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Entrepreneurship
Introduction: Mission, Problem and Solution

The Fertilizers Market size is estimated at 381.7 billion USD in 2024, and is expected to reach 541.2 billion USD by 2030 CITE. This is not surprising because

What if we could reach that optimal point without the need for fertilizers? For example, by manufacturing genetically modified seeds that produce crops capable of fixing their own nitrogen? In addition to saving farmers billions and saving thousands of lives

Mission

At NitroBLAST, we are focusing on transforming global agriculture. Our ultimate goal is to create sustainable, nitrogen-fixing crops that help farmers rely less on chemical fertilizers, while still ensuring strong crop yields. By doing this, we aim to empower farmers to embrace our technology and contribute to reducing the environmental impact of fertilizers.

  • Innovating for sustainability: We’re working to develop crops that naturally fix atmospheric nitrogen and reduce the need for synthetic fertilizers.
  • Empower farmers: Our solution is designed to lower farmers’ costs by reducing the need to buy fertilizers.
  • Reduce environmental impact: By cutting global CO2 and nitrogen emissions, we hope to reduce the harm caused by over-fertilization and promote a more sustainable future.
Problem

Farms across the country are losing massive amounts of nitrogen—74% of what they apply, or about 312,000 tons, is wasted each year. This nitrogen, meant to nourish crops, instead ends up polluting our air and soil. Most of it comes from over-f

For staple crops like cereals and maize, up to 40% of farm operating costs go toward fertilizers CITE, and the rising costs have triggered protests across Europe, with Dutch farmers at the forefront CITE. Attempts to curb nitrogen emissions in the Netherlands have only deepened the tension.

The consequences of over-fertilization are profound, leading to environmental devastation such as harmful algal blooms, air pollution, and serious health risks. Yet, despite its harmful effects, fertilizer is critical for boosting crop yields at a time when the global population is growing and food demand continues to skyrocket. Tackling this problem is not just a national issue—it’s a global priority that demands immediate and innovative solutions.

Solution

At NitroBLAST, we’re taking an exciting first step toward solving the nitrogen crisis by giving crops the ability to naturally fix atmospheric nitrogen.

Our research centers on incorporating a nitrogen-fixing organelle, called the Nitroplast, into eukaryotic cells, and eventually plant seeds. This innovation is inspired by a natural partnership between the cyanobacteria UCYN-A and the marine alga B. bigelowii. Using synthetic biology, we’re working to introduce this nitrogen-fixing capability into important crops like wheat, corn, and rice, much like how peas and beans already partner with nitrogen-fixing bacteria.

The result? Crops that can fix their own nitrogen directly from the atmosphere, reducing or even eliminating the need for synthetic fertilizers. This means lower costs for farmers and fewer environmental problems caused by fertilizer overuse. Unlike traditional fertilizers, which require energy-intensive production and contribute to greenhouse gas emissions, NitroBLAST crops would work naturally inside the plant, helping to minimize agriculture’s carbon footprint.

We imagine a future where crops can thrive in nutrient-poor soils without harmful chemicals, supporting sustainable farming that protects water sources, reduces pollution, and preserves biodiversity.

Business Plan

Over the course of this iGEM project, the TU Delft iGEM team has worked on entrepreneurial ideas for the commercialization of our product, NitroBLAST seeds. In this section, we will highlight this product, its use in today's world and the commercialization strategy that we could potentially adopt.

NitroBLAST Seeds

In the ideal scenario, our marketable product would be a NitroBLAST seed, which would give rise to a plant that fixes its own nitrogen, thus omitting the need for fertilizer. After discussion with experts in the field of plant seeds technologies, we discovered that the realization of such a seed would involve incorporation of the nitroplast at the embryonic stage or in the meristem. Once we have completed research and development, our product would be a NitroBLAST seed. Our target buyers would be seed companies, with a focus on plant breeding. For every subsequent sale made by these companies on NitroBLAST seeds of any generation thereafter, our company would receive a fixed share of the profits made by this sale.

Commercialization Strategy

For the realization of our product, we developed a commercialization strategy, through which we could make the transition from the lab to the market. In this section, we have outlined steps for the commercialization of our product.

  1. Research and development: In this stage, we will continue refining our technology as a start-up, ensuring it is scalable, reliable, and can be applied not only to eukaryotic model organisms, but also to a range of crops and agricultural systems. Following this, we will conduct pilot studies and field trials with focus groups of key stakeholders (e.g. innovative farmers or research institutions like TU Delft AgTech Institute, which is already a sponsor of our project) to validate the effectiveness of nitrogen-fixing crops in real-world conditions. Our final challenge in this phase would then be to navigate regulatory hurdles, such as getting approval from agricultural and environmental agencies. We would hire an expert to guide us through this (more on this in the skill gap analysis). Along with the hiring, we will ensure that our product complies with biosafety standards and obtain certifications needed for commercial agriculture using these seeds in The Netherlands, for example the seed certification of the Netherlands General Inspection Service for Agricultural Seeds and Seed Potatoes (NAK), which ensures that GM seeds meet standards related to safety, quality, and environmental protection.
  2. Intellectual property protection: In this phase, we will aim at securing strong intellectual property protection for our technology, including patenting our unique genetic engineering methods or products such as the fusion with which we incorporate the Nitroplast in a cell, as well as the resulting seed itself. For these steps, we will also make use of our skill gap analysis and hire the corresponding experts on intellectual property law. Following this, we will refine and protect the brand identity of our technology through trademark registration, which is the legal process of registering a specific symbol, name, or logo to obtain exclusive rights to use it. This can be done for any other distinctive signs that identify and distinguish our product.
  3. Building partnerships: In this phase, we will focus on our engagement with potential early adopters of our technology. These would generally be individuals and institutions that are open to experimenting with sustainable agricultural methods. These could be, for example, leading academic and research institutions that can aid us in acquiring additional R&D funding, or agrochemical and AgTech companies whose large customer bases will allow us to scale-up faster. Some examples of potential partnerships, from different backgrounds, could be Wageningen University and Research (WUR) Center, Bayer, BASF, or KWS. Throughout our project, we had conversations with representatives of some of these entities (more on this in the Human Practices page), and we also took into consideration some of their suggestions about patenting for our project.
  4. Market release: In this phase, we would start out initially by selling directly to progressive, innovative farmers and farmers’ collectives focused on sustainable and organic farming. Following this, we would license our technology to larger seed companies that can integrate the nitrogen-fixing capabilities into their crop offerings. This would scale our impact quickly without managing the entire production and sales process ourselves.
  5. Marketing: Following this, we would develop a strong content marketing strategy focused on educating farmers from all around the world about sustainable farming practices, nitrogen-fixation technology, and long-term cost benefits, while partnering with agricultural influencers (Justin Rhodes, Tyker Froberg, Peter, etc.), thought leaders, and industry publications to spread awareness of our product’s environmental and economic benefits.
  6. Revenue model: Our revenue model would first focus on the direct sale of the seeds to farmers and farmers’ collectives. Once we have licensed our technology to large seed producers, we would also acquire a share of the profits made by them via NitroBLAST seeds.
  7. Scaling and growth: At first, we would start by selling to the Dutch market. This is because although the nitrogen crisis is a global issue, its effect is particularly pronounced in the Dutch landscape. Following this, we would leverage partnerships with global agricultural companies and NGOs to expand into new territories and collaborate with global sustainability initiatives to align with regional regulations and goals. This includes government subsidy programs for sustainable agriculture that will further aid in reducing the financial burden on farmers.
Lean Canvas Business Model

The Lean Canvas is a simplified, one-page business model framework designed to help entrepreneurs and startups quickly outline and validate their business ideas. The focus is on identifying key assumptions, problems, and solutions early on, allowing for faster testing and iteration. We have designed the following Lean Canvas for our business:

Customers and Competitors
Identity of Customers

Our seeds would not be limited to traditional farmers alone. In addition to them, there is a large group of entities that could be interested in obtaining our products or acquiring part of the company’s shares in the event it goes through an IPO (more on this in the Exit Strategy section). The growing concern for a more sustainable future and the restrictions on CO2 emissions and nitrogen derivatives are driving large groups to seek greener solutions. If, in doing so, they can also save the money and time involved in using fertilizers, the possibilities increase even further. Among the potential consumers, we can find:

  • Agricultural Cooperatives and Associations, like the Coöperatieve Rabobank U.A. from the Netherlands or The Nationals Farmer Union from the UK.
  • Agrochemical and Fertilizer Companies, like BASF or Yara International.
  • Environmental Organizations and NGOs, such as WWF and Greenpeace.
  • Government and Regulatory bodies, like the EFSA (European Food Safety Authority) or the Ministry of Agriculture, Nature and Food Quality from the Netherlands.
  • Academic and Research Institutions, like the WUR (Wageningen University and Research) or the TU Delft AgTech Institute.
  • Food and Beverage Companies, such as Unilever or General Mills.
  • Farmers and Agricultural Producers, like the Sunkist Growers or the Southern States Cooperative, both from USA.
  • Consumer and Advocacy Groups, such as Consumers International or Friends of the Earth.
  • Investors and Venture Capitalists, like AgFunder and Pontifax AgTech (USA).
Existing Competitors/Alternatives
  • Precision Agriculture: Uses technology to respond to variability in crops and soil conditions, optimizing the need for fertilizer.
  • Biological Nitrogen Fixation: Utilizes natural bacteria in plants to fix nitrogen, minimizing the need for fertilizers.
  • Genetically Modified Crops: Existing GM crops are designed for higher efficiency and reduced reliance on fertilizers and chemicals.
  • Cover Cropping: Plants cover crops to improve soil health and reduce nitrogen loss.
  • Crop Rotation: Alternates crops to naturally restore soil nitrogen levels and reduce fertilizer dependency.
Business Plan: Market Analysis and Scalability
SWOT: Strengths, Weaknesses, Opportunities and Threats analysis

A SWOT analysis, is a strategic planning tool that identifies a company's internal strengths and weaknesses, as well as external opportunities and threats. This framework helps us to understand our competitive position and develop strategies to leverage strengths, address weaknesses, capitalize on opportunities, and mitigate threats.

Strenghts
  • Self-Sustaining Nitrogen Fixation: Our plants fix nitrogen independently, reducing or even eliminating the need for fertilizers.
  • Cost Savings for Farmers: With less fertilizer requirements, farmers can save a significant amount of money on fertilizer purchases and application costs.
  • Environmental Sustainability: By eliminating the need for synthetic nitrogen fertilizers, we would be reducing the environmental damage caused by them, such as eutrophication and greenhouse gas emissions.
  • Innovative Technology: Introducing an organelle in a cell and creating genetically modified seeds with this characteristic would be a unique technological advancement, which would position our company as a major innovator.
  • Simplified Farm Operations: Farmers using our seeds would no longer need to apply fertilizers, saving both labor and reducing the time spent in the fields.
Weaknesses
  • High R&D Costs: Both developing genetically modified organisms and ensuring their safety and efficacy involves high research, development, and regulatory approval costs, as well as a lot of time that we are not considering in these analyses.
  • Regulatory Challenges: GMO products often face strict regulations in many countries. Our seeds may face pushback from regulatory bodies, especially in regions with strict GMO laws, like the European Union or, in our case, the Netherlands.
  • Public Perception of GMOs: Despite all their benefits, GMOs still face a rather negative public perception, which could impact the reception of our product. Both consumers and farmers may be skeptical of our new genetic modifications.
  • Long Development Time: Compared to fertilizer companies or those offering nitrogen-fixing bacteria, our company's time-to-market might be longer due to the complexities of developing and testing genetically modified seeds.
Opportunities
  1. Disrupting the Fertilizer Market: Our product has the potential to disrupt the fertilizer market by reducing, or even eliminating, dependence on synthetic fertilizers. With the later increase in regulations and taxes on nitrogen fertilizers, our technology is an attractive alternative for those who want to save some money while still getting optimized results.
  2. Environmental Policy Alignment: Governments and international organizations are increasingly pushing for sustainable agricultural practices. Our seeds align with these policies, which could make our company eligible for subsidies and other incentives.
  3. Global Expansion: Our product could have significant appeal in regions with poor soil quality or limited access to fertilizers, helping to improve harvests in developing countries and regions with difficult farming conditions.
  4. Potential Partnerships: Partnering with big agricultural companies could rapidly increase our market share and credibility. Additionally, by partnering with environmental organizations, such as WWF or Greenpeace, we could promote our product as a new sustainable farming practice, fighting against the previously mentioned negative public perception of GMOs.
  5. Technological Evolution: As the technology evolves, we could incorporate other beneficial traits into the plants that could also improve the harvest and facilitate the farmers' work, such as making them drought-resistant, pest-resistant, etc.
Threats
  • Competition from Fertilizer Companies: Fertilizer companies are well-established and may counter our product with innovations of their own that optimize their use or pollute less.
  • Competition from Nitrogen-Fixing Bacteria: Companies that sell nitrogen-fixing bacteria, such as Rhizobium inoculants, also offer a natural solution to nitrogen fixation. These products are already on the market, well-understood, and cheaper to produce compared to GMO seeds.
  • Consumer and Farmer Resistance: Resistance towards GMO consumption and prejudices, particularly in regions that are skeptical of genetic engineering, may slow the adoption of our product. Additionally, farmers might be hesitant to switch from known methods unless they see clear and immediate benefits, as they usually imply an initial investment that not everyone can afford.
  • Uncertain Long-Term Impact: Long-term ecological impacts of genetically modified seeds with nitroplasts are still unknown. Unforeseen consequences in the ecosystem or resistance to the technology could become a challenge in the long term.
  • Market Entry by New Products: Startups or biotech firms with competing nitrogen-fixing solutions, such as CRISPR-engineered plants or enhanced microbial inoculants, could enter the market and offer better conditions, results, or a more competitive price.
PESTEL Analysis

PESTEL is a strategic framework used to analyze and monitor the external environment factors that might impact an organization. It stands for Political, Economic, Social, Technological, Environmental, and Legal factors. This analysis helps us understand the macro-environmental influences on our business, which is crucial for strategic planning and future analysis.

Total Addresable Market: TAM
Serviceable Available Market: SAM
Serviceable Obtainable Market: SOM
Business Plan: Execution Plan
Skill Gap Analysis
Exit Strategy
Intellectual Property Strategy

One promising approach to balance the need for fertilizer and the welfare of the environment, is the development of plants that can fix atmospheric nitrogen independently. This innovation would not only reduce the need for synthetic fertilizers and manure but also help mitigate climate change and the nitrogen crisis. To this end, we first need to better study the nitroplast, how it interacts with the host organism and how it could be potentially introduced into other cells.

It has been discovered that, to ensure the endosymbiotic relationship, several proteins that are essential to UCYN-A are expressed in the host, B. bigelowii, and imported into the symbiont, similar to chloroplasts and mitochondria, though to a lesser extent [11]. Many of these proteins possess specialized localization peptides that direct their cellular function. In UCYN-A, these peptides are usually a C-terminal extension and are known as the “uTP” (UCYN-A Transit Peptide), although not yet identified [11]. Our first aim was to employ bioinformatics analyses to identify the characteristic motifs required for a protein to be imported by UCYN-A. For this, we made use of host (B. bigelowii) and nitroplast (UCYN-A) genome data as well as the proteomics data published by Coale et al.. We identified 2 putative uTP sequences with high likelihood, which we named uTP1 and uTP2.

To understand the functioning of the UCYN-A import mechanism, we attempted to identify the proteins involved in translocating host-encoded proteins into UCYN-A. First, we located genes in the host genome that are potentially involved in the translocation, based on their similarity to proteins in other import mechanisms such as from Paulinella chromatophora (UCYN-A analogue for photosynthesis). Potential chaperones analogous to heat-shock proteins were also included in the search. These chaperones are hypothesized to bind to proteins tagged by the uTP and keep them from folding, allowing translocation through the UCYN-A membrane. We then followed this by obtaining the tertiary structure of all candidate proteins using a structure prediction tool, and used docking tools to select candidate proteins likely to bind the previously identified transit motifs.

In addition to in silico experiments, we also aimed to investigate the transport mechanisms of UCYN-A in vivo. Instead of making use of plants as target organisms, we opted for using single-cell model eukaryote organisms, namely the yeast S. cerevisiae and the green alga C. reinhardtii. The initial in vivo characterization of the UCYN-A transport system involved examining the expression and localization of the UCYN-A transit peptides in these eukaryotic model organisms to test whether uTP would have any unexpected effect on cell viability and would not target any other organelle. To this end, we designed vectors, cloned them using Gibson-assembly, transformed bacteria, purified expression plasmids, and transformed those into S. cerevisiae and C. reinhardtii. We expressed uTP-tagged fluorescent proteins, together with controls targeting other organelles, and localization was assessed using fluorescence microscopy. Our preliminary results indicate that uTP did not target any other organelle and did not lead to alterations in cell morphology.

Studies have demonstrated the insertion of bacteria into cells by engineering endosymbionts in S. cerevisiae using either E. coli or S. elongatus [12]. Another study successfully inserted Azotobacter strains into C. reinhardtii [13]. Building on this research, we initially aimed to develop a reliable protocol for transplanting a nitroplast into C. reinhardtii and S. cerevisiae as a proof-of-concept for transplantation into other eukaryotes, using polyethylene glycol (PEG) fusion protocols. However, due to time limitations, we started out with the model eukaryotic bacteria, E. coli, and refined a protocol for its fusion with S. cerevisiae.

We obtained B. bigelowii as a gift from Dr. Kyoko Hagino (Kochi University, Japan) and cultured it according to the protocol. For the transplantation of the nitroplast into a different model organism, we were also required to isolate this organelle from its host. Our project also implemented the protocol for nitroplast isolation from its host, according to the protocol and suggestions provided by Dr. Tyler Coale (University of California, San Diego, USA). Isolated UCYN-A could potentially be used for PEG fusion with other eukaryotes.

Finally, we have also wondered how our project would affect society. For this, our human practices team has actively worked to understand the possible consequences of our project. Also, wider acceptance of our idea goes hand in hand with educating the general population. Last but not least, we have also assessed the potential economic benefits of our idea and the omission of fertilizer use in agriculture. For this, we made an economic analysis and business plan.

Our project lays the foundation for the transplantation of nitroplast into eukaryotic hosts. The emergence of nitrogen-fixing plants could lead to a significant drop in the demand for fertilizers, and consequently in both carbon emissions and nitrogen pollution.

Long Term Impact
Would We Truly Eliminate the Need for Fertilizers?
How Much Money Would We Save?

Being a team from the Netherlands, we have actively followed the unfolding of the nitrogen crisis and seen the farmer's protests on the news. While nitrogen deposition is incredibly harmful to the environment, the Dutch agriculture sector is a big driving factor behind its economy, with agricultural exports being worth 124 billion euros in 2023 alone [8].

The Netherlands is also considered one of the front runners in terms of food and agriculture technology. Given the leadership of The Netherlands in this field, why not leverage synthetic biology to address the nitrogen crisis? We were inspired by previous iGEM teams such as Wageningen 2021 [9] and Stony-Brook 2023 [10] that have tackled similar challenges, alongside a recent publication in Nature in April 2024 [11].

EXTRA! Entrepreneurship Dictionary

Being a team from the Netherlands, we have actively followed the unfolding of the nitrogen crisis and seen the farmer's protests on the news. While nitrogen deposition is incredibly harmful to the environment, the Dutch agriculture sector is a big driving factor behind its economy, with agricultural exports being worth 124 billion euros in 2023 alone [8].

The Netherlands is also considered one of the front runners in terms of food and agriculture technology. Given the leadership of The Netherlands in this field, why not leverage synthetic biology to address the nitrogen crisis? We were inspired by previous iGEM teams such as Wageningen 2021 [9] and Stony-Brook 2023 [10] that have tackled similar challenges, alongside a recent publication in Nature in April 2024 [11].

References
  1. Nitrogen - WUR. url: https://www.wur.nl/en/research-results/dossiers/file/nitrogen.htm
  2. The nitrogen strategy and the transformation of the rural areas — Nature and biodiversity — Government.nl. url: https://www.government.nl/topics/nature-and-biodiversity/the-nitrogen-strategy-and-the-transformation-of-the-rural-areas
  3. National Oceanic US Department of Commerce and Atmospheric Administration. “What is eutrophication?”. https://www.noaa.gov/what-is-eutrophication
  4. Jeff Elhai. “Engineering of crop plants to facilitate bottom-up innovation: A possible role for broad host-range nitroplasts and neoplasts”. In: (Apr. 2023). doi: 10.31219/OSF.IO/NY2RC. url: https://osf.io/ny2rc
  5. Toename stikstofoverschot in landbouw door droge zomer 2022 — CBS. url: https://www.cbs.nl/nl-nl/nieuws/2024/05/toename-stikstofoverschot-in-landbouw-door-droge-zomer-2022
  6. AR4 Climate Change 2007: Mitigation of Climate Change — IPCC. url: https://www.ipcc.ch/report/ar4/wg3/
  7. Protesting farmers close roads and borders in nationwide campaign - DutchNews.nl. url: https://www.dutchnews.nl/2019/12/protesting-farmers-close-roads-and-borders-in-nationwide-campaign/
  8. Statistics Netherlands. “Dutch agricultural exports worth nearly 124 billion euros in 2023”. In: (June 2024). url: https://www.cbs.nl/en-gb/news/2024/10/dutch-agricultural-exports-worth-nearly-124-billion-euros-in-2023
  9. Team Wageningen 2021 homepage. url: https://2021.igem.org/Team:Wageningen_UR
  10. Team — Stony-Brook - IGEM 2023. url: https://2023.igem.wiki/stony-brook/
  11. Loconte V. Turk-Kubo K.A. Vanslembrouck B. Mak W.K.E. Cheung S. Ekman A. Chen J.H. Hagino K. Takano Y. Coale T.H. and T. Nishimura. “Nitrogen-fixing organelle in a marine alga”. In: Science 384 (2024), pp. 217–222.
  12. Angad P. Mehta et al. “Engineering yeast endosymbionts as a step toward the evolution of mitochondria”. In: Proceedings of the National Academy of Sciences of the United States of America 115.46 (Nov. 2018), pp. 11796–11801. issn: 10916490. doi: 10.1073/PNAS.1813143115/SUPPL{\ _ }FILE/PNAS .1813143115 . SM02 . MP4. url: https://www.pnas.org/doi/abs/10.1073/pnas.1813143115
  13. N.H. Nghia et al. “Uptake of Azotobacters by Somatic Fusion of Cell-wall Mutants of Chlamydomonas reinhardii”. In: Biochemie und Physiologie der Pflanzen 181.5 (Jan. 1986), pp. 347–357. issn: 0015-3796. doi: 10.1016/S0015-3796(86)80008-7.
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