{% extends "layout.html" %} {% block title %}Project Description{% endblock %} {% block lead %}"Research is to see what everybody else has seen, and to think what nobody else has thought." — Albert Szent-Györgyi{% endblock %} {% block page_content %}
Our iGEM team MSP-Maastricht is dedicated to addressing coastal eutrophication through a sustainable and circular approach. This problem is particularly relevant to our team, as alleviating it is a step towards overcoming the nitrogen crisis faced by the Netherlands. Nitrogen pollution from agriculture, transport, and industry sectors has become a global concern. With agriculture being a major economic contributor of the Netherlands, excess fertilizer run-off has led to pollution of our many waterways. This then results in its spread into seawater, causing ocean acidification, anoxia, and algal blooms, which further exacerbate climate change (UNEP, 2019; KVK, 2023). To minimize damage from fertilizer leaching we propose nitrate assimilation from coastal waters, where the concentration is the highest, for single-cell proteins (SCPs) production. For this to be achieved, our team will utilize a genetically modified strain of Vibrio natriegens (V. natriegens), which thrives in high-salinity environments. Through the genetic introduction of a nitrate transporter, nitrate reductase (Nas), and nitrite reductase (Nir) derived from Klebsiella oxytoca, the engineered organism can convert nitrates to ammonium, reducing the nutrient levels that cause harmful algal blooms and restoring ecosystem balance (Figure 1). To further extend the product's value chain, the resulting ammonium will then be converted into nutrient-rich products, utilizing bacterial native enzymatic machinery, through assimilation into amino acids, and subsequently into SCPs for agriculture as seen in Figure 1 (Long et al., 2017; Reihani & Khosravi-Darani, 2019; Zhang et al., 2021; Bojana Bajić et al., 2022; Zeng et al., 2022; Ding et al., 2023; Zeng et al., 2023; Zhang et al., 2023). This then facilitates upcycling while reducing agricultural production, as SCPs can be used to enhance livestock diets, reduce the need for additional fertilizers, and improve organic fertilizers' quality.
For prevention of the organism’s spread, potentially resulting in ecosystem imbalances, it will be encapsulated in a semi-permeable membrane chamber facilitating flow through whilst containing the GMO. This design aspect further facilitates the ease of harvesting our upcycled products.
With the rapid and sustained increase in population growth in the last 50 years, the agricultural sector has implemented the use of synthetic fertilizers to keep up with crop production (Figure 2 and Figure 3).
A major component in such fertilizers is nitrates. With this conventional intensification, accompanied by depleted soil from monocropping to sustain food and fodder demand, a large portion of fertilizer is unabsorbable. Their runoff into water bodies leads to eutrophication as the nitrates provide nutrients for algae, facilitating bloom (Figure 4). As such, the Netherlands’ position as a global agricultural powerhouse, with 54% of its surface area used as farmland, has led it to develop the worst water quality in the EU (Fraters et al., 2021). Runoff from agricultural areas has spread through the highly interconnected river system and into the Dutch coastline (Ærtebjerg et al., 2001). Furthermore, this has led to the development of marine dead zones due to the hypoxic and toxic conditions related to nitrate-induced algal bloom and decomposition (Figure 5). This leads to a loss of coastal biodiversity, while subsequently posing dangers to human health as well (Sanseverino et al., 2016).
As a team, we were primarily inspired by seeing the severe algal bloom state of our local water bodies, in particular, the pond situated next to our university library (Figure 6).
We immediately saw a need for new innovative solutions to manage and mitigate eutrophication. Noting the severity of the problem, our team was further inspired by freshwater eutrophication projects of previous iGEM teams (Dusseldorf 2020, Wageningen 2021, Anatolia 2022, Wego-Taipei 2022, Bonn-Rherinbach 2023). The decision to focus on coastal areas was made for a more global approach, while simultaneously giving our team a concrete location for our GMO. As this is both a local and a worldwide problem, this decision gives the less acknowledged area of marine eutrophication bioremediation the attention we believe that it deserves. Prevention of the coastal spread is essential for marine biodiversity which directly faces the consequences of eutrophication such as harmful algal blooms (HABs), hypoxia, and ecosystem degradation (Marine, 2024). This disrupts food chains and the overall health of marine environments. These algal blooms can also have detrimental consequences for human health such as exotoxins both in marine accumulation and airborne (Sanseverino et al., 2016).
V. natriegens is a gram-negative facultatively anaerobic marine bacterium (Thoma & Blombach., 2021). First isolated in 1958 from marsh mud from Sapelo Island in Georgia, it has only recently garnered interest from the scientific community due to the exceptional doubling time of less than 10 min (Payne, 1958). The optimal growth conditions for V. natriegens are 15 g/L NaCl, 37 °C, and a pH of 7.5 (Thoma & Blombach., 2021). While Na+ is essential for the proliferation of V. natriegens, the bacterium is still able to maintain metabolic activity in its absence (Thoma & Blombach., 2021). Over the years, several iGEM teams, including Marburg 2018 and SCU-China 2021, have used V. natriegens as a tool for accelerating the development of innovative solutions in synthetic biology.
Deciding on application in marine environments, we looked to V. natriegens as our primary chassis organism. Not only can it survive in high salinity environments but is also a fast-growing bacterium gaining attention in synthetic biology applications, set to rival the more traditional E. coli. The rapid growth rate and potential for using waste substrates into high-value products inspired us to explore its application for addressing nutrient pollution and for the production of SCPs (Bojana Bajić et al., 2022).
At the core of our project lies the concept of promoting circular economies within synthetic biology research. Rather than merely focusing on reducing nutrient inputs, we wanted to convert this waste into high-value products. We aim to engineer V. natriegens into a single-cell protein powerhouse by turning waste nitrates from polluted water systems into ammonia, which is integral for amino acid production. These amino acids make SCPs a versatile supplement for animal feed. This not only helps in reducing the nutrient load in water bodies to reduce algal blooms, but also adds economic value.
Ærtebjerg, G., Carstensen, J., Dahl, K., & Hansen, J. (2001). Eutrophication in Europe’s coastal waters. European Environment Agency.
Altieri, A. H., Harrison, S. B., Seemann, J., Collin, R., Diaz, R. J., & Knowlton, N. (2017). Tropical dead zones and mass mortalities on coral reefs. Proceedings of the National Academy of Sciences, 114(14), 3660–3665. https://doi.org/10.1073/pnas.1621517114
Bojana Bajić, Vučurović, D. G., Đurđina Vasić, Rada Jevtić-Mučibabić, & Siniša Dodić. (2022). Biotechnological Production of Sustainable Microbial Proteins from Agro-Industrial Residues and By-Products. Foods, 12(1), 107–107. https://doi.org/10.3390/foods12010107
Cosme, N., & Hauschild, M. Z. (2017). Characterization of waterborne nitrogen emissions for marine eutrophication modelling in life cycle impact assessment at the damage level and global scale. The International Journal of Life Cycle Assessment, 22(10), 1558–1570. https://doi.org/10.1007/s11367-017-1271-5
Ding, H., Li, J., Deng, F., Huang, S., Zhou, P., Liu, X., Li, Z., & Li, D. (2023). Ammonia nitrogen recovery from biogas slurry by SCP production using Candida utilis. Journal of Environmental Management, 325, 116657–116657. https://doi.org/10.1016/j.jenvman.2022.116657
Erisman, J. W., Sutton, M. A., Galloway, J., Klimont, Z., & Winiwarter, W. (2008). How a century of ammonia synthesis changed the world. Nature Geoscience, 1(10), 636-639.
Filatova, A. (2024). Engineered Nitrate Assimilation Pathway in V. Natriegens [Biorender].
Fraters, B., Hooijboer, A. E. J., Plette, A. C. C., van Duijnhoven, N., & Rozemeijer, J. C. (2021). The 2020 Nitrate Report with the results of the monitoring of the effects of the EU Nitrates Directive Action Programmes. National Institute for Public Health and the Environment: Ministry of Health, Welfare and Sport. https://www.rivm.nl/bibliotheek/rapporten/2020-0184.pdf
Jiang, X., & Jiao, N. (2016). Nitrate assimilation by marine heterotrophic bacteria. Science China Earth Sciences, 59(3), 477–483. https://doi.org/10.1007/s11430-015-5212-5
KVK. (2023, August 1). Measures to reduce nitrogen pollution in various sectors. https://www.kvk.nl/en/sustainability/nitrogen-emissions-in-the-netherlands-what-do-we-know/
Long, C. P., Gonzalez, J. E., Cipolla, R. M., & Antoniewicz, M. R. (2017). Metabolism of the fast-growing bacterium Vibrio natriegens elucidated by 13C metabolic flux analysis. Metabolic Engineering, 44, 191–197. https://doi.org/10.1016/j.ymben.2017.10.008
Marine, W. (2024, June 7). Eutrophication. Water.europa.eu; European Union. https://water.europa.eu/marine/europe-seas/pressures-impacts/nutrient
Ohashi, Y., Shi, W., Takatani, N., Aichi, M., Maeda, S., Watanabe, S., Yoshikawa, H., & Omata, T. (2011). Regulation of nitrate assimilation in cyanobacteria. Journal of Experimental Botany, 62(4), 1411–1424. https://doi.org/10.1093/jxb/erq427
Payne, W. J. (1958). STUDIES ON BACTERIAL UTILIZATION OF URONIC ACIDS III. Journal of Bacteriology, 76(3), 301–307. https://doi.org/10.1128/jb.76.3.301-307.1958
Reihani, S. F. S., & Khosravi-Darani, K. (2019). Influencing factors on single-cell protein production by submerged fermentation: A review. Electronic Journal of Biotechnology, 37, 34–40. https://doi.org/10.1016/j.ejbt.2018.11.005
Sanseverino, I., Sofia, D., Luca Pozzoli, Srdjan Dobričić, & Lettieri, T. (2016). Algal bloom and its economic impact. European Union. https://doi.org/10.2788/660478
Thoma, F., & Blombach, B. (2021). Metabolic engineering of Vibrio natriegens. Essays in Biochemistry, 65(2). https://doi.org/10.1042/ebc20200135
UNEP. (2019, October 22). Why nitrogen management is key for climate change mitigation. https://www.unep.org/news-and-stories/story/why-nitrogen-management-key-climate-change-mitigation
van Heeswijk, W. C., Westerhoff, H. V., & Boogerd, F. C. (2013). Nitrogen assimilation in Escherichia coli: putting molecular data into a systems perspective. Microbiology and Molecular Biology Reviews, 77(4), 628–695. https://doi.org/10.1128/MMBR.00025-13
West, P. C., Gerber, J. S., Engstrom, P. M., Mueller, Nathaniel. D., & Brauman, K. A. (2014). Excess nitrogen from croplands, 2009 (K. M. Carlson, S. Siebert, E. S. Cassidy, D. k. Ray, G. k. Macdonald, & M. Johnston, Eds.). Our World in Data. https://ourworldindata.org/grapher/excess-nitrogen
Zeng, D., Jiang, Y., Su, Y., & Zhang, Y. (2022). Upcycling waste organic acids and nitrogen into single cell protein via brewer’s yeast. Journal of Cleaner Production, 369, 133279–133279.https://doi.org/10.1016/j.jclepro.2022.133279
Zeng, D., Wang, S., Jiang, Y., Su, Y., & Zhang, Y. (2023). Recovery and upcycling of residual lactic acid and ammonium from biowaste into yeast single cell protein. Separation and Purification Technology, 314, 123632. https://doi.org/10.1016/j.seppur.2023.123632
Zhang, B., Ren, D., Liu, Q., Liu, X., & Bao, J. (2023). Coproduction of single cell protein and lipid from lignocellulose derived carbohydrates and inorganic ammonia salt with soluble ammonia recycling. Bioresource Technology, 129345–129345. https://doi.org/10.1016/j.biortech.2023.129345
Zhang, L., Zhou, P., Chen, Y. C., Cao, Q., Liu, X. F., & Li, D. (2021). The production of single cell protein from biogas slurry with high ammonia-nitrogen content by screened Nectaromyces rattus. Poultry Science, 100(9), 101334. https://doi.org/10.1016/j.psj.2021.101334