Human Practices

Why NitraNix?

When brainstorming for ideas in which direction this year’s project should be headed, a few things came to our minds. We all agreed on wanting to create something environmentally and socially beneficial for the world. As our team consists of people of many different nationalities, it was especially important to us that our project will have an international impact. This is why what stood out to us was approaching ways with synthetic biology to tackle nitrate pollution, being a problem affecting the environment and the people not only in Germany, but worldwide [1].

In our project, a biological alternative for denitrification is the main inspiration for designing a tool to combat the issue. Denitrifying bacteria are organisms from which we planned to isolate enzymes that catalyze reactions degrading NO₃^-. The model offers a cell-free alternative in which the enzymes connected with a linker are the main players in the synthetic approach.

Getting to Know the Problem

To ensure that our project will address the needs and be relevant to current developments regarding nitrate pollution, the most important thing was to inform ourselves about the ways nitrate pollution is caused, what consequences it has, how it is tackled presently, and which opportunities there are in aiding the issue using a synthetic product.

According to law, EU Members are obligated to prevent nitrate concentrations from exceeding 50 milligrams per liter [2]. In 2018, Germany was found guilty of violating the EU Nitrates Directive for failing to find a solution for reducing nitrate concentrations [2]. This shows just how critical it is to find a solution for nitrate pollution.

Apart from scientific research, we therefore wanted to get to know how the environment and people are affected by the problem, which ethical questions developing our product have to be considered, and what information current stakeholders regarding the problem have to offer and what they think of our approach.

First Draft and Project Goals

Our initial plan for dealing with the nitrate excess problem was to overexpress the proteins catalyzing the several steps of denitrification (see project description) in E. coli. Like this, the bacteria could have been inserted into water with high nitrate levels to stimulate the reduction of NO₃^- (nitrate) to N₂ (atmospheric nitrogen), thus reducing the amount of nitrate and releasing the nitrogen into the air. However, as we did not like the idea of inserting GMOs into the water which are then contained within groundwater and likely even drinking water, we modified our idea to use electroenzymatic nitrate reduction, providing just the needed proteins in a cell free system.

The denitrification method is done via enzymatic electrosynthesis, a challenging approach that might give further scientific insights and pose a suitable alternative to current methods of denitrification applied in water treatment, as these often bear disadvantages. Current methods of denitrification include chemical, physical (often via osmosis or electrodialysis), chemical-physical (done by ion-exchange), and biological denitrification. From these, only the last two are usable on a large scale. However, chemical-physical denitrification using ion-exchange has the problem of removing relevant minerals from the water, which is not wanted for drinking water and involves higher costs [3,4].

Therefore, our product aims to provide an easier and cheaper alternative, leaving no unwanted byproducts. Often wastewater treatment facilities include treatment for denitrification, but to tackle high nitrate levels in groundwater, in situ solutions, which simulate denitrification directly inside aquifers, are additionally needed. This is where our product will come into action as well [4].

Although our product is meant to aid in exceeding nitrate levels and thus prevent health risks and environmental consequences, societal awareness and action are much more essential. As an example, farmers can only produce food sustainably regarding nitrate contamination if they receive further education and are equipped with the necessary technical equipment. However, this means additional costs that can only be borne with the support of the government and customers who are willing to pay higher prices for sustainably produced products.

Consulting Experts in the Field - Interview with Erftverband

After much research of our own, we already had a clear idea of how we could approach solutions with a synthetic product. However, to get a deeper understanding of the current situation, the biological processes within the nitrogen cycle, and a general opinion on our ideas and suggestions for improvement, we contacted Dr. Nils Cremer from Erftverband, an association managing water treatment near our home cities, Bonn and Rheinbach.

Erftverband is a water treatment association in North Rhine-Westphalia state, Western Germany, administrating over 1400 water measuring points and 31 sewage treatment plants [5]. Because nitrate pollution is one of the biggest problems in their region regarding water quality [3], different ways of reducing nitrate levels, both preventive and counteractive, are used.

Dr. Nils Cremer, who studied hydrogeology and hydrochemistry, is responsible for all questions related to water chemistry, groundwater sampling and quality management in the area administered by the Erftverband, where he’s been working for over 19 years now [3]. A detailed report on nitrate in the area of responsibility of the Erftverband written by Cremer [6] presenting data on nitrate levels for individual measuring points in the Erftverband’s area, the causes and consequences of nitrate pollution, and possible strategies to tackle the problem especially drew our attention. Therefore, we contacted Cremer and asked for an interview.

Some information we gained from the interview is listed below. The full interview transcription can be viewed here.

How does water treatment work?

To achieve high quality standards for water that customers may use in a variety of ways, including as drinking water, several steps regarding water treatment have to be taken. One example is the removal of iron and manganese via oxidation and filtration, which otherwise lead to clogging and brown water color. Other steps include softening and deacidification. Denitrification, however, is rarely applied due to its high costs and complexity [3].

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What are the causes of nitrate pollution?

Referring to the location and measured data of individual measuring points in the Erftverband’s area, the main cause of nitrate pollution is agriculture, which correlates with the general situation worldwide. The growth process of crops heavily depends on nitrate fertilization. In most cases, however, much more nitrate is injected into the ground than the plants can take in. When the fields are uncultivated after harvesting, the water in the ground is not absorbed by plants anymore. Therefore, it seeps into deeper levels of the groundwater during the winter, taking minerals and other substances, including the remaining nitrate in the ground and from decomposed crop leftovers with it. This process is called groundwater recharge, and leads to increased nitrate levels in the groundwater if excessive nitrate remains on the fields [3]. Reducing the amount of fertilization therefore helps to reduce nitrate concentration levels in the groundwater [3]. However, nitrate cannot be fully abandoned in the plant growth process as it is needed to produce proteins or chlorophyll [3,4].

Another major source of nitrate seepage is excrements of animals in livestock breeding, which is injected into the groundwater after decomposing in the same way as crop leftovers during groundwater recharge [3].

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Preventative methods to reduce nitrate levels

Since nitrate pollution is mainly caused by seepage of excessive nitrate into the ground, the most obvious way of reducing the concentration of nitrate in the groundwater is to fertilize more efficiently. To achieve this, Nmin samples, specific soil measurements to estimate how much nitrogen fertilization is needed, can be taken. Additionally, multiple measurement locations lead to more specific knowledge of nitrate levels in the ground and thus salvage the possibility of precision farming, injecting fertilizers in the ground only where they are needed [3].

Another effective method to reduce nitrate intake into the ground is to dispose of crop leftovers and manure from the fields. To prevent excessive nitrate already in the ground from seeping into deeper levels during the winter, catch crop cultivation can be used. This means that after the harvest, a new crop is planted to absorb the remaining nitrate. The grown crops can then even be used as fodder or mineralized to serve as fertilizer for the next years’ crops [3].

However, without external support, farmers are often not able to apply these methods because they imply additional costs and require measuring equipment. Cremer states that society accepting to pay higher prices for sustainably produced food regarding nitrate pollution and subsidizing precision farming and measuring equipment would help to address this issue [3].

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Methods for biological denitrification

Biological denitrification has been approached using straw for the supply of carbon dioxide or utilizing molecular hydrogen, but has been abandoned in most cases due to higher costs and technical complexity in comparison to preventative measures [6]. In the area of responsibility of the Erftverband, only one water treatment facility applies denitrification using a heterotrophic method with acetic acid, and that is just because they have no other alternatives [3,6]. Usually, searching for water with lower nitrate levels in other locations, building wells in deeper groundwater storeys or blending with low nitrate water are much easier and cheaper alternatives [3].

A natural example of denitrification that can be observed when pyrite is contained in the ground is the reaction of this pyrite with nitrate, reducing the nitrate to nitrogen gas. However, this also releases iron into the water. Sometimes, biological substances in the groundwater can also oxidate nitrate, but cause unwanted side effects by releasing uranium, which is often absorbed by such organisms during the reaction [3].

Using a cell-free system, we want to aid these problems and provide a simpler and cheaper solution for biological denitrification, avoiding the side effects of the mentioned denitrification processes. Still, advances in precision farming and the reduction of livestock breeding remain issues of highest priority.

Responsible and Positive Impact

To evaluate the advantages of using Nitranix as a denitrification method, we must first identify the main problems caused by nitrate pollution, and understand the impact of nitrates. After extensive period of literature research, we managed to determine three main areas, threatened by high nitrate concentration:

It is crucial to recognize that the existence of in situ denitrification methods should never serve as an excuse to neglect other essential measures aimed at reducing nitrate intake. Instead, it should be viewed as a complementary strategy to provide lower nitrate levels for both people and the environment. Therefore, while agricultural practices are adapted to reduce general nitrate intake, methods such as catch crop cultivation must remain the highest priority. In this context, in situ denitrification via bioelectrosynthesis will serve as a valuable tool to expedite and support the process of reducing nitrate levels.

Ensuring access to safe and clean drinking water is a fundamental human right. Nitrate pollution challenges this right, as contaminated water sources may necessitate costly treatment measures or alternative water supplies for affected communities. With the help of NitraNix, the lowering of nitrate will reduce the incidence of chronic health conditions such as cancer and cardiovascular problems, as well as the methemoglobinemia mentioned previously.

Project's Impact and End Users

With the growing problem of nitrate pollution, our aim was to address the need for developing better counteractive measures to be used in water treatment facilities, which is our main target group. As much as nitrate pollution can be reduced, water is still being polluted and needs active treatment. With our project, we hope to facilitate further research on the counteractive measures, such as biological denitrification. After more thorough research and experiments are done on this method, it would be possible to implement a more cost-efficient process of nitrate removal from water in the real world.

Clean water is needed everywhere, and we believe that everyone should have access to water free of nitrate pollution. Consequently, we designed our project to target many end-user groups, both governmental and private:

Project Implementation

Speaking of the practical execution of our idea, we are talking about denitrifying the wastewater in the first place due to its increased exposure to nitrate. Denitrification in modern facilities usually takes place in the aquifer (in situ) as this is the cheapest option [13]. However, applying protein electrosynthesis in an open system might be more difficult due to potential clogging by bigger particles and an unstable pH.

Electroenzymatic denitrification might also be held in bioreactors (enclosed systems) in order to avoid biomaterial spillage. Modeling the bioreactor and integrating it into the industry is a huge challenge itself due to the novelty of electro biosynthetic technology. Electrodes and potentials must be optimized for different proteins and environments in the treatment facility situationally. Both the material and surface area of the electrode play a role in the efficiency of the electroenzymatic reactions and thus will affect denitrification as well [14].

However, electrosynthesis might be beneficial over the classic biological denitrification due to the opportunity to automate the process through solar cell usage, which cuts the expense of electricity. The bioreactor could be built in a way that uses renewable energy to consistently provide the necessary voltage for the denitrification process, which would help the project be economically feasible.

An enclosed bioreactor would also protect workers from interacting with biomaterial, while the electrosynthesis itself would allow them to quit using toxic substances such as ammonia to reduce the nitrate in the water. With microbial proteins being the driving force of nitrate reduction, the need to use other chemicals to drive the reaction will be eliminated, and people working in the water treatment field won’t have to face every day both high concentration of nitrates and toxic denitrifying substances.

Microbial protein synthesis, in contrast to electroenzymatic reactions, is already widely used and well known. E. coli is one of the most famous, cheap and controllable microorganisms to handle; however, it has its own downsides, which make it not suitable for involvement in the food industry.

Alternative microbial cell factories, that could be used for denitrifying protein production, are lactic acid bacteria (LAB). Being gram-positive, they do not possess endotoxins (typical for gram-negative bacteria), such as E. coli, which makes them better candidates to use in modeling bioreactors for the food industry. LAB are harmless to the human organism, and a lot of studies nowadays are highlighting the potential of those bacteria as recombinant protein producers [15]. Thus, by switching to another microbial protein producer, we could also ensure safety when using biological denitrification in the food industry and private households and house projects.

Conclusion

Over the course of almost a year, NitraNix underwent many changes and improvements before being formulated in its final form. Our team members dedicated themselves to literature research and contacted professionals in the field in order to get closer to the subject of the study and understand the problem of nitrate pollution in detail. Our project is aimed at affordability and provides a safe and customizable solution for water denitrification, protecting water treatment facility workers and people all over the world from the harmful effects of nitrate.

We believe that improving water quality will improve life conditions for millions of people all over the world, and we are focusing on denitrification as the first step to achieving it.

References

1. Bijay-Singh, & Craswell, E. (2021). Fertilizers and nitrate pollution of surface and ground water: An increasingly pervasive global problem. SN Applied Sciences, 3(4).
   https://doi.org/10.1007/s42452-021-04521-8
2. Umweltbundesamt. (2023, September 15). Indicator: Nitrate in groundwater. Umweltbundesamt.
   https://www.umweltbundesamt.de/en/data/environmental-indicators/indicator-nitrate-in-groundwater#assessing-the-development
3. iGEM Bonn-Rheinbach. (2023, June 2). Interview with Dr. Nils Cremer. personal.
4. Jimi Sol. (2021, January 28). Understanding our soil: The nitrogen cycle, fixers, and fertilizer. YouTube.
   https://www.youtube.com/watch?v=A8qTRBc8Bws
5. Erftverband. (2023, April). Das Unternehmen - Erftverband.
   https://www.erftverband.de/wp-content/uploads/2023/05/20230510_ev-in-zahlen-2023_web_mit-korr-ohne-fsc-logo.pdf
6. Cremer, N. (2015, October). Nitrat im Grundwasser - Konzentrationsniveau, Abbauprozesse und Abbaupotenzial im Tätigkeitsbereich des Erftverbands.
   https://www.erftverband.de/wp-content/uploads/2021/08/210624_ev_infofluss_321_web.pdf
7. Erisman, J. W., Van Grinsven, H., Grizzetti, B., Bouraoui, F., Powlson, D., Sutton, M., Bleeker, A., & Reis, S. (2014, October 8). The European nitrogen problem in a global perspective. European Commission.
   https://publications.jrc.ec.europa.eu/repository/handle/JRC57902
8. European Commission. (n.d.). Nitrates. European Commission.
   https://environment.ec.europa.eu/topics/water/nitrates_en#:~:text=Excess%20nitrogen%20from%20agricultural%20sources,through%20runoff%20from%20agricultural%20fields
9. Butterbach-Bahl, K., Baggs, E. M., Dannenmann, M., Kiese, R., & Zechmeister-Boltenstern, S. (2013). Nitrous oxide emissions from soils: How well do we understand the processes and their controls? Philosophical Transactions of the Royal Society B: Biological Sciences, 368(1621), 20130122.
   https://doi.org/10.1098/rstb.2013.0122
10. Minnesota Department of Health (2022, August 12). Nitrate in drinking water. Minnesota Department of Health.
    https://www.health.state.mn.us/communities/environment/water/contaminants/nitrate.html
11. Ward, M., Jones, R., Brender, J., de Kok, T., Weyer, P., Nolan, B., Villanueva, C., & van Breda, S. (2018). Drinking water nitrate and human health: An updated review. International Journal of Environmental Research and Public Health, 15(7), 1557.
    https://doi.org/10.3390/ijerph15071557
12. Dahm, J. (2022, July 11). Germany approves nitrate compromise to avoid hefty fines. Euractiv.
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13. Soares, M. I. M. (2000). Biological Denitrification of Groundwater. Water, Air, and Soil Pollution, 123(1/4), 183–193.
    https://doi.org/10.1023/a:1005242600186
14. Enzmann, F. (2020). Development of a scalable reactor for bioelectromethanogenesis and other bioelectrochemical applications (dissertation).
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15. García-Fruitós, E. (2012). Lactic acid bacteria: A promising alternative for recombinant protein production. Microbial Cell Factories, 11(1), 157.
    https://doi.org/10.1186/1475-2859-11-157