using both Chlamydomonas and other microalgae. Indeed, the prospects of microalgal biofuel production led the United States Department of Energy (DOE) to conduct an extensive research program called the Aquatic Species Program from 1978 to 1996, which resulted in a 0.1 hectare pond system being established in Roswell, New Mexico. Substantial effort was put into collecting and evaluating more than 3000 different algal strains, which in itself can be seen as a great scientific achievement. However, in their summarizing report from 1998, the DOE concluded that even though they had seen up to 90% efficiency in CO₂ utilization and could reach a biomass yield of 50 gram biomass per day, the system could not yet be considered economically viable. ^[16]

It all comes down to productivity. We live in an economy which favors efficiency and economic growth, often at the expense of more sustainable alternatives. And so far, microalgal biomass production has not reached the threshold of productivity required to become a competitive alternative to more established and lucrative systems. Limitations include low productivity due to e.g. culture self-shading, potential contamination of unwanted microbial species, energy usage, nutrient and water supply as well as high harvesting costs. If algal cultivation is to replace e.g. other wastewater treatment systems, fossil fuels, fertilizers or feedstock, it needs to become more economically feasible.

We suggest addressing this challenge from several fronts, integrating both creative infrastructural engineering as well as targeted synthetic biology. As seen above, the production cost of Chlamydomonas biomass can easily be reduced by growing the algae on a wastewater medium in direct sunlight (open raceway pond) or a glass container (photobioreactor). Furthermore, if commercial strains are adapted to higher CO₂ concentrations^[15], the bioreactor could simultaneously be connected to an exhaust system from an industry or other CO₂ emission source. This would effectively eliminate the costs of both energy, carbon and nutrient supply at the growth stage, as well as provide simultaneous purification of both effluent gas and water. We thereby hope to maximize the incentive to invest in such a system, preferably in close proximity to the polluting source.

However, the challenges of biomass yield and harvesting still remain to be addressed. The initial recovery of suspended cells in a culture has been estimated to account for up to 30% of the total cost in microalgal biomass production and is one of the main setbacks of its implementation. ^[17][18] One commonly applied harvesting technique involves addition of chemical flocculants, substances that will disrupt or neutralize the negative charges of the algal cell walls in order to make cells adhere to each other in “flocs” that sediment to the bottom of the container. However these chemicals can potentially introduce more harmful or toxic substances into the water which may subsequently be hard to remove^[19]. Cells can also be collected mechanically by centrifugation, though this is a highly energy consuming and expensive procedure^[17]. Yet another approach that has gained more attention over the last 15 years is the rotating algal biofilm reactor (RABR), where the cells are attached as a biofilm to some rotating surface suspended in a liquid medium^[20]. However, such systems require co-cultivation with bacterial strains that can produce the necessary compounds for biofilm formation^[21], which could potentially cause contamination or competition in the culture as well as likely reduce the total photosynthetic efficiency and carbon uptake since these bacteria are heterotrophic.

This is where the iGEM Uppsala 2025 team project comes in! We want to use synthetic biology to create transgenic Chlamydomonas with the ability to express extracellular proteins which can enable cell-cell and cell-substrate contacts. These proteins will induce flocculation and/or adhesion to improve biomass recovery without the need of harmful chemicals, nor requiring any substantial energy input. Our hope is that a self-flocculation system can simplify microalgal harvesting and increase the yield to such a degree that industries and communities find both economical and environmental incentive to invest in these sustainable biomass systems.

And Now?

OUR GOALS: FROM IDEA TO PROOF-OF-CONCEPT

With our project, we are not trying to single-handedly solve the global energy crisis or wastewater problem - not yet. What we are aiming for is to lay the molecular foundation for a new type of self-flocculating microalgae. Our goal is to demonstrate that Chlamydomonas can be genetically equipped with surface proteins that enable adhesion, either to neighboring cells or to artificial substrates.

This means that throughout the project we set out to:

• Design and build genetic constructs for expressing extracellular adhesion proteins in both E. coli (as a testing platform) and Chlamydomonas reinhardtii (as our final host).
• Test whether these proteins retain their function when expressed in a heterologous system, using fluorescent tags and microscopy as our main tools for validation.
• Compare different adhesion strategies, including protein pairs that mediate cell-cell interactions (Z17/Z18), and proteins that allow anchoring to surfaces (GP1 and FLO1).
• Assess whether the engineered strains show signs of clustering or flocculation, which would provide a first proof-of-concept for our design.