euphoresis in a forest

Our Journey

Our journey started in January 2023, when our team met for the first time. Our brainstorming process started with more than 20 ideas. At the beginning of this year, we were just perfect strangers with different interests and backgrounds but with common principles and priorities regarding environmental protection. The majority of the proposed projects shared the same goal of restoring environmental conditions. The ultimate goal was to create a project that would assist the ecosystem in regaining its lost balance.

Choosing the challenge we would be tackling in and out of the lab for the better part of a year was a challenge in and of itself! Environmental protection, which had piqued the interest of a significant portion of our team from the beginning, created our own mini-black hole, and its gravity kept winning people over until the last man or woman standing had to admit it is really one of the biggest challenges that our country, Greece, faces: the loss of thousands of hectares (1 hectare = 10.000 m2) of forest land, the loss of "Greece's lung" to destructive wildfires.

Picture of greek forest fire
Picture of greek forest fire
Figure 1: Forest fires in Greece

The immediate result of the aforementioned wildfires is huge habitat and biodiversity loss. This year’s wildfires destroyed huge areas of virgin forest land in Rhodes and Dadia, resulting in severe biodiversity loss and threatening a variety of animal species.1, 2 Greece has a total of 1,249 protected areas, comprising 803 sites designated under national laws, 446 recognized as Natura 2000 sites (one of which is Dadia) and 614 species protected under EU law. Forest ecosystems cover 42% of the country. In Greece, the group of protected habitats with the largest number consists of forests, accounting for 31.4% of the total.3

Pie graph of the persentage of terestial in greece that is designated as protected
Figure 2: 34.9% of terrestrial area of Greece is designated as protected areas 3
Forest biodiversity
Figure 3: Forest biodiversity

Having until now observed this unfathomable disaster, we decided to focus on the post-fire period, the natural regeneration of burnt forests using synthetic biology. An outline of our gaps before taking the problem heads-on would be as follows: the impact of wildfires on the environment; the challenges that arise; any and all possible approaches available to solve said challenges; the difficulties of reforestation and where they stem from; novel approaches to forest regeneration and where we should focus to find them.



The Problem

Understanding the problem meant combining our personal research with the opinions of experts like Professor Giorgos Zalidis. This gave us a holistic view of the aftermath of forest fires.

Wildfires are always unforeseen phenomena, brought on by human action or natural causes, that devastate the environment and human well-being.4 It is indicative that 119 million hectares (1 hectare = 10.000 m2) of trees were reportedly lost due to fire over a period of twenty years, making fire responsible for 27% of tree cover loss worldwide.5, 6

Every year, in our country, Greece, wildfires are a common phenomenon, especially during the summer period. Our country has ranked in Europe’s second place, regarding the percentage of burned area from 2008 up until 2022. 7

Moreover, data shows that the number of wildfires in Greece increased by approximately 60%. This translates to more than 46 fires breaking out every year and more than 30,000 hectares of land being burned.8 To this situation, add this year’s percentages of catastrophic wildfire incidents that shocked people inside and outside the country. Among the most memorable ones are the wildfires in Mati (2018) that resulted in 100 lives being lost and the Evia wildfires (2021) that led to huge ecological damage.9, 10

Nevertheless, this year’s summer, the summer of 2023, was a catastrophic period compared to 2008-2022, with multiple wildfires in numerous places, making Greece take Europe's first place in 2023. 11, 12

Persentage of country's land burnt between years 2008-2022
Figure 4: Persentage of country's land burnt between years 2008-2022
Persentage of country's land burnt in year 2023
Figure 5: Persentage of country's land burnt in year 2023

We need to look into the reasons that make those forest fires so destructive. Under certain conditions, fires may actually be beneficial to the ecosystem. Ashes' nutrients can stimulate plant growth; they aid in the eradication of diseases; and small fires can even help prevent large wildfires. 13, 14 Sadly, the consequences of climate change and human interference negate these advantages. The intensity and frequency of wildfires have increased due to rising temperatures, longer droughts, and hot weather. Because of how detrimental those conditions are, natural reforestation cannot keep up. As a result, the formation of a new forest before the next massive wildfire is an unlikely scenario; there is not enough time for natural compensators to act.15, 16, 17

Soil quality restoration is one of the fundamental issues brought on by wildfires and a component of forest restoration failure. Numerous organic and inorganic compounds can burn and volatilize as a result of the massive consequences of a strong fire, resulting in a significant loss of soil organic matter and nutrients.18, 19 To the same extent, a sizable loss of biomass has been found in the soil microorganisms that are involved in the life cycle of the forests.20 The degradation of the root system, soil disaggregation, and its increased hydrophobicity after a wildfire can lead to soil erosion. As a result, floods, landslides, rockfalls, and debris flows, all of which are linked to erosion and the consequential soil’s inability to absorb water, can contribute to additional mass movement and soil loss.21

The remaining organic matter is transformed into complex hydrophobic structures. More specifically, increased temperatures promote the formation of aromatic compounds, polyphenols, tannins, and lignin-like structures that reduce both the soil’s hygroscopicity and nutritional value. This situation, combined with the lack of various nutritional substances such as nitrogenous compounds, sulfur, and potassium in burned soils, can lead to the shaping of an infertile land, unable to boost a forest’s rebirth. Another factor that, in some cases, obstructs forest regeneration is the lack of seeds in the soil. Due to high temperatures during a wildfire or due to recurring fires in the same area, the available seeds are drastically reduced.18, 19, 20, 22

Figure 6: Concequences of forest fires to soil's health

These factors establish an inhospitable setting for the growth of new vegetation. Upon our bibliographic research, we found that, in a 5-year span, 46% of burned areas in the Mediterranean showed a lower plant cover compared to the situation before the fires because of soil erosion, harsh conditions, and repetitive wildfires in the spots of interest.23 This poses, now more than ever before, the threat of a permanent desertification of once-alive and perfectly balanced ecosystems if things continue to be the way they are. 24, 25

Figure 7: Permanent desertification of forest land after an intense forest fire


Our Vision

While educating ourselves about the problem and getting in contact with scientific experts, we focused on the hydrophobicity of the soil and the need for organic matter and nitrogen in an available form for the growth of the plants and the soil’s microbiome, which can lead to the irreversible infertile situation: desertification. The next step was to conceptualize it practically and design our solution.

After contacting experts in this field like Dr. Maria Doussi and the Forestry Department of Thessaloniki, we realized that the proposal to intervene in nature is demonstrably controversial. Much of the scientific community believes that nature should be left alone after any major disturbance, such as a forest fire, and that any interference would result in a major disruption of the balance of the ecosystem. On the other hand, it is common knowledge that these phenomena have become more frequent in recent decades and that nature alone is sometimes unable to overcome and keep up with them. In search of the golden ratio, we decided to propose a solution as minimally invasive as possible that respects nature and its rhythms. Our goal is to strengthen the regenerative mechanisms of the soil by improving useful properties that are necessary.

Reversing the established phenomenon of desertification is a difficult and expensive process. The practices to avoid it focus mostly on prevention: preventing the loss of the first soil layer in more practical and mechanical ways, such as reduced tillage, green mulch planting, and crop rotation. 26 Regarding forest soil rehabilitation, after discussing with the Forestry Departments of Thessaloniki and Istiea, one of the most common measures is the use of logs in sloping fields to avoid soil subsidence. These measures are not directly invasive, but they do not directly benefit the soil properties. In this context, Mr. Tsaligopoulos informed us about soil transplantation, another technique to restore the soil, but it is very difficult and very expensive. Another commonly used technique is the widely known practice of reforestation, in which new small plants are introduced into burned soils. This technique is also invasive and can prove harmful if not applied correctly, leading to issues like a decrease in biodiversity.27 Our project's approach aims to differentiate due to its focus on changing the soil's characteristics after a strong fire so that water runoff and erosion are limited, soil fertility is enhanced, and the soil promotes its own forest’s regeneration.

A further major inspiration source was the UN’s 2015-launched sustainable development agenda. 28 Sustainable Development Goal 15.3 of the United Nations highlights the significance of combating desertification and restoring degraded land and soil. 29

Figure 8: Euphoresis and Sustainable Development Goals

In light of all the above, we created “Euphoresis”, the first synbio approach for forest regeneration. It is an innovative, environmentally friendly bio-product that enhances the water absorbability of soils, combats erosion, and establishes a conducive and fertile microenvironment for seed germination. The name comes from the combination of the two Greek words “euphoria”, which means fertility, and “regenesis”, which means rebirth or creation.

Figure 9: The orientation of the name "Euphoresis"

The rebirth of the destroyed ecosystem (and consequently of the society linked to it) is a recurring theme in ancient Greek mythology, while pre-revolution Greece revealed the idea of a Phoenix rising up from the ashes! Our proposed solution might not give birth to a mythical creature however it involves a three-level approach: a biodegradable hydrogel hosts a bacterial consortium while also encapsulating plant seeds.



Euphoresis: a synbio soil-ution for forest conservation

The Hydrogel


Realizing that one of our first priorities should be to solve the problem of hydrophobicity of the soil, we started looking at hydrophilicity-enhancing ideas we could bring to life or improve with the help of synbio. Professors Giorgos Zalidis and Dimitris Bouranis informed us about polymeric resins that are being used in agriculture for their high water absorption ability. So we focused our research on polymeric resins, specifically in the category of hydrogels.

Hydrogels as soil conditioners were introduced in urban farming in the 1950s. They are used for their capacity to retain water, prevent water runoff, minimize soil erosion, etc. 30, 31 At first, the hydrogels were made from synthetic polymers such as polyacrylamide, but in recent years, their toxicity has steered research towards biodegradable polymeric hydrogels that do not render a later removal from the soil necessary. 32New EU regulations on soil health also demand the use of biodegradable soil conditioners. 26 For these reasons, we decided to create a hydrogel-forming biopolymer. Our polymer will exhibit a high swelling capacity, enabling it to absorb rainwater effectively, thus mitigating water runoff and contributing to improved resistance against erosion.33

Our hydrogel formulation matrix consists of three ingredients: two polysaccharides, pectin and chitosan, and a synthetically engineered antimicrobial peptide serving as a crosslinker agent. 34, 35 This unique combination will ensure the stability and safety of our product.

Pectin was introduced to us by Professors Dimitris Bouranis and Christos Chatzidoukas. It is an anionic, water-soluble polysaccharide constituting the cell walls of most plants. It has high solubility and high swelling ability in aquatic environments. Pectin hydrogels are characterized by their biocompatibility, biodegradability, low toxicity, and the ability to immobilize cells, proteins, drugs, or growth factors.36, 37, 38

Chitosan is a natural, nontoxic, biocompatible, cationic polymer. It's biodegradable, has antimicrobial and antifungal properties, and promotes plant growth.39 Pectin and chitosan hydrogels combine the beneficial properties of both of the polysaccharides, while the cross-linking ability between the two increases the mechanical stability of the final copolymer.39, 40

Figure 11: Crosslink between pectin and chitosan

For the production of our final product, we decided to follow the principles of circular economy and contribute to the zero world waste goals. This approach also falls under Sustainable Development Goal 12 and contributes to a lower-cost production, making future implementation one step closer.

Pectin and chitosan, the two polysaccharides required for euphoresis, will be extracted from waste products of the food industry, specifically of the fruit and crustacean industries, in an effort to promote environmental well-being through waste recycling. 28, 41, 42 Chitin will be taken out of crustacean shells and converted into chitosan. Crustaceans contain approximately 40% meat, with the remaining 60% being inedible. 43 It is obvious that there is a need to make use of the 6–8 million tons of shell waste (such as crab, lobster, and shrimp) produced by the crustacean industry each year.44 Finally, pectin will be extracted from fruit industry waste, mostly from orange peels. The majority of the fruit waste produced by the fruit processing industries is disposed of in landfills, contributing to greenhouse gas emissions.45

Figure 12: Extraction of pectin and chitosan from citrus and crustaceans waste and formation of bio-copolymer

After researching the potential bio-co-polymeric hydrogels' characteristics, we came to the conclusion that we should aim to maximize both the stability of the hydrogels as well as their life expectancy. Subsequently, we decided to include an engineered peptide. We used an existing peptide with antimicrobial properties against gram-positive bacteria as a template and designed our own peptide to be physically crosslinked via charge interaction with chitosan, while it can also form "covalent dimers" via the cysteines in its structure. By choosing an antimicrobial peptide, we are aiming to add an extra biosafety layer as a complementary step to the other biosafety measures against the release of our GMMs.



Figure 13: The final bio-copolymer whith crosslink between chitosan, pectin and the engineered peptide

Microspheres


We have built a system of microspheres that are trapped inside our hydrogel and serve as hosts for our bacterial consortium. They consist of alginate and pectin, a co-polymeric system that has been previously used for bacteria encapsulation with adjustable pore sizes, which depend on the ratio of the two polysaccharides. By changing the pore sizes, we can permit the release of the desired enzymes while preventing bacterial leakage.46 These microspheres thus serve a dual purpose by improving the biosafety measures of our suggested material and fostering a favorable microenvironment for the bacterial consortium.

Figure 14: The layers of euphoresis; Inside the hydrogel there are microspheres that host the microbial consortium


Microbial Consortium


The bacterial consortium within our hydrogel comprises two distinct species: WB800N, a strain of Bacillus subtilis, and Nostoc Oryzae TAU-MAC 2710, a strain of filamentous cyanobacteria. These bacteria have been carefully engineered to communicate with each other using a quorum-sensing system. 47, 48 This mechanism ensures harmonious coordination among the bacteria, allowing them to function co-dependently while controlling the kill switch system and safeguarding against leaks from the hydrogel.

Figure 15: The microbial consortium that communicates through a quorum-sensing mechanism inside the microspheres


Bacillus subtilis

Bacillus subtilis has been engineered to constantly produce extracellular laccases, which will be transferred into the soil through the pores of our microspheres. Laccases are enzymes that degrade hydrophobic phenolic compounds and lignin-like structures that are abundant after a strong wildfire. The degradation of those compounds into smaller structures is necessary because of their limited bioavailability, as a scarcity of soil microbial species contains their degradation enzymes. 49, 50, 51 The addition of those enzymes tackles both the hydrophobicity and the limited organic matter on the soil’s surface. 52

Figure 16: The engineered Basilus subtilis expresses extracellular laccases that break down the hydrophobic compounds formed after a forest fire

Nostoc oryzae

Having in mind soil fertility, we have engineered filamentous cyanobacteria to create a unique biofertilizer. These modified cyanobacteria over-express a specific gene that promotes their differentiation in heterocysts, resulting in an enhanced nitrogen fixation process.53 This genetic modification increases the ability of the cyanobacteria to assimilate and provide nitrogen, thereby improving the soil's nutrient content.

Figure 17: Nostoc oryzae is engineered to form more heterocytes that facilitate the nitrogen fixation process

The two modified microbial strains are facilitating soil improvement by increasing the concentration of available organic matter and nitrogen. Without the desired concentration, it is impossible for new vegetation to grow and the soil microbiome to increase. Euphoresis’ desired result is the creation of fertile land.

We also applied two kill switch mechanisms, one of which is coordinated by the quorum sensing system. This is our primary and strongest biosafety layer against the release of genetically modified organisms into the environment when the degradation of our product starts. You can find more information in our Design page! Previously proposed kill switches, available in research papers or past iGEM projects, did not have activation systems that could be easily modified for Euphoresis’ conditions (the main problem being in vivo parameters). We had to propose a new, combinational communication system to link the survival of our cyanobacterium with the survival of our B. subtilis, the latter having many available activation systems.

Picture of greek forest fire
Figure 18: Nostoc oryzae kill-switch alive state
Picture of greek forest fire
Figure 19: Nostoc oryzae kill-switch deceased state

Seeds


In the final stage of our project, we made an effort to make each situation's solution more comprehensive. As stated before, the soil’s seed banks are not always readily accessible. When there are frequent fires or fires with high intensity, new forest plants cannot vegetate, so our product will include seeds. With the guidance of Professors Andreas Drouzas, Ioannis Tsiripidis, and Athanassios Mavromatis we decided to include a variety of local or endemic species’ seeds, chosen carefully for each environment, to ensure the maintenance of biodiversity and raise the success rate of vegetation rehabilitation. We will include both endemic leguminous and forest plants, and we will create a plan for every terrain that we would apply our project on. The plan would have a mosaic pattern, in order for the new forest to include plants more resistant to fires, ensuring in this way their protection from this phenomenon.

Safety layers of our project. Biodegradable hydrogel with group 1 bacteria and a kill switch system
Figure 20: Seeds are included inside the hydrogel when needed



Review

We worked arduously for nearly nine months to attempt to cover every aspect of our project. We made an effort to intervene as little as possible; the ecosystem's balance depends on respecting the barriers that nature erects. Despite starting with a problem that directly affected us—forest fires—we were still able to design a solution with a wider range of uses. Every area at risk from desertification, drought, and infertility can utilize Euphoresis, the proposed soil conditioner and biofertilizer. With further tweaks, we hope to increase its potential applications.


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