Human Practices
Our project aims to help and sustain society as we know it today. The ever-growing world population poses a major challenge towards the food industry and agriculture as the space available to harvest food decreases. Simultaneously, the number of mouths to feed grows every single day.
Only if society has the feeling of security and being looked after, it can achieve greater things. If insecurities and doubts about the future exist, there will be turmoil and uprisings. To prevent such disaster, the satisfaction of the basic needs, namely food and clean water, must be ensured.
Our project aims to provide both, as we filter the water and therefore clean it while extracting a substance which is crucial to decent plant growth – phosphate. With our sustainable approach of a reusable, light-induced phosphate binding protein, we fight against the phosphorous shortages and for a cleaner, greener and certain future.
Integrated Human Practices
Science communication is essential in both ways. Not only do we have to educate the people that don’t know about our project and the background information about the phosphorus problem. We also must learn from experts that have been practicing in this field longer than us and therefore are experienced. Additionally, we need to talk to the people who are affected by our project and listen to their concerns and demands.
During our project, we had meetings with different experts, professionals, mentors, and advisors. They helped us to modify our project and gave us their opinions. Our advisors helped us to choose which optogenetic tools are suitable, how we can design them, and which immobilizing method would work the best.
Expanding our project’s application possibilities, we had interviews with a sewage treatment plant, two urban water management experts and two farmers. They told us how they assess the current status of phosphate, which problems and possible solutions already are existing and which demands our product has to meet in the area of application.
In the process of optimizing our approach for a possible third engineering cycle, we had interviews with an optogenetic expert and an environmental engineering expert. With their point of view, we will discuss in our outlook how a third engineering cycle could be implemented and which candidates are suitable for a switch and phosphate binding protein.
Counseling meetings with our advisors
- Counseling interview with Prof. Lars Blank
After we determined our project to phosphate recycling, we spoke to Prof. Blank, who is an expert for applied microbiology. He noted the changed laws about phosphate recycling from wastewater. We learned that the molecule polyphosphate is more worthy for the industry than phosphate for the environment, plants and animals. We discussed the possibility to use the wastewater from purification plants, but here the volumetric flow rate is really high and therefore, we have to use a smaller approach in the laboratory. So we thought about the phosphate recycling methods and how to generate polyphosphate. Prof. Blank helped us to get in touch with Prof. Wintgens.
- Counseling interview with Prof. Thomas Wintgens
Prof. Thomas Wintgens is an expert in the field of water- and environmental technology and is currently working at the institute of sanitary environmental engineering. He told us that the phosphate concentration of wastewater of purification plants is 10 mg/L and that 1.8 g phosphate per citizen end up in the wastewater every day. In the sewage sludge, the phosphate concentration is 50-100 mg phosphate per Liter, therefore, to use our project at purification plants is unlikely. Instead of using wastewater from purification plants, we were advised to use wastewater from the food industry, as the phosphate concentration is higher.
- Counseling interview with Prof. Ulrich Schwaneberg
Prof. Schwaneberg, an expert in the field of directed evolution and translation research of interactive materials, noted to work as close as possible to real conditions, which includes the phosphate concentration of wastewater. We also brainstormed about some disruptive substances in the wastewater.
Figure 3: Counseling interview with Prof. Ulrich Schwaneberg
- Counseling interview with Prof. Wolfgang Wiechert
Prof. Wiechert, an expert in the field of computational systems biology and cell simulation for microcolonies, helped us to get in touch with Thomas Drepper and Ulrich Krauss, as well as Dörte Rother. He also recommended to create a cyclic process and supported us majorly in the development of our hardware project.
Figure 4: Counseling interview with Prof. Wolfgang Wiechert
Visit at the Forschungszentrum Jülich - Meeting with Dr. Drepper, Dr. Krauss and Prof. Dr. Rother
We had the opportunity to visit the Forschungszentrum Jülich and to be consulted by Dr. Ulrich Krauss, Dr. Thomas Drepper and Prof. Dr. Dörte Rother in the process of developing and improving our optogenetic approach of a PBP.
Dr. Ulrich Krauss is a leader of a research group dealing with light-activated domains, such as LOV.
Dr. Thomas Drepper is head of the research group Microbial Photobiotechnology at the Institute for Molecular Enzyme Technology at the Research Center Jülich. His working group is involved in the development and optimization of new fluorescent proteins for anaerobic applications.
Prof. Dr. Dörthe Rother is the head of the research group biocatalysis at Research Center Jülich and her group builds complex products from renewable raw materials using synthetic enzyme cascades. For this purpose, they combine enzymes that do not occur together in nature in a modular and flexible way.
At the first meeting, they shared their professional opinions about the project after the presentation of our ideas. With their years of experience, they also gave us some first impressions on how we could possibly approach the laboratory part in our project. Additionally, they showed us the experimental setup of their optogenetic approach and proposed that we can follow it. At the second meeting, we were given the opportunity to look at our selected proteins in virtual reality as part of the Digifellowship project of the Forschungszentrum Jülich. With Dr. Krauss and Dr. Drepper, we took a look at different PBPs, their ability to bind phosphate and the potential switches to control the phosphate binding and release with a light signal. With virtual reality, we were able to decide on how to fuse our PBPs with the light switches as best as possible. During our process of the gene design, they helped us with many issues which arose in succession of designing a functional light switchable protein.
The visit of the Forschungszentrum Jülich was one decisive step in our journey to develop and improve our project. We are glad to have been given the opportunity to be a part of the Digifellowship-project, which helped us to understand how our protein works.
Interview with Botanical Department of the RWTH Aachen University
We had the opportunity to meet Prof. Dr. rer. nat. Joost van Dongen to discuss the relevance of phosphorus to plant growth. Prof. van Dongen is the head of the botanical institute at RWTH Aachen University. His working group researches the stress tolerance of plants.
Prof. van Dongen explained that phosphorus is a part of cell walls and DNA. Because of that, phosphorus is essential for all living organisms. Plants also need phosphorus to create blossoms, whereby a lack of phosphorus hinders plant growth.
Together, we discussed that a lack of phosphorus will probably have a bigger impact on plant species with smaller seeds because there is phosphorus stored in seeds. As there is no experimental data about our thesis, we decided to start our own experiment.
In order to examine the impact of phosphate on plants, we decided to grow different kinds of plants under the same conditions but one with and one without the addition of phosphate.
For this purpose, we were provided with a cell in the greenhouse of the botanical department of RWTH Aachen University under the supervision of Prof. van Dongen. His coworkers helped us to select the seeds and the right composition of fertilizers. We chose to plant Solanum lycopersicum, Cichorium endivia, Pisum sativum, Triticum spec., Arabidopsis thaliana and Glycine max on rockwool and water them every 2-3 days with the prepared solutions.
The composition of fertilizers can be seen in table 1.
Table 1: Composition of the solutions with which the plants were watered. The solvent was demineralized water. With phosphor use $KH_{2}PO_{4}$ without phosphor use KCl instead.
| Salts | Concentration (mM) |
|---|
| $Ca2xNO_{3}$ x4 $H_{2}O$ | 2.5 |
| $KNO_{3}$ | 2.5 |
| $KH_{2}PO_{4}$ / KCl | 0.5 |
| $MgSO_{4}$ x7 $H_{2}O$ | 1.0 |
| Micronutrients | 1 mL per 1 L |
| Fe-EDTA | 0.06 |
| NaOH | Titration until pH = 5.6 |
For the analysis, we decided to use only the plants of S. lycopersicum, as they were the most informative and significant.
The evaluation showed that the plants which were watered with the phosphate-containing solution had a growth advantage of more than four times compared to the phosphate-deprived plants. This can be seen in figure 1, where the height is plotted against the time in days. To quantify the data, a linear trend line was drawn to determine the slope.
Figure 5: The relative height of two S. lycopersicum plants plotted against the time in days. The orange-marked line represents the plant irrigated with phosphate; the blue-marked line derived from the plant without phosphate addition.
The results clearly show that phosphate has a significant positive effect on the growth of the plants. We proved our thesis right, that a lack of phosphorus has a bigger impact on plants with small seeds than on plants with big seeds.
Figure 6: Time lapse of plants with and without phosphate
Interview with Tim Riedling
To get an expert opinion on the use of phosphate in agriculture, we had the chance to interview a farmer. Tim Riedling works on a farm that breeds both plants and animals. He told us that phosphate plays a big role on this farm. On the one hand, it is used in the specially mixed fertilizer for silo maize, but it is also added to the animal feed for dairy cows. He also said that people working in the agricultural sector are aware of the major problem of phosphate oversupply and the resulting environmental damage, as well as the shortage of phosphate. He also sees a very big problem in the future if a method to recycle phosphate is not found soon. According to him, the cultivation of food and the resulting yields would then drop significantly, so that a global famine cannot be ruled out. He was therefore very interested in our project and confirmed to us that there would be a great deal of interest in agriculture. In addition, our product, the pure phosphate in powder form, is very good and easy to use for farmers.
As can be seen from this summary of the interview, we were able to obtain a lot of information and a specialist opinion and are very happy about it!
Interview with Phillip Krainbring
Phillip Krainbring works as a farm manager on an arable farm in the Magdeburg Börde. Even as a child, he was enthusiastic about agriculture. For us, this conversation was of great importance, as it gave us the opportunity to perceive our project from a farming point of view.
At the beginning of the discussion, the importance of phosphate was once again emphasized by the farmer. Nitrogen-phosphate fertilizer is most used alongside organic fertilizer, such as manure. Phillip Krainbring focused above all on the tense political situation of fertilizer use. Particularly important here is the Fertiliser Ordinance, which regulates the application of the amount of phosphate.
He also cited the Russia-Ukraine conflict as the cause of price increases for phosphate fertilizers. At this point, our project would provide a remedy, because local production of phosphate in Germany would lower the price. Farmers are aware of the problem of washing out phosphate. However, not all regions of Germany are equally affected, which is why a distinction within the Fertiliser Ordinance is necessary. Through our project, at the end of which is the production of polyphosphate, offers the advantage that a granular fertilizer can be applied to the fields. This would be much less odorous, partially removing the social pressure from farmers. There would then be fewer complaints about odor nuisance.
Phillip Krainbring encouraged us in our actions as he also attaches great importance to the phosphor issue in agriculture and nutrition for the growing world population, respectively. He said that an economical phosphate-fertilizer in the form of powder would meet the requirements in his area of application the best. Furthermore, he sees a great future in the recycling of phosphate, because its a finite resource and the agriculture depends on it. All in all, he thinks our project to be very promising and the solution to many problems. We were highly motivated to pursue our approaches as the polyphosphate production at the end of our production cycle is exactly what the farmers need. The dialogue with Phillip Krainbring turned out to be essential for our product development.
Figure 7: Interview with Phillip Krainbring
Interview with Prof. Schäfer
Prof. Heinrich Schäfer is currently working as a division manager of wastewater technology for the Erftverband. This company has 31 sewage treatment plants and therefore Prof. Schäfer holds a lot of valuable information about how our project can be implemented into the sewage treatment plant process.
He introduced us to various ways to make our project usable for wastewater treatment plants. One possibility would be to place our proteins directly into the aeration tanks. The requirements of our proteins on their environment must be considered. The problem of how to remove the proteins from the basins so that a controlled release of the bound phosphate is possible would also have to be solved.
Another possibility is to build large columns behind the aeration tanks to be able to bind phosphate from the wastewater there. However, the large volume flows (12,000 $m^{3}$ per day in a sewage treatment plant) must be considered. Costs and effort are probably also very high here.
Small volume flows and a higher concentration of phosphorus are found in wet sludge. At this point, an implementation of our project is maybe possible.
Another possibility could be the purification of phosphate from the ash. In connection with the planned mono-incineration plants, economic advantages could also arise here. The installation would only be carried out decentral on a few mono-incineration plants, which would reduce costs and effort.
Early purification of the sewage sludge would have the advantage that the sewage sludge could then be reused, for example in the cement industry.
Our approach requires a closed system because our molecular switch works via blue light. Such conditions would have to be provided first, which would also be a cost factor.
The concentration of phosphorus in wastewater is about 10 mg per liter. This value has been taken under dry weather conditions. About 95% of the phosphorus can be filtered out of the wastewater using the conventional method of iron chloride precipitation.
2.7 kg of iron chloride per precipitated kilogram of phosphorus is required. With our project, we want to provide a replacement for the precipitant.
Another possibility to place our project in the system of a sewage treatment plant would be to place it into the so-called sludge path. After thickening (the medium is still flowing), there are smaller volumes. After thermal disposal, the medium is in granular form. Again, the crucial question is whether our approach can deal with these conditions. An upscaling is possible after the follow-up clarification.
The sewage treatment plant has a pH value between 6 and 7.5. Temperatures between 20 °C and 35 °C prevail before digestion. 35 °C can also be assumed before the digestion tank. In the digester there are methane bacteria that work anaerobically. The water temperature of the clarifiers is about 10 °C in winter and 20 °C in summer.
This talk with Prof. Schäfer was a fundamental step in our product development process. Getting a new perspective on application approaches and discussing the prevailing conditions in detail significantly drove our implementation further.
Figure 8: Meeting with Prof. Schäfer from the Erftverband
Interview with Dr. Mayer
Dr. Brooke Mayer is an associate professor at the Marquette University in the field of civil, construction and environmental engineering. She is an expert for regaining phosphate out of wastewater using phosphate binding proteins. Also, she is specialized in pathogen treatment in wastewater.
During our talk, she recommended to test our proteins with polluted water, to see if other organic materials have an influence at the binding capacities of our protein. She also advised us to use dirty water to test the absorption of red and blue light. This would allow us to judge which switch system would be more suitable.
Furthermore, she encouraged us to use the ash as source material to regain phosphate. This would have the advantage, that the phosphate would be far more concentrated, and we would not have to deal with the huge volumes of water like in a sewage treatment plant.
After all, she encouraged us in our project. She could not recommend any dimeric phosphate binding protein what supports our research so far. Nevertheless, she gave us useful advice for the following steps and the outlook of the project.
Figure 9: Interview with Dr. Mayer
Interview with Prof. Möglich
We had the opportunity to talk to an expert for optogenetics, Prof. Andreas Möglich. This interview helped us to get important insides about the use of an optogenetic switch and what other possibilities can be explored. Prof. Möglich studied biochemistry at the University of Chicago and got his PhD in biophysics at the University of Basel, after which he started to work in the synthetic biology segment and focused on optogenetic switches. Now, Prof. Möglich is working at the University of Bayreuth in the department of photobiochemistry.
He told us about the various utilization opportunities of photosensitizers. As these increasingly become the focus of science, more and more optogenetic switches are being discovered, newly synthesized and the studies of their mode of operation improve. We discussed the pros and cons of our used photosensitizer and optogenetic switch and afterwards focused on optogenetic switches which could improve our project. These optogenetic switches provide shorter excitation and reversion times, improving the applicability of the switches. In addition, they have the ability not only to be switched on like the VVD domain we use, but also to be switched off by illumination with light of a different wavelength. Since different wavelength-operating optogenetic switches could be used, we discussed whether blue-light activated photo switches or red-light activated photo switches would be better for our project. But as this is depending on the actual application of our project, we could not come to a final decision.
Since Prof. Möglich showed us many different aspects of optogenetic switches that we did not know yet, we are very glad that we had the opportunity to talk to him! He contributed a valuable part to our outlook.
Figure 10: Interview with Prof. Möglich
