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Euphoresis’s baby steps into the real world: From project to product.

Before considering implementing our idea in the real world, it is important that we define how Euphoresis would look and function as a final product. This is necessary for the industrial scale-up of the project, but mainly for the people to get a first-level understanding of our goals and envision the various potential applications only by touching and feeling a final Euphoresis’ bead! Here, we present you with our three different Euphoresis design “versions”:


  Euphoresis – the original

This is the core version of our product, aiming to deal with soil erosion, increase soil organic matter, and enrich soil with nitrogenous compounds. Euphoresis’ particles obtain the form of spheres or disks with a 10 mm diameter, to be compatible with drone spreaders. Euphoresis consists of the following parts:

The Hydrogel

A biodegradable biopolymer consisting of the polysaccharides pectin and chitosan. Both compounds will be obtained from green sources, sustainably. Specifically, pectin will be extracted from fruit peels, while chitin, a precursor chitosan compound, will be extracted from crustacean cells. A designed peptide will serve as a cross-linker, securing a swelling capacity of approximately 80% . Moreover, the peptide will have antimicrobial properties, ensuring product safety. The biopolymer will absorbbe absorbing water and contribute to the soil’s hygroscopicity

The microorganisms

Inside the biopolymer, the modified Bacillus subtilis strain WB800 will overproduce and secrete laccase enzymes that break down hydrophobic lignin-like compounds, whereas the genetically enhanced filamentous cyanobacterium strain will undergo intensified differentiation into specialized heterocyst cells. These heterocysts will facilitate nitrogen fixation, ultimately enriching the soil with nitrogenous compounds. For biosafety reasons, two kill switch mechanisms, one induced by the absence of IPTG in the medium and the other being associated with a quorum sensing system between the two species, will be implemented in Bacillus subtilis and in the cyanobacterium, respectively (read more abput this system in our Design page). The bacteria will be encapsulated in alginate-pectin nanoparticles that will exist within the hydrogel.

Euphoresis product: the combination of hydrogel, peptide and microbial consortium
Figure 1:Engineered bacterial consortium inside the microspheres and the hydrogel matrix

  Euphoresis plus – seeds in action

Our second version will carry the same elements as the original Euphoresis product. The main difference will be the addition of seeds, putting our enriched Euphoresis product into the reforestation game . Euphoresis plus will be useful in forests that have been burned twice in a period of ten to fifteen years, and where seeds have been eliminated.

After reaching Mr.mr. Vlachonasios Konstantinos, Professor of Plant Molecular Physiology at the Aristotle University of Thessaloniki (see Integrated Human Practices), we understood that the seeds we will use should be seeds from the respective endemic species. Therefore, flexibility in the design of Euphoresis plus is crucial since each reforestation case would require a different cocktail of seeds.

The hydrogel-microorganisms entity forms a safe and hospitable environment for the seeds to grow, as they would have access to water and nutrients even in soils that are fully degraded. Additionally, engineering Bacillus subtilis to produce hormones, such as gibberellins, under certain conditions, could promote controlled plant growth.1


  Euphoresis hydryad ­– a step to the future

In recent years, some attempts have been made to introduce Genetically Modified trees in forests or in agriculture to promote faster growth, or to absorb greenhouse gases.2,3 Those ideas could potentially match with the Euphoresis project, to create Euphoresis hydryad, the ultimate SynBio version of our product that will contain GMO seeds, carefully engineered to promote, for instance, a more effective reforestation and enhanced forest fire resistance. However, the strict EU Policy for GMOs, as pointed out by Mr. Vlachonasios too, is a core problem for the implementation of such a concept, at least in the European countries. However, Biotechnology and Synthetic Biology have seen incredible progress in the recent yearsperiod, so much that we can expect the possibility of this application in the future.


Application in soils, post-fire

As we already mentioned, wildfires, in the context of climate change, are of great intensity and frequency, which has a significant impact on soil quality. Water absorption is difficult and leads to soil erosion, organic matter decreases, and hydrophobic lignin-like structures are being formed, preventing new plants from growing, and nutrients, like nitrogen, are absent (read more in our Description page). The Euphoresis series is designed to provide solutions to all of the above issues. However, addressing numerous 'When', 'Where' and 'How' questions for an impactful application of Euphoresis in burned forest regions is of great essence.


At first glance, Euphoresis can be applied to every forest area that has suffered a severe wildfire. Nevertheless, more considerations should be taken into account: According to the European Commission’s “Guidelines on Biodiversity-Friendly Afforestation, Reforestation, and Tree Planting” (2023), afforestation of wetlands should not happen, as should any interventions in areas with low vegetation 4. Moreover, application should be careful in forest areas that were previously rich in mosses and lichens, as well as mushrooms, as they may be negatively affected by Euphoresis’s nitrogen fertilization function 4.

Furthermore, experts from Thessaloniki’s Forest Department (visit Human practices page) explained to us that our final product would be even more effective in forest areas that were burned twice in a 15-year period, and which have higher chances of desertification.

Regarding the terrain, we understood upon discussing with the foresters that it would make more sense to apply Euphoresis in areas of extreme ascents, since it is difficult to reach, so that drone application would be useful. This is where the suggestion of Mr. Christos Tsantilas, agroscientist and soil scientist, becomes relevant. Mr. Tsantilas pointed out that areas of high slope are the most vulnerable to soil erosion due to phenomena such as heavy rains or strong winds, so Euphoresis’s contribution would be essential and critical.


As the experts from the Forestry Department explained to us, reforestation procedures start two 2 years after a wildfire incident, but until that moment, the soil’s stability is crucial to be restored. This means that the original Euphoresis product would help with prevent soil erosion duringin that period. It also leads us to the conclusion that EuphoresisUPHORESIS plus could be a considerable alternative.


Mr. Tsantilas recommended the application of Euphoresis should be done in a period of mild rainfall. In this way, hydrogel will be capable of absorbing the desired amounts of water without being destroyed (as in the case of thunderstorms). Upon evaluating precipitation data from Greece’s National Weather Service, we realized that the spring months are the ideal period for application 5.


We expect our material to last for 21-35 days before degrading, considering chitosan:pectin ratio (3:1) combined with the peptide crosslinker 6. pH conditions in soil play an important role in degradation rates, with Mr. Tsantilas suggesting that we should design our product to function at a range of pH 5-8.


Researchers conducted experiments using different hydrogel concentrations in semi-arid regions and discovered that 0.27% and 0.33% w/w concentrations (hydrogel/soil) increased soil aggregation, which reached 35%, and soil available water by 49% 7. Those could be good reference points for further investigation, in order to clarify the ideal Euphoresis concentrations for each purpose.


As mentioned before, Euphoresis needs to be applied in large areas that often include several ascended spots, which are difficult to reach. To address those challenges, we will employ modern technology: drones. Those flying machines that seem to have emerged from science-fiction movies, will now have the duty of dropping Euphoresis bombs in every terrain in need.

Even a simple internet search will reveal that drone spreaders used for seed or pesticide pellets on agricultural fields are already an implemented concept8,9. Moreover, the Greek Fire Department has the know-how on the use of drones, using them on various firefighting operations10. That is why we believe Euphoresis drone spreading operations have a realistic basis.

Agricultural drones come out in different sizes and with variable features. They can spread spherical or disk-like particle, up to 1 cm in diameter, in a distance between a few centimeters and even close to 30m8,9.


Several soil preparation methods can be applied before reforestation progresses. Some of them include tillage, conditioning with organic or inorganic materials, and terracing. However, those techniques seem to be rather expensive for large areas and do not have any positive impact on the soil’s properties and hydrophilicity11.

Euphoresis overpasses those problems and could be a highly effective solution for soil restoration after wildfires, because of the following reasons:

  • It increases soil quality and water absorption.
  • It has a zero footprint in ecosystems because of the gradual polymer biodegradation. It does not intervene in nature’s processes as it is a closed system that prevents microorganisms from interacting with their surroundings.
  • It is produced under the principles of Circular Economy and Sustainability, with green methods (see Cost).
  • It is based on Synthetic Biology, a scientific field that emerges and provides flexibility in modifying or reconstructing the entire product to cover different needs. This is highly important because it makes way for Euphoresis to be enhanced with new features and be applied in the whole spectrum of forests and ecosystems, as well as to applied in multiple fields, other than post-fire soil restoration.

Other Applications

1. Urban green spaces


Greece occupies the last place in Europe when it comes to urban green spaces, with just 2 m2 to 10 m2 per capita. Reversing this situation holds significant importance, since green spaces in cities improve quality of life and even help manage CO2 emissions12. Officials from the Central Macedonia Region (see Integrated Human Practices) also pointed out to us that creating and preserving green spaces in cities is one of their top priorities; therefore, it would be useful to them if a product contributed to this effort.

Euphoresis can increase soil stability and water absorption in urban areas as well, resulting in less use of water, rapid growth, and enhanced preservation of the plants that make our cities beautiful and sustainable. Moreover, cyanobacteria are capable of capturing CO2 emissions, and with further engineering, we could design a novel Euphoresis product that will specialize and aim to improve urban microclimate13.

Illustration of urban green spaces
Figure 2: Urban Green spaces

2. UFORESIS – Getting ready for colonization of Mars


Illustration of urban green spaces
Figure 3: Colonization of Mars with the implementation of ugoresis

The climate crisis and human intervention have caused serious environmental problems on Earth. Biodiversity is being threatened, and the availability of resources is something highly questionable for the future. That is why many people hope that the colonization of the red planet will create potential for new sources of food and energy and for the continuation of human activity and progress. Such plans, of course, seem rather ambitious, since people who would first inhabit Martian lands should ensure water, oxygen, and food production, as well as the creation of a hospitable environment for themselves and for those who will be inhabiting Mars in the future.

Cyanobacteria could thrive in the extreme Martian conditions, requiring just CO2, water, nitrogen, and the Martian regolith to survive. If engineered correctly, they could provide in exchange oxygen, nitrogenous compounds, fuel, and even food and building materials14,15. On the other hand, it has been proven in research that hydrogels could promote efficient plant growth in Martian conditions16. These two combined prove that Euphoresis could stand as an ideal Martian startup kit and start conquering the uUniverse! And who knows, maybe it will be dispersed by flying saucers…

3. Artificial forests


Artificial forests are designed and created by humans for various reasons: fFrom collecting wood to fixing CO2 emissions and from entertainment reasons to art17,18. One characteristic example is Tree Mountain in Finland, an artificial forest that serves as a work of art, as well as a symbol of human responsibility and sustainability, since 11,000 people planted the 11,000 trees18. Moreover, according to Mr. Mavromatis Athanasios, an Agricultural Science professor at Aristotle University of Thessaloniki who is part of the ‘’Genetics and Plant Breeding Lab’’, who we conducted, artificial forests could be implemented in desertified environments and such plant could include the addition of genetically modified seeds, in order for the new plants to withstand the harsh conditions.

Our team wants to contribute to such efforts, and we think that Euphoresis could help establish more artificial ecosystems like this and promote the of sustainability.

Illustration of urban green spaces
Figure 4: Creation of artificial forest with the proper design of euphoresis

4. Agriculture


Illustration of urban green spaces
Figure 5: utilization of euphoresis in agriculture

Hydrogels have been used in agriculture over the past decades19, while both cyanobacteria and Bacillus subtilis strains have been successfully studied for their potential use in sustainable agricultural units20,21. Euphoresis unites their powers in one single particle, which we hope can reach the agricultural market and be recognized as a versatile product that aims for sustainable crop development.

Safety

Our product consists of a biopolymer and a set of microorganisms encapsulated in biodegradable microspheres. We want to ensure that all of Euphoresis’ levels do not interfereintervene within their surroundings in a negative way and that they are non-toxic for humans or animals.

Pectin and chitosan, our hydrogel’s polysaccharides, as well as alginate, the nanoparticle ingredient along with pectin, are non-toxic compounds that come from natural resources and cause no harm to a human or animal that may accidentally consume them22,23. As mentioned before, they will be completely degraded in a maximum of 35 days, meaning that they will not stay permanently in the environment and not cause any harm. As for the antimicrobial peptide-cross linker, further experiments shall be conducted in order to determine whether it is harmful for the external microflora.

Regarding our microorganisms, we designed two kill switch systems. For Bacillus subtilis, production of the lethal BsrG toxin will occur the moment the IPTG inducer is no longer available in the medium inside the microsphere or in the surrounding environment in general.

As for the cyanobacterium, we developed a toxin-antitoxin system, the operation of which depends on a quorum-sensing system between our Bacillus subtilis and cyanobacteria strains. Bacillus subtilis will be producing small AHL molecules that will reach the cyanobacteria and promote the production of the antitoxin. When Bacillus subtilis dies, due to the absence of IPTG, no more AHL molecules will be produced, causing the antitoxin production to shut down. Elevated toxin concentrations will cause the death of the cyanobacterium. The IPTG concentration in the medium will be regulated so that the bacteria die before the hydrogel degradation. This will ensure that our genetically modified microorganisms will do their job without ever getting to touch the soil or being set free in the air.

An issue that needs to be addressed is the toxicity of the metabolites of our selected microorganisms. Apparently, toxins produced both by cyanobacteria and Bacillus species can cause gastrointestinal-related symptoms24,25. Therefore, we need to ensure that microorganism concentrations are kept at a low level that would not cause any problems in the event of accidental consumption. (Read more about our project's safety considarations in our Safety and Design pages!)

Illustration of urban green spaces
Figure 6: Our project's safety measures

Cost

  The Hydrogel

Pectin extraction

Various methods, both conventional and non-conventional are available to extract thefrom fruit peels our desired hydrogel molecule from fruit peels. Non-conventional methods seem to be the “greener” alternative, since they do not require such high amounts of chemicals, but they do have their disadvantages, whichthat are mainly related to the usage of electricity and scale-up feasibility. Among those methods, we can find microwave-assisted, ultrasonic-assisted, enzyme-assisted and subcritical water methods.

Pectin can be extracted in both green and cost-effective ways with the use of organic acids, such as citric acid. Researchers conducted an Economic Feasibility Study and proved that the production cost of pectin extraction under this method could be $9.8 USD/kg, lower than the estimated selling price of $13.6 USD/kg, concluding that, economically speaking, this method has a positive outcome. As for the further reduction of the environmental footprint, they suggest choosing acids that are strong in terms of maintenance and originate from green sources. They also suggest using a combination of this method with the subcritical water method for further solvent reduction, which practically means a reduction in economic and environmental costs.26

Chitosan production

Chitin extraction from crustaceans’ cells can be achieved using the same techniques as with pectin27. Economic Viability Analyses for chitosan production in Spain and Ecuador claim that production costs for chitosan could, respectively, be $14 and $8.4/kg, with a positive return rate and a price point starting even at $5. The impact on the environment should undergo further investigation; however, it is said that it is relatively low, compared to other scenarios28.

  The Consortium

Cyanobacteria cultivation

Cyanobacterial growth requires the use of photo-bioreactors (PBRs). The cost of 1 m2 of PBR can vary from $10 to $3000. Production of 1 tone of cyanobacterial biomass can reach $3000, meaning $30 per kg. The cost can be further reduced with the use of cheap water sources, such as agricultural wastewater29].

Bacillus subtilis cultivation

Bacillus subtilis’ use as a cell factory has seen an increase in recent years due to its unique features as a microorganism. Therefore, industrial fermentation is an existing and applicable concept30. For instance, researchers achieved industrial-scale fibrinolytic enzyme production by Bacillus subtilis WR350 by fermenting the strain in a 100-liter fermenter. With the utilization of cheaper raw materials in the nutrient, they managed to reduce fermentation production costs from $59.04 to $13.7831. This gives us a clear indication that we could achieve low-cost and effective Bacillus subtilis WB800 biomass production.

  Antimicrobial peptide production and extraction

E. coli strains have been successfully used as cell factories too, with a known example being human insulin production. Therefore, someone could imply that scaling up antimicrobial peptide production will be successful as well. This is confirmed by research, as a team achieved a cost of $45.8/mg, which is, as they claim, a more economically sustainable method compared to the chemical ones, as well as a fair step for starting a scaling-up process32.

End Users

The vast effects of wildfires seem to affect a significant percentage of Greece’s lands and ecosystems, as well as many citizens whose lives and properties are threatened, and their quality of life rapidly changes for the worse. (link human?) Therefore, dealing with the situation is mainly a matter of public interest. Our goal is to provide the state authorities, which oversee preventing or dealing with the consequences of natural disasters, with an extra weapon to protect forests and face the “monster” of climate change in general.

Our first step in promoting Euphoresis as a novel solution to the public sector, includes addressing two Greek ministries that have “fire”, “forests'' and “climate change issues” as parts of their daily agenda: the Ministry of Environment and Energy and the Ministry of Climate Crisis and Civil Protection . The first one is in charge of Greek Forest Departments, while the second supervises fire departments. Both agencies serve as our proposed end-users since they have the capability of assessing which areas are most in need and dispersing our product to the burned areas.

As soon as we reach an agreement with the governmental factors, our next step be to create a network, along with local authorities, such as the Decentralized Administrations, Regions, and Municipalities, for the distribution, storage, and usage of Euphoresis. Our aspiration is to be next to our partners as an active part of the solution, rather than just focusing on the earnings, especially when dealing with major environmental issues. For this purpose, we will constantly stay in touch with regional fire and forest departments, in order to obtain the necessary data and information. Since every forest has unique features and each wildfire event has a different impact on the respective ecosystems, we want to use those data to provide every region in need with a Euphoresis solution that fits them the best, regarding both the extent of each problem and the different soil properties. That being done, we will go on and settle the necessary product amount that is to be thrown, in partnership with the above authorities. Finally, after providing our partners with know-how information on storage and use, we will be conducting them on a common basis so as to get feedback on the product’s effectiveness, as well as for safety issues.

The public sector may find Euphoresis useful in dealing with other challenges too: Our bacteria-enriched hydrogel could effectively contribute to the management of desertification, which is a major threat to the environment and often closely related to wildfires’ intensity (see Description). Moreover, cities’ mayors and governors could find Euphoresis useful in promoting urban green spaces incorporation.

Euphoresis goes abroad: Our ambitious plan includes developing our product into a global trend in postfire soil restoration and beyond. As the problems we have described and which concern Greece are also of a global nature, we hope Ministries of Environment and the relative authorities worldwide will find Euphoresis an interesting soi-lution!

Agriculture: Both Bacillus subtilis and cyanobacterial species have been previously studied and/or used as biofertilizers. Now farmers can have the opportunity to use a 2- in- 1 product containing both bacteria in full biosafety conditions and captured in an agriculture’s good-old friend: the hydrogel biopolymer.

Space: Space agencies (Hellenic Space Center, ESA, NASA) could use Euphoresis to conquer the infertile soils of Mars in the not-so-distant future…

Future Challenges

1. The EU doesn’t love GMOs as much as we do

The EU member states apply very strict measures regarding the release of Genetically Modified Organisms into the environment, without any layers that prevent those organisms from getting in touch with the environment.33 This means that the release of a Euphoresis product that contains GMO seeds is practically inapplicable at this moment. Moreover, we should ensure that our kill switch systems function perfectly, since the accidental release of our genetically modified microorganisms would cause us legal problems, apart from a potentially significant environmental impact.

2. More experiments on the way

After research, experimental procedures, and talking with experts, we came to the conclusion that Euphoresis is a promising project with great potential in forest and soil restoration. However, what we have done for the iGEM Competition is only the beginning. More experiments should be conducted, including experiments on burned soils, in order to reassure that Euphoresis can be implemented successfully. Dr. Nikolaos A. Peppas (visit Human practices ), Professor & Director of the Institute for Biomaterials, Drug Delivery and Regenerative Medicine at the University of Texas at Austin, suggested, for example, the utilization of the Time-Temperature Superposition method to determine our polymer’s swelling and degradation rates in more detail.

3. Cost is always the issue

Euphoresis consists of many different ingredients, that can be obtained in various ways. Some of them are cheaper, but others require the use of expensive equipment or media. If we want Euphoresis to be an affordable solution, we need to address in more detail all the potential economic requirements that are needed for its industrial production. On the other hand, we should always preserve our “green” identity and look for methods that won’t harm the environment. Our goal is a relatively economic product, which will, however, highlight, through its features, our ideas on Ecology and Sustainability.

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