2023: NitraNix Project Description

Project Description

Nitrate serves as the primary form of nitrogen absorbed by plants and plays a vital role in crop growth. However, excessive utilisation of this compound, primarily found in fertilisers, can contribute to nitrate pollution. This issue has garnered increasing attention from the scientific community due to the associated health risks posed by the consumption of contaminated drinking water. Contrary to conventional beliefs, recent studies have demonstrated that nitrogen pollution is a significant problem in developed countries, including Europe, highlighting its substantial impact [1].

This year our project focuses on developing a method to solve the nitrate pollution problem in groundwater. Agriculture, industry, sewage, septic tanks and landfills are the main pollution sources [2] . The WHO quality standard of 50 milligrams nitrate per litre has been exceeded by Germany since 2008 every year at almost one in six measuring points. In 2018, the European Court of Justice found Germany guilty of violating the EU Nitrates Directive [3]. Nitrate poses a health hazard, contributing to the development of various cancers, anaemia, and cardiovascular diseases, among other diseases where unfortunately the most affected are pregnant people and children [4]. 

Currently, most approaches to combat nitrate pollution involve preventing nitrate leakage and diluting elevated nitrate levels with regular water, a technique known as “Wasser Bildung.” Another method involves denitrification, which is rarely utilised due to the high cost of the chemical process [5]. The effectiveness of current microbial bioremediation methods has been limited by the characteristics of the microbes employed, primarily anaerobic denitrifying bacteria, which are challenging to maintain. Additionally, aerobic denitrifying bacteria exhibit less efficient reduction due to competition from a more favourable electron acceptor, oxygen. Moreover, these bacteria often require substantial amounts of glucose as an energy source, leading to a negative cycle that demands more land for cultivation and increased fertilizer usage [6].

We aim to develop a biosynthetic technology that transforms nitrate in a novel water denitrification approach. The critical elements in this new approach would be the enzymes, nitrate reductase, nitrite reductase, nitric oxide reductase and nitrous oxide reductase which are naturally produced by native denitrifying bacteria, such as pseudomonas stutzeri, Azotobacter vinelandii, Paracoccus Denitrificans, Pannonibacter, etc,  which convert nitrate into atmospheric nitrogen using electrons from the electron supply chain. As shown in figure 1.

Figure 1. Nitrate reductase (napA) in green in complex with cytochrome in cyan ( napB) showed as a cartoon representation and surface of the protein complex. The image was taken using the pdb accession 3ML1 in Pymol.

The Denitrifying cycle consists of a series of reactions that lead to the production of nitrogen gas, the biochemical pathway is a four-step process where first nitrate (NO3 – ) is reduced to nitrite (NO2– ) by nitrogen reductase, the catalytic active part expressed by the gene napA, then nitrite is further reduced to nitric oxide (NO) by nitrite reductase (nirS), nitric oxide is reduced to nitrous oxide (N2O) , which is a potent greenhouse gas, by the enzyme nitric oxide reductase (norB), finally the limiting step of the pathway is the reduction of nitrous oxide into nitrogen gas (N2 ) by the enzyme nitrous oxide reductase (nosZ). As shown in equation 1.

Equation 1. Schematic representation of the denitrifying pathway, showing the reaction catalyzed by the enzymes and also the valence of the nitrogen in its different states.

In order to avoid the release of Genetic Modified Organism into the water stream, our main focus will be to create a cell-free denitrifying cycle by isolation of the catalytic active periplasmic enzymes from Paracoccus Denitrifican, a model organism in understanding aerobic denitrification, then test its activity by different biochemical assays where we will detect the production of N2 by adding certain auxiliary enzymes like cytochromes to deliver the electrons. Finally, our approach to making the process more efficient will rely on a previous work of groups of scientists in Leipzig, where they could introduce a peptide linker into a well know cytochrome in order to supply the delivery enzyme with electrons by attaching it to an electrode [6]. We are aiming to reproduce this approach by modifying napB a cytochrome that is a crucial player in the delivery of electrons in the whole periplasmatic denitrifying pathway. By this approach, we aim to create a cheap and novel biocatalytic process.

Considering the projected future with increased fertiliser usage due to population growth and expanding urban areas resulting in a higher number of sewage systems, our project holds promising potential in biotechnology. It has the capacity to save numerous human lives by addressing the challenges posed by nitrate pollution.

References

  1. Abascal, E., Gómez-Coma, L., Ortiz, I., & Ortiz, A. (2022). Global diagnosis of nitrate pollution in groundwater and review of removal technologies. In Science of The Total Environment (Vol. 810, p. 152233). Elsevier BV. https://doi.org/10.1016/j.scitotenv.2021.152233
  2. Ogrinc, N., Tamše, S., Zavadlav, S., Vrzel, J., & Jin, L. (2019). Evaluation of geochemical processes and nitrate pollution sources at the Ljubljansko polje aquifer (Slovenia): A stable isotope perspective. In Science of The Total Environment (Vol. 646, pp. 1588–1600). Elsevier BV. https://doi.org/10.1016/j.scitotenv.2018.07.245
  3. Wilke, S. (2022, May 12). Indicator: Nitrate in groundwater. Umweltbundesamt. https://www.umweltbundesamt.de/en/data/environmental-indicators/indicator-nitrate-in-groundwater#at-a-glance
  4. 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. In International Journal of Environmental Research and Public Health (Vol. 15, Issue 7, p. 1557). MDPI AG. https://doi.org/10.3390/ijerph15071557
  5. Dahab, M. F. (1991). Nitrate Treatment Methods: An Overview. In Nitrate Contamination (pp. 349–368). Springer Berlin Heidelberg. https://doi.org/10.1007/978-3-642-76040-2_26
  6. Zernia, S., Frank, R., Weiße, R. H.-J., Jahnke, H.-G., Bellmann-Sickert, K., Prager, A., Abel, B., Sträter, N., Robitzki, A., & Beck-Sickinger, A. G. (2018). Surface-Binding Peptide Facilitates Electricity-Driven NADPH-Free Cytochrome P450 Catalysis. In ChemCatChem (Vol. 10, Issue 3, pp. 525–530). Wiley. https://doi.org/10.1002/cctc.201701810