Team:NAWI-Graz - 2021.igem.org

Team:NAWI-Graz

Human Practice – A Story about the future

The project finding process was like asking a question that intrigued you, but you did not know the answer yet. It really had to be a good question, we really wanted to be excited about it, so we took our time with it. This is a very firm description of that process, which took us 3 months of meetings and presentations by team members and their ideas. We evaluated a lot of topics during our early team’s formation period at the end of 2019. Around mid-February of 2020 we defined our final ideas all around the recently appearing problems due to global farming. Many things we thought about really don`t get that much public exposure, that needed to be changed!

All these ideas may have emerged, because some of our team members originally are from the more rural Styrian areas around the city of Graz. And by rural origins we mean the most classical and stereotypical picture one could possibly have. Many of us have some type of farming background due to parents and grandparents, mostly working on the farm when having some spare time from university, being used to milk cows, mowing huge patches of grass and sowing pumpkin seeds for the delicious and famous pumpkin seed oil. And just like Werner Heisenberg has formulated great questions when he had gone on one of his countless creativity walks in nature. So did we, and the question we wanted to find answers to was right in front of our eyes when we walked by the natural little farms in the cozy and hilly landscapes of east Styria. We had it in front of us the whole time walking and driving by fields full of crops every day.

We knew that we wanted to tackle the world's inefficient food production issues not knowing on which path or direction exactly we were heading. After speaking with some local farmers, mostly some family friends and relatives of some of our team members , we had a much clearer definition of the problem: The inefficiency arises mostly throughout the fact that significant amounts of important nutritional uptake from fertilizers isn’t making it into the plant's roots, which is mainly causing some serious challenges on many different levels as will be discussed further down. After consulting our principal investigator, we finalized this process around just one of these nutrients which seemed to be the most critical one to deal with. Phosphorus ! [1]

Unfortunately, the pandemic made it impossible to take part in the 2020 competition, therefore we decided to participate one year later. Also, this came in very handy since it bestowed us some time to elaborate more on the scientific background of the project. We were able to read a lot of publications, speak to a lot of academics that were working in the field of life sciences and beyond, and we planned our practical work in very much detail.

After the bio bricks arrived in the summer of 2021, we were able to identify the whole complex background of this project, because things seem very interconnected on so many levels. Of course, it was an ongoing and more than not never-ending process of constant reevaluation and new question formulation. The following text describes our final way of thinking about the whole project and why it is so critical and relevant for future generations to not let it slide:

The molecule phosphorus is deeply related with the formation of life as we know it on planet earth. Phosphorus accomplishes its life-giving properties, inter alia, throughout its biochemical ability to form high energy compounds, serving as an energy currency for a vast variety of biochemical events relevant to life. Since all these reactions are linked with the gain or loss of energy in some way, which on the other hand relates to phosphorus containing molecules it must be assumed that phosphorus almost always played a crucial role to the formation and maintenance of life from its earliest beginnings on. [2]

In the biosphere this atom can be found mostly in oxygen compounds such as phosphates. It undergoes a natural pathway or cycle, which therefore is called the phosphorus cycle. It's divided into four distinct steps: (i) the tectonic uplifting of phosphorus containing minerals to the earth's surface, (ii) its physical and chemical degradation into more soluble phosphates,(iii) the aqueous transport of these phosphates into rivers and lakes, plus its ability of uptake into the lifecycle , (iv) and finally its sedimentation linked with the association of organic matter with mineral matter containing a diversity of phosphorus derivatives. [3]

Neither the role of phosphorus as an essential life-giving compound can be denied, nor the fact that its natural limitations are setting up future threats to society, which made itself highly dependent on phosphate as a fertilizer component since the beginning of the last century.[4] Hence the minable phosphorus levels world wide determine our ability to grow food, the resource phosphorus cannot be produced artificially, and thus it is essential to life [5]

Phosphorus has no substitution in food production and only a few rock types contain minerals that are rich enough in its phosphorus levels to be able to meet nowadays standards of modern fertilizers, which is still the main source of the world's phosphorus supplies. Using phosphate fertilizers since the 50s of the last decade had boosted crop yields immensely and helped to feed vast numbers of the fast-growing world population, which in turn had built a high dependency on a nonrenewable resource in the form of phosphate rock. Throughout this process phosphate rock is farmed and released much faster into the economical phosphorus cycle than it would naturally, also leaving aquatic and terrestrial ecosystems suffering from severe pollution. [6].

Whilst the ever-growing phosphorus concentrations in nature caused by mankind in the last few decades are giving rise to a high degree of destruction of diverse wild ecosystems of yet unknown extent, and beyond that the finite resource phosphate rock is estimated to be depleted in approximately 50 – 100 years. The expected global demand of phosphorus in agricultural and technical applications is expected to peak around the year 2030 increasing the price of phosphorus on the world market, whilst the quality of mined phosphate in the phosphate rock is decreasing simultaneously. [7]

If no changes are made to the recent fertilizing and degradation strategy the growing demand and price for phosphorus in combination with the contra-rotating lowering of available phosphate rock levels is certainly setting up a huge threat for future generations in relation to this critical element. [8]. Mostly modernized farming since the 1950s and an over usage of nutrients such as phosphorus in fertilizers mobilized the excess of nutrients into the environment causing severe damage such as eutrophication and the formation of dead zones in the recent decades.[9]. Modern approaches to tackle this negative ecological dependence on nonrenewable resources aim for an increase of more organic phosphate sources which already exist as side products of global farming and wastewater volumes just to name a few ones instead of using phosphate rock. [10] Also, ideas of implementing a higher educational level in more plant-based diets for future generations and consequently a global change in everyday diets could deliver sustainable solutions to the phosphorus problem in the long term. [11].

The concept of higher efficiency in manuring the crop is a logical consequence of these thoughts. The latest approaches aim for the same crop yield standards as usual but by maximizing the uptake of nutrients from the soil into the plant roots and ultimately being able to decrease the amount of necessary phosphate concentrations to achieve this yield. These concepts are highly researched on and one of the main topics of interest. [12] [13] [14] [15]

We picked up these strategies and claim that a well-performing molecular biological implementation could be leading to less phosphate contamination of waste waters oozing into groundwater supplies causing the above-mentioned problems by entering the biosphere. Over and above that the amounts of phosphate in fertilizers can be minimized without the expense of losing potential crop yields, hopefully prolonging the usage of phosphate rock supplies , until different and enhanced ways of phosphate fertilization can be discovered.

Integrated Human Practice

After we had finally perceived that our project will be based upon the phosphorus problem, we`ve had to define all relevant parameters. Having plenty of ways to formulate a statement or a question leaves one really thinking before heading into different kinds of directions… In the first chapters of our iGEM career we had to face severe problems formulating a scientific approach towards this question we asked ourselves in the beginning. How can we improve crop yields?

We found so much evidence of work that was already done by others, we could not find our way through everything that has been done before on this topic. We really attempted to achieve something unique, hanging on ideas already made by others was surely not the way to go, but the fact that we wanted to start from scratch really was nerve-wracking. This “how to start”- frustration caused us quite a headache until we at some point even started to doubt our whole approach so far and asked ourselves why we went with it in the first place. Why do we want to improve the crop yield? It took us some time to realize that we had just found the answer to it, only the point of view had to be adjusted. First, it should not be a qualitative “how can we”-question at all. The first approach should be why do we want to improve crop yields?

Two substantially different yet closely connected layers of this question arise when asking this question. The ethical facets on the one hand and on the other hand the economical aspect this project also clearly brings to the table. The relatedness becomes clear when considering both issues in detail.

In equal measures, the ethical aspect of the phosphate problem veers towards sustainability. [16]. Even though increasing crop yields is beneficial and can be accomplished with just increasing the phosphate concentrations in fertilizers, an excess of dissolved phosphate leads to an irrepressible biological growth and water-quality issues caused by eutrophication. Furthermore, phosphate minerals, the large-scale main phosphate source, is claimed to be ending somewhere around the turn of the century. This is also affecting the economical point of view when considering the future.

And this indeed was our next big challenge to start working on. After reviewing many things, we found ourselves discussing everything. We needed to cut all irrelevant and off topic parameters and discern the relevant aspects without omitting anything either. Compromise must be achieved without neglection, we really defined our core principles around relevancy. Neither can we stop the extinguishing of phosphate rock, nor change the dependency on any other raw material. It is basically not our field and would also go far beyond the scope of what is possible in our situation. Also, we cannot change the economic situation, which is always aiming for growth even by causing destruction in other areas. It is simply too anchored in society, changing these things may take longer than the resources could last. Since phosphate is an important growth factor and the quality demand has a high relevance for major companies the over-usage of valuable phosphate won't come to an end soon. This fact was hard to realize and accept. But making just small changes can lead to big wins in the future. A trade-off can be gained by focusing on our lab work. In detail we first considered.

The first design we planned our project with was driven by our imaginations. It was the design for a system, that let us utilise the bacteria in the soil, to make the inorganic fixated phosphate in the ground either more accessible for plants, or to let the bacteria gather the phosphate in the soil and bind it, so that it doesn't get washed out by rain that quickly. To bring the cells to do such things we wanted to use a quorum sensing system with one receiver and a sender cell.

Due to unexpected hurdles we had to scale our goals down. The approach we tried to realize was a quorum sensing induced system to detect and display phosphate concentrations in the soil where the sender cell detects phosphate levels, excretes quorum sensing molecules like HSL and the receiver cell perceives this and expresses a marker like GFP.

There were several reasons for why we were not able to realize our original plans.

First and foremost, the pandemic and the actions to prevent its spread made it very hard for us to get a safe place to do our lab work. Another problem was that we underestimated the time it would take to realize what we planned on paper. The several stages of our project were much harder to reach and the lab methods like PCR or cloning needed much longer to be successful that we had to do this downscaling.

We had a a long time to figure out project and plan for the lab work, long before we even started working. We were not able to do much face-to-face human practice, since travel was restricted, even inside our country up until late September. We focused on remote options to gather information and perspective on the problem we were trying to solve.

PODCASTS

We were planning to travel to the surrounding teams to do interviews about their iGEM projects. Since the Covid pandemic was restricting our access to travel we had to find another way to further the understanding of synthetic biology topics in the community. Our social media team came up with the idea to publish a podcast on Spotify. We sent out invitations to teams and asked for volunteer teams on social media and many teams contacted us. We collaborated with seven other iGEM teams from all around the world and released a total of nine episodes to date. The teams were meant to use the podcast to showcase their project through other than written media.

The content is a casual conversation about the topic of research of the interviewed team. The core concept of each episode was “explain it to me like I'm five”, to expand the reach of these projects beyond academic circles. The created podcasts were shared by our universities and partners. It was a great experience to work together with other iGEM teams and find out about their projects and challenges by providing a platform for all other teams that participated in our podcast to draw attention to their questions and on how they are trying to find their answers to it.

TU-STREETECH

Since covid made it impossible to visit schools or hold information panels we collaborated with our university TU-Graz. In the past years the Streetech festival was held at Graz´s main square, but due to the circumstances was moved online. It is an event that is meant to showcase student teams that work at the university and explain their projects to the public.

We were asked to show an experiment that could be done by people watching at home and was related to microbiology. We decided to make kombucha from scratch to show our passion for microbiology in a relatable way.

During the three-day online event we were answering viewer questions and had some interesting conversations about synthetic biology. We even got requests to join our team or how to organize an iGEM team for next year's competition.

LAKE BLED

We also visited lake Bled in nearby Slovenia. Lake Bled is in the Julian Alps in northwest Slovenia with less than 20 km of distance away from Austria and is the country’s second biggest lake. Lake Bled is best known for its island with a church in the middle of the lake. This natural gem was first mentioned around 1000 years ago and is now one of the main tourist attractions in Slovenia. It was deeply impacted by the problem we are trying to solve with our project. The fertilizer runoff in the regions surrounding the lake was promoting the bloom of cyanobacteria. These blooms are predicted to increase in frequency and intensity in response to climate change and eutrophication, even though nowadays we can talk about Bled Lake as a success story it hasn't always been like that. In the 60s and 70s it was rapidly dying out due to severe pollution. Algal blooms were occurring annually, the water was not mixing, and the local authorities had to intervene. To understand how this was solved and what role water monitoring was/is playing in the health of the lake we talked to some of the local people.

We set out to interview the local water analyst and microbiologist Špela Remec Rekar and Zina Raunic, a molecular biologist, both living in proximity to the lake. Since we have members that are Slovenian nationals and speak the language fluently, we were able to conduct a thorough interview and translate it to English. A small part of the team was able to travel at the end of September 2021 and stayed 2 nights at the house of said team members. Here are both of the interview`s transcripts (the interviewer always was Jernej Juric due to its native level with the Slovenian language):

Jernej Juric: “Hi, we are here at Bled Lake. And today Zina is joining us. Could you please present yourself?”

Zina Raunic: “Hi I'm Zina. I live in Bohinjska Bela which is a village 2km away from the lake. I studied microbiology and now I’m working in the pharmaceutical industry. In my free time I like to do sports, such as yoga.”

Jernej Juric: “It is great that we found a local person. Can you tell us a legend or a story about the lake?”

Zina Raunic: “When I was in primary school, I really loved the legend about the origin of the lake. It is about magical fairies and farmers who used to live here. In the old times there was no lake, only farmland. And the farmers didn't allow the fairies to dance on their fields. There were many arguments between them and then one day the fairies, out of anger, summoned all the rivers and streams from the surrounding mountains and filled the valley with water. That is how the lake came to be. “

Jernej Juric: “Thanks. How big is the lake?”

Zina Raunic: “To go all around it, it is approximately 6km. “

Jernej Juric: “Have you ever run around it or maybe went around with a bicycle?”

Zina Raunic: “Yes, I did, although in summer it is quite hard to go around, because there are so many tourists. So, usually locals do it in the off season. “

Jernej Juric: “And how long does it take?”

Zina Raunic: “Well, I usually need around 40 min. though I’m not the fastest person around. “

Jernej Juric: “Quality of water?”

Zina Raunic: “-You can’t drink the water, but it seems okay. When we were kids, we used to spend all our days at the lake. -There were some algal blooms in recent years. -But in general, the quality is good. “

Jernej Juric: “Do you know who is doing the water monitoring?”

Zina Raunic: “I think ARSO is taking care of the monitoring.”

Jernej Juric: “Yes, and let me take this opportunity to present our next interviewee -- Špela Remec Rekar. Thanks Zina. Could you please present yourself?”

Špela Remec Rekar: “I am Špela Rekar and I work for the Slovenian Environment Agency (ARSO). I’m responsible for the monitoring of surface water, especially lakes. We monitor all the lakes in Slovenia.”

Jernej Juric: “The lake wasn’t always clean and healthy. There was severe pollution of the water in the 1960s and 1970s. What is the difference between now and the 1960/70s, where the eutrophication events happened, and the lake was basically dying? “

Špela Remec Rekar: “The Radovna water drainage pipeline was constructed in the year 1964, which means that already at that time the people saw that the lake has had some problems. It even overreached oneself that the lake's water did not circulate anymore. A chemocline formed at 18 metres depth. We can see that from the data taken already before the Second World War. An engineer published measurements of temperature and in three years, the deepest layer of the lake had a temperature higher than 4 degrees and the lake was not holomictic anymore. It mixed only down to the chemocline. This is nowadays occurring on the Velenjsko lake.”

Jernej Juric: “Is chemokine like thermocline? “

Špela Remec Rekar: “They are similar, although the water density in the chemocline is higher due to chemical compounds. Usually those are produced by anaerobic organisms in the sediment layers. “

Jernej Juric: “Can you tell us other relevant things to understand this problem on a deeper level?”

Špela Remec Rekar “The hypolimnion water drainage pipeline and a deepened Radovna stream were/are benefiting the lake's health. Built in 1964, designed by Prof. Risman. The occurrence of algal blooms throughout the years, main actors were/are the Planktothrix rubescens algae. In summer it floats in the lower levels of the lake. The sewage system that was built in 1987 also benefited the health of the lake to a large degree. “

Jernej Juric: “Did the water levels change throughout the lake`s history?”

Špela Remec Rekar “Thousands of years ago, the lake level was 75 meters higher than today. All the surrounding hills were at that time under water. Also, the Grajska cliff! The lake was way higher, yes. “

Jernej Juric: “Our team is trying to lower the amount of phosphate in the soil, from where it can be flushed out into water bodies. How polluted is or was the lake with this element? And what are the boundaries of phosphate in water bodies before it becomes detrimental for the environment? The main factor affecting the phytoplankton is phosphate, right?”

Špela Remec Rekar: “Yes, it is a limiting factor. You cannot build a house without building blocks. When there is an algal bloom, we know it happens because of high phosphorus amounts in the water although I must mention it, we measure total phosphorus amounts, because there is almost no soluble P form. The soluble form is used up instantly. The whole strategy of measurements depends on these planktonic organisms. They can be found surviving on very different layers of the lake, they can survive darkness and bright light earth further on they have a mechanism called luxury uptake which helps them to accumulate phosphorus for generations.”

Jernej Juric: “How does the concentration of phosphorus change throughout the year?”

Špela Remec Rekar: “We monitor the yearly concentration of phosphorus, since 1980 we began monitoring total phosphorus concentrations in the lake`s water. At that time the concentrations went up to 80 [mg/L]. This means the lake was already hypertrophic.”

Jernej Juric: “And what are the boundaries of phosphate in water bodies before it becomes detrimental for the environment?”

Špela Remec Rekar: “There is no boundary although at 80 [mg/L] of algal bloom is quite common. The year 1990 was a breakthrough, the water drainage system, and the deepened river end of the Radovna stream lowered the concentration of phosphorus under 20 [mg/L]. It is very important that the measuring method is adapted to the lake and the phosphorus concentrations in the water. Otherwise, it can happen that your measurements are irrelevant.”

References

[1] https://taurus.ag/importance-of-phosphorus-to-crops/
[2] CHICAGO: GULICK, ADDISON. “PHOSPHORUS AS A FACTOR IN THE ORIGIN OF LIFE.” American Scientist 43, no. 3 (1955): 479–89. http://www.jstor.org/stable/27826652.
[3] doi: 10.1016/B0-08-043751-6/08153-6
[4] doi: 10.1002/ejic.201400115
[5] doi:10.1016/j.resconrec.2015.09.013
[6] doi: 10.1146/annurev-environ-010213-113300
[7] doi: 10.1016/j.gloenvcha.2008.10.009
[8] doi: 10.3390/su3102027
[9] doi: 10.2166/9781780401805
[10] doi: 10.1002/jsfa.4650
[11] doi: 10.1093/ajcn/78.3.660S
[12] doi: https://doi.org/10.2134/asaspecpub26.c2
[13] doi: 10.1007/s11104-011-0907-7
[14] doi: 10.1016/j.chemosphere.2011.01.065
[15] doi: 10.1007/s11104-011-0950-4
[16] https://doi.org/10.1021/acs.est.9b02464