Water is the most abundant resource worldwide, and is also the most used. We not only need water to live, but to keep our economy afloat, our industries running and, most importantly, our ecosystems thriving.

Water scarcity is one of the biggest challenges facing mankind in the 21st century.

As of today, 2 billion people (1 in 4) worldwide lack safe drinking water. Our reservoirs are at an all-time low, but an even greater issue is the poor quality of the remaining water.

The current and predominant agricultural model is responsible for leaving us with poor quality water. Industrial livestock farming with its excessive excrement from macrofarms and industrial agriculture, with its massive use of fertilizers are poisoning our most precious resource.

According to the Catalan Water Agency, the problems associated with the masses, such as the high density of livestock and agriculture in the area, put at risk the achievement of the environmental objectives and the 2027 milestone would be virtually unattainable unless the nitrogen inputs to the soil were substantially reduced.

It is estimated that in the period 2016-2021 the municipalities and county councils of Catalonia, at least, have made an investment valued at 19,101,169,8 €. In Europe, according to the European Council, the economic cost is equivalent to losses of between 13 and 65 billion euros per year, which is unacceptable.

Trends in the agricultural sector reveal an increasing demand for NPK (nitrogen, phosphorus and potassium) based fertilizers. With a growing population and increasing demand for food, ensuring the sustainability of the agricultural industry is of paramount importance.

In view of all this, we understand the need to implement a product like ours in the market. In order for this to become a reality, we are first considering a business-to-consumer (B2C) business model. Where we would sell our final product, powdered biomass rich in cytokinins, inside compostable sachets for the farmer. (see more in the page).

In the long term, we are thinking of implementing a business-to-business (B2B) business model in the initial stages. AlgaGenix would provide the raw biomass rich in cytokinins to be used by biostimulants manufacturing companies. We have already spoken with some companies that fit in this category, and thought about future collaborations. In this way, AlgaGenix would benefit from the facilities, know-how and distribution network of the manufacturers and the manufacturers will have access to the raw biomass of our algae to scale up the production of these biostimulants. The finished products could then be sold to the users of the product, the farmers, through normal B2C transactions.

AlgaGenix plans to improve the microalgae model organism Chlamydomonas reinhardtii through synthetic biology so that it removes nitrates from polluted waters and turns them into cytokinin-rich biomass –which can later be used as a fertilizer– while improving water quality. This way, we are cleaning polluted water whilst producing value-added products, embracing the circular economy.

The genetic modifications will be conducted through MoClo and consist in the overexpression of 4 endogenous genes aimed at enhancing nitrate uptake and cytokinin biosynthesis, as well as the introduction of an exogenous Glutamine Synthetase from Synechocystis sp. PCC6803 to avoid the endogenous regulation of its homologous protein in C. reinhardtii and to ensure the link between the uptake and the production parts of the pathway. (see more in project description or engineering).

Our algae is designed to fit in a bioreactor right within highly-polluted nitrate waters, from where our engineered Chlamydomonas will transform said nitrates into biological matter, enriched with cytokinins.

The efficiency of the process depends on many factors (species, geographical position, etc.), but most importantly, on the photobioreactor (PBR).

Tubular PBRs are the most efficient; however, they are expensive. The cheapest but least efficient system is the open circular pond. However, we cannot use it because Chlamydomonas need constant agitation, and it is very inconvenient to manually agitate them constantly. They are also highly susceptible to contamination, as they are open to the atmosphere, and they are known to have high water requirements due to evaporation.

In the long term, we could consider using a tubular system. But currently, owing to space and financial requirements, we cannot use this system, although it would be the most efficient for our algae.

Once uptake and biosynthesis in the algae are completed, a very important step is the filtration of water to separate the cells from the medium. There are two main methods for this purpose: filter filtration and centrifugation.

Centrifugation is better, but more expensive because of its high energy cost. On the other hand, filtration is not as efficient, but it is cheaper. Nevertheless, conventional filtration does not work for Chlamydomonas as they are mobile, and with conventional filters, Chlamydomonas escape from filtration.

After talking to a researcher, we came up with a different approach that could work: sand filters with differential pores. Sand filters work with silica grains of different sizes. After pouring water through the sand, the sand retains the biomass because of its electrical charge, and then the biomass is recovered by backwashing.

Once the algae are collected in a semi-liquid state, the biomass is recovered by backwashing, and the algae are then lysed with UV light, so as not to commercialize live GMOs.

Then, we will have to dehydrate them if we want our final product to be a powder. There are two main methods for this purpose: freeze-drying and heat-drying.

Freeze-drying is a drying process that preserves the properties of the microalgae better, and because of the extreme temperatures, it will kill any algae that have escaped radiation.

Nevertheless, it is a more expensive process because of the liquid nitrogen and the apparatus. In contrast, heat-drying is a cheaper option, but it may affect the quality of the synthesized molecules, such as cytokinins, which would be of no interest to us. That said, although it is more expensive, to preserve our product, we will freeze-dry our algae.

Once the algae have been dehydrated, we will obtain a cytokinin-rich, concentrated biomass, which can then be processed and sold as a solid, powdered product.

The farmer or final consumer can then dissolve the product until the desired concentration is obtained. We will provide a table with the weights of our product to prepare different formulations.

Moreover, after talking to farmers, they told us that their level of trust, and therefore adherence to the product, would increase if they could manipulate the product themselves and know what they are actually putting on the crops.

Liquid fertilizer producers will have to use larger means of transport to transport the bottles, which increases the cost and causes more pollution.

On the other hand, AlgaGenix product would be sold in compostable plastic sachets, thus reducing pollution, storage space, and transport.

We also worked early to identify the safety issues that would need to be addressed during the final application of AlgaGenix in the real world. We anticipate that our products will be applied primarily and directly in the field. However, although these products do not contain Genetically Modified Organisms (GMOs), it is necessary to note that the components of our product were produced in GMOs.

In order to determine how to tackle this issue the best, we decided to talk to Manuel González del Valle, I+D Manager at G2G Algae Solutions, who is working closely with microalgae, GMOs, and their regulations. After an extensive discussion, he told us that we did not have to worry about complying with the regulations concerning the introduction of GMOs into the environment, since they were not present in the product we would be introducing into the open field. In the EU, there are a couple of directives concerning GMO, one being the deliberate release directive (Directive 2001/18/EC) and the contained used directive (Directive 2009/41/EC). Because we would work under the contained used directive, as the algae are closed in the photobioreactor, it is not required to have our organism authorized. This is only needed when the GMO is released into the environment.

Nevertheless, it is necessary to notify competent authorities before beginning contained use activities. Installations are verified by authorities to ensure that they are suitable for the activity and that there is no risk to human health and the environment. If the activity involves moderate or high risk, such as Class 3 or 4, the authority must be consulted before the activity can proceed.

The levels of containment and protective measures were assigned to contained use activities from 1 to 4.
- Activities in class 1 pose no or negligible risk
- Activities of class 2 are low-risk in nature
- Activities in class 3 are moderately risky
- Activities classified in class 4 are considered high-risk

In our case, the activities involved in producing biomass from algae were categorized as class 1.

As we are not implementing our GMOs for food or animal feed purposes, and our end-product will not contain any GMO, most regulations do not apply to our product. However, we must ensure the protection of workers from risks related to exposure to biological agents at work, according to Directive 2000/54/EC8. The requirements for this directive are to perform a thorough risk assessment and then assign the activities to an appropriate risk class similar to that for Directive 2009/41/EC. Likewise, the risks associated with our lab work are categorized as class 1 risks, similar to those assessed for the contained used directive. Moreover, we ensure that everyone who is working in the laboratory is provided with appropriate clothing and protective equipment. All of us completed a safety and security assessment and training, providing us with knowledge of any risk scenario.

If we were to implement our project in another country, we would need to check whether it imposed additional restrictions.

The main application of our project is in the field, on farms.

The primary source of nitrogen on farms comes from manure, which consists mainly of animal faeces and urine.

According to regulations, every livestock farmer is responsible for the waste they generate. Most farms are tied to a specific land base for manure application (the number of hectares is related to the size of the farm), and each year they must report to the authorities how much manure they have generated and where they have applied it.

Some farmers, however, do not have enough land to dispose of all the slurry they generate. These farmers have to take the manure to an approved treatment plant.

A small minority of farmers, on the other hand, treat the manure on their own farm using an authorized method recognized by the authorities (solid-liquid separation, NDN, etc.).

After talking to BonArea cooperative, we were told that our market niche would have to be farmers who either have to bring their manure to a treatment plant or have their own systems, should our alternative be more economical.

Our market niche would expand if we process slurry from other animals such as chickens or cows, not only from pig manure. In addition, pig slurry is highly concentrated, so it would be very difficult to work with.

As a part of our university studies, some of our team members were taking a course on Biochemistry and Plant Physiology where the laboratory part was focused around the use of a biostimulant in Solanum lycopersicum, a tomato variety. We saw an immense opportunity in this, since we could be able to test the effect of CK in this plant.

We thought about the different ways of using kinetin, a type of CK, to benefit our project, and after some literature reading, we found that they were associated with salinity-response stress mechanisms, so we decided to follow this path. Our ultimate goal was being able to use moderately-salty water (as a result, for instance, of the mixture of fresh water and seawater) to irrigate crops in areas of high water stress. This idea developed from our visit to the Comunitat de Regants Sindicat Agrícola de l’Ebre, whose main issue was high salinity.

This was, therefore, a clear application of our final product in a way that further contributed to the recirculation of our resources and that directly tackled the concerns of a local community severely affected by freshwater scarcity.

In the implementation of our project, naturally, we are expecting to face many challenges. These will both include the ones that are already apparent, and the unforeseen challenges. Here, we have listed some of the greatest challenges we expect to face in our implementation.

Upscaling our production

For our solution to be implemented, it is vital to optimize and upscale the production of our cytokinin-rich biomass. We still haven’t assessed CK production in wild-type Chamydomonas under the nitrate conditions found in manure water. Once our algae are fully transformed, we can then compare CK production in normal vs our modified algae. Nevertheless, we have estimated CK biosynthesis based on published experiments with algae in normal media. This said, a lot of preparation would be required to produce small amounts of hormones, and this is unsustainable if we wish to implement our project. Luckily, once we introduce the modified algae in the bioreactor, scalability will be easier, as we will be working with larger volumes and more cells. Moreover, thanks to our collaboration with EOD Europe (see future steps), we hope that the efficiency of the process is significantly improved.

Therefore, it can be assumed that there is a possibility for effective production, and a potential for high-yield upscaled production, once we overcome the challenge of optimization and upscaling.

Production costs

Goes hand-in-hand with scalability issues. The main steps to obtain our product include:
- algae cultivation in the bioreactor
- sand filter filtrations
- freeze-drying
- processing

The more volume we have, the more expensive the process of obtaining the product becomes.

These seem to be unavoidable costs, as they are essential steps. After many meetings and discussions, we have chosen the best techniques that ensured the integrity and good quality of the samples, and at the same time, are cheap in cost. In the section AlgaGenix in action reasons for choosing this workflow are mentioned.

To address this, we have started to look for funding and sponsorship opportunities (see future steps), so that we can scale up, and once we have consumers, the production costs will be offset by the cost of our product.

Unfortunately, our iGEM journey ends in November, but this is not the end of AlgaGenix! We currently have several lines open to continue with several key aspects of our project.

Seeking financial support

Firstly, no project would be possible without financial support, so part of our team is working on establishing contact with stakeholders and companies interested in financing our project. Especially companies with an interest in meeting the Sustainable Development Goals (SDGs), which were set for 2030 and are far from meeting.

In this context, a few companies have expressed their interest in our bioremediation project. For example, after a meeting held with the Catalan Water Agency, they were so interested in the project that they proposed us to do a pilot test on a farm in Osona, a region of Catalonia, once we had demonstrated that our project would work.

Establishing collaborations

The 20th of September, we were invited to Bayer´s Demo Day at the innovation hub in Barcelona. We were able to speak with many stakeholders and startups from the agricultural sector, including Bayer representatives.

In one of these encounters, we spoke with Henna Niskakoski, the CSO of EOD Europe. EOD ® Europe is a Finnish company that develops and manufactures innovative applications based on nanobubble technology. They have developed a Nanoboost device that increases the amount of oxygen in the irrigation water and makes the plants stronger. The amount of oxygen in the water is increased through nanobubbles. Adding oxygen to the water promotes root growth, improves plants’ stress tolerance and immune system, and enhances the absorption of nutrients.

Their technology has already proven to be very beneficial for plants, and they now want to try their nanobubble-based technology on other organisms, like microalgae.