Human Practices

Introduction

The overuse of synthetic nitrogen-based fertilisers has caused significant damage to ecosystems across the globe. Our project aims to use synthetic biology to engineer E. coli to produce ammonia naturally, in order to decrease the fossil fuels and nitrogen pollution impact of the fertiliser industry. SOILutions aims to develop an oxygen-sensing feature that allows the bacteria to activate only under low oxygen conditions, increasing the longevity and usability of these engineered microorganisms.

With the potential to increase ammonia production in soil, this synthetic biology solution may be the last piece of the puzzle for creating a consistent and far more sustainable supply of fertiliser for farms across the world. A renewable and sustainable way of producing nitrogen-based fertiliser, through our engineered bacteria, ensures the continuous production of modern crops, without causing ecological damage from nitrogen overuse.

The Problem with Nitrogen

The overuse of nitrogen, particularly within fertilisers, creates major environmental and health concerns. Globally, the significant increase in usage of nitrogen from 11 million tons in 1961 to 119 million tons in 2018 (Imran, 2021) represents an increase in usage of over 1000%.

This results in the accelerated rate of algae bloom, release of nitrogen-based greenhouse gases, and increase of cancer risk, as nitrogen is a procarcinogen (Halliwell, 1999). This ultimately results in an economic cost, outlined by Sutton et. al. (2011) as amounting to between 70 billion and 320 billion euro annually for the EU alone.

The SOILutions iGEM team is composed of 21 young people from Sydney, united by a concern for our planet. We found it concerning that Australia has a disproportionately large emission of nitrogen, which means we contribute to many of the issues outlined above.

However, our unique challenge was to also recognise the rural population, for whom, nitrogen-based fertiliser is crucial to their livelihoods. Australia’s strong agricultural sector acts as the backbone to its economy, so unsurprisingly, farmers commonly use large quantities of nitrogen to fertilise crops.

Our mission, as human practices, is to engage with these stakeholders reliant on nitrogen, and investigate current practices in the present to make farming and fertilising more sustainable. To the SOILutions iGEM team, our integrated human practices meant we were responsible for understanding the relationship between science and industry, and where synthetic biology may fit in this complex equation.

Only after we extensively interviewed numerous experts in various fields, from soil chemistry to agriculture, could we have meaningful conversations that informed us on how we can best implement solutions to solve problems. Discussions with essential stakeholders like farmers and fertiliser experts highlighted the current issues with fertiliser and sustainable farming. Our work as human practices illuminated the numerous perspectives on soil health, sustainable nitrogen usage and its application in the real world, driving the centre of a balanced and realistic approach to excessive nitrogen emissions.

Designing Framework

Engaging with Those Affected

Before designing our solution towards lowering the emissions of NO2 and excess Nitrogen within the soil and atmosphere, the SOILutions team worked to understand the state of this issue, as well as empathise with stakeholders involved with and affected by the use of nitrogen-based fertilisers.

Upon interviewing stakeholders involved in varying aspects of the usage of nitrogen-based fertilisers, we have come to understand some of the numerous perspectives surrounding the issue and its relation to the detriment towards the global warming crisis.

Independent Consumers

We decided to interview farmer and cattle buyer, Dave Payne, as a way to understand the perspective of a farmer currently in use of nitrogen-based fertilisers via poultry manure. Through Mr Payne’s extended experience as a farmer, our team felt that he would be a suitable representation of a potential end user of our product. His perspective on the market was a farmer’s view, demonstrating our consumers' response to our solution. The interview with Mr Payne raised questions about the accessibility of our product, could it be easily understood? Our team was provoked to think about the people within our market, even for people within our team, the concept of our solution was difficult to comprehend.

As a result, our team considered the end users of our product in the real world and how we could raise more awareness of the problem, and communicate our product effectively. We create more public engagement strategies, such as social media, a podcast, and a comic to help convey the importance of our project, encouraging our potential users to become more informed and willing to make a small scale difference.

As a potential consumer of a solution towards greener practices instead of nitrogen-based fertilisers, Mr Payne found it “hard seeking out advice and information that is reliable from an independent organisation, as the companies currently out in the market are profit driven, without the soil in their key focus.”. Furthermore, we have come to understand that the cost of being sustainable as an independent farmer causes the possibility of implementing greener practices to become a lot more difficult. Upon hearing this, our group understood that our solution needs to account for the difficulty of being sustainable as independent farmers, in order to create an easily accessible solution.

Companies producing alternative solutions

Previously, our team had conducted background research into a range of companies producing alternative solutions to sustainable fertilisers. Our business department was able to contact Charles Day, the CEO of Jupiter Ionics, a renewable energy expert in commercialising technology used for sustainable agriculture.

SOILutions chose to contact the company as their production of green ammonia can be implemented into large scale sustainable agriculture. Their green ammonia created from renewable energy can decarbonise fertiliser, similar to what we were aiming to produce. Our interview with Jupiter Ionics provided invaluable insight into the market that our project would be entering into.

During the interview, Mr Day introduced the concept of a “green premium”, a problem that meant that our solution is not able to be distributed on a large scale on commercial farms. As such, our team realised the challenges of implementing our project into the real world. Instead, the SOILutions decided that the final product would be more suitable to a smaller audience of farmers aiming to be sustainable. This however posed questions for our team as since the usage of our product would be reduced, our impact on the environment may not be as great.

After understanding the rising concerns towards the increase of excess Nitrogen and NO2 released into the atmosphere, our group aimed to prioritise values and synthetic biology throughout developing a good solution to reduce this damage.

Understanding the Problem

Overview of the Problem

The rise in nitrogen usage, particularly within fertilisers, poses a significant environmental challenge. From 11 million tons in 1961, global nitrogen consumption has soared to an alarming 119 million tons by 2018, marking an increase of over 1000% (Imran, 2021). While nitrogen is pivotal for plant growth, plants have a notoriously inefficient absorption rate. Consequently, a large proportion of nitrogen tends to leach from plants.

This unchecked release of nitrogen into our environment instigates harmful phenomena like algae blooms. These blooms not only consume vital oxygen but also pollute our waterways, culminating in the demise of numerous marine species. Moreover, the leached nitrogen is a key contributor to greenhouse gas emissions, notably N2O – a gas 300 times more efficient than CO2 in trapping heat.

Further complicating matters, nitrogen is identified as a procarcinogen (Halliwell, 1999). This means that once inside the human body, there's a potential for it to transform into a carcinogenic agent. Such significant emissions pose a disproportionate threat to global human health. Beyond environmental and health concerns, the economic implications are also daunting. As per Sutton et. al. (2011) the European Union bears a financial strain between 70 billion [euro] and 320 billion [euro] annually due to nitrogen pollution.

Approaching Social Attitudes to Science & Synthetic Biology

The realm of environmental sustainability, especially as it intersects with scientific advancements, elicits diverse reactions from society. For instance, take the example of genetically modified organisms (GMOs) in agriculture. Their introduction brought a plethora of benefits such as increased crop yields, reduced farming costs, minimised pesticide use, and enhanced food quality and nutrition. However, the tides of public opinion have gradually turned against GMOs. Many are plagued by fears of potential health risks, the emergence of monocultures if GMO adoption becomes too widespread, and the unintended consequences of tampering with natural genetic processes. While some of these concerns might be debatable, the prevalent societal trepidation towards GMOs underscores the importance of gauging public sentiment, particularly when rolling out products rooted in synthetic biology.

Our interview conducted with Dave Payne provided us with valuable insights in regards to outside perspectives, providing us details on the views of potential opposition to synthetic biology in fertiliser technology. In particular, a view proposed by Dr Christine Jones regarded GMOs as disruptive to the natural processes, and that it is better that they are left out of nature. Presenting at the Tocal Agricultural College in May 2023, Dr Jones was the keynote speaker of the presentation, and was keen to express the unnecessity of using synthetic biology in order to produce fertiliser.

Interviewing Key Stakeholders

In a bid to align our endeavours with the pulse of the community, our team, SOILutions, undertook extensive outreach. Through various interviews with stakeholders and experts, we deepened our understanding of the intertwined environmental, economic, and ethical paradigms shaping our mission. For instance, insights from Jupiter Ionics highlighted the market challenges our product might face, especially the concept of a “green premium” making it less viable for large-scale commercial farms. Furthermore, through the lens of agricultural stalwarts like Dave Payne, we glimpsed the perceptions of potential end-users. Their feedback instigated a strategic shift, prompting us to focus on niche audiences that valued sustainability over mere cost-efficiency.

Moreover, workshops, such as the one at Tocal Agricultural College, illuminated the mosaic of perspectives on leveraging soil biology to revolutionise agricultural practices. Experts like Dr. Christine Jones emphasised the significance of natural processes, reminding us of the multifaceted viewpoints within the very community we aim to serve. Given the intricate fabric of the problem, from soil degradation, food safety to the broader societal implications, engaging with affected communities is not just essential; it's foundational to our mission.

Another effort to engage with key stakeholders was performing various tests upon 12 different samples of soil. These samples range from agricultural locations, local households, construction sites and even some store-bought ones. This was done in conjunction with various communities members of the group were connected to, in order to test how much potential soil had to be for effective growing plants – or conversely, how damaged they were. Only after the conferencing we had performed with affected individuals, could we understand the extent of the issue.

Defining a Solution

Values and Objectives

While each stakeholder expressed their own unique concerns, the overarching theme and the primary concern for everyone involved was the sustainable health of the soil and its ability to support crops. Each individual, community, and industry stands to face significant setbacks should the quality of soil diminish due to excessive nitrogen emissions from fertilisers.

We had 5 main objectives during the design and development of the project as explored below.

Sustainable Use

The ability to self regulate their function allows the microbes within the organic fertiliser to survive with less human intervention, reducing the frequency in which fertiliser has to be re applied as opposed to conventional chemical fertilisers which require regular maintenance and attention.

The nature in which diazotrophs operate provide crops with the appropriate amount of ammonia, eliminating the issues of excess nitrogen pollution caused by chemical fertilisers, thus providing aid in long term soil fertility, a factor emphasised in our interview with Dave Payne that is necessary for the function of farms.

Reduce Nitrogen Emissions

To reduce nitrogen leaching through a self-regulated microbe product involves harnessing the power of beneficial microorganisms to mitigate the release of excess nitrogen from agricultural and environmental conditions. Jupiter Ionics recommended crop rotation as a suitable solution for nitrogen leaching, involving planting crops in sequential plots to improve soil fertility and health to combat leaching in nitrate. Additionally, conducting soil samples on a weekly basis will also aid the integration of the microorganisms into the soil through the monitoring of their activity, allowing for alterations to soil composition to be made.

Improve Soil Health

Short Term

Our soil has self regulated microbes that can quickly start nitrogen fixing to increase ammonia levels within soil without creating harmful excess, subsequently leading to a positive impact to soil health and lead to immediate improvement to the agricultural industry.

Another factor is the soil with use of specific microbial strains which can benefit agricultural plants to become more resilient against natural disasters like drought, pests, and temperature fluctuations, which can lead to positive effects for the agricultural industry as countries can plant crops in difficult environment conditions.

Long Term

With our self-regulating microbes we can help plants and other crops to maintain a balanced nutrient cycle over a long term. Our advancements in integrating oxygen sensors within the diazotrophs allows them to enter a sleep-like state when in the presence of high concentrations of oxygen, which is dangerous to their function. This ultimately allows the beneficial bacteria within the soil to survive continuously within the soil without manual replenishment, thus providing the benefits of fertilisation for a longer period of time.

Maintain Financial Sustainability

As punctuated by Charles Day in our interview with Jupiter Ionics, a primary focus in selling fertilisers is maintaining a consistent, non-volatile price whilst transitioning from a lab scale production to larger commercial levels. Factors such as heat transfer, mass transfer and material impact will play heavily into a commercial transition when entering the market. We needed to ensure that our solution would be feasible in the face of the “green premium” challenge that we were soon to encounter whilst entering the commercial market, establishing vital connections with early adopting stockholders to counteract it’s negative effects.

Safe for the Surrounding Ecosystem and People

Our product is able to mitigate the issues of excess nitrogen pollution caused by conventional chemical fertilisers. Such issues would normally lead to runoff that pollute waterways, disrupting local ecosystems and causing algae blooms. The diazotrophs eliminate this issue through the processes which produce only the necessary amount of nitrogen to the crop without any excess amount. This controlled output in turn reduces the severity of carcinogens present, benefiting the people who work closely with our product.

Synthetic Biology as a Solution

The potential of Synthetic Biology to transform the ammonia market is very much real, with Green ammonia already projected to grow to 28.4 thousand tons by 2030 compared to its 94 tons in 2021. We believe synthetic biology will play a significant role in it’s rise, with our product being able to deliver ammonia in both a sustainable manner and in suitable amounts to crops.

The leap to using fertilisers using synthetic biology is no doubt a risky one for our end users, and requires their belief in our product to ensure success in the real world. As seen in our interview with Jason Simmons, independent farmers in Australia do express their doubts in regards to modern advances in fertiliser, being hesitant to adopt replacements to reliable fertilisers that have proven useful over many years. This being evident in green ammonia only occupying 1% of the industry, the reliance and dominance of conventional fertilisers are seen in the funding of numerous universities for furthering such technology, making it more difficult to stand out as an equal to current technology.

In any industry, developing a more sustainable product comes with a “green premium”, with the early adopters having to tolerate significant increases in price in order to have a decreased impact on the environment. Only with significant investment and adoption of a more sustainable solution can an industry achieve a more renewable environmental impact.

Our interview with David Payne expanded on our understanding of the risks provided towards independent farmers, especially with the cost associated with achieving sustainable farming. As stated previously, this widespread adoption cannot happen without early adopters who are willing to pay the “green premium” for early products. In the case of our genetically modified E.coli, we would have to find a large testing group of farmers who are willing to test and trial our “SOILution”.

Ideating and Desiging the Solution

Wet Lab Experts

Insights from a professional perspective within the wet lab stemmed from Ari Drogo, an advisor who had experience in previous iGem competitions, who guided us in our documentation and presentation of our findings within the lab. He helped develop our understanding of graphical presentation, streamlining the process of displaying our data in various graphs.

Additionally, the complex process of Golden Gate Assembly was broken down and streamlined so that we could understand this crucial process of data transfer. Any shortcomings or flaws that would’ve manifested themselves in our documentation were mitigated through his advice regarding the organisation of our content.

Further, his wet lab expertise emphasised the importance of quantitative results tailored to iGem requirements. Originally, levels of fluorescence and absorption were measured qualitatively, however after consulting Ari, he suggested that we measure it quantitatively instead to better meet criteria.

Dry Lab Experts

When designing our solution, it was crucial that we kept the perspectives of our target market at hand, as the prevalence of current methods of fertilisation would make it difficult to stand out to them. Punctuated in our interview with Jason Simmons, less progressive farmers express their main concern towards newer products being reliable in delivering their purpose, instead using chemical fertilisers which have proven to be consistent in their delivery of nutrients to the soil. Therefore, we recognised the importance of testing and proving the reliability of our project, as it would be paramount to success in our emergence in the fertiliser market.

Implementation

Real World Application

To gain a general consensus right at the beginning of our implementation, we may send samples to farms with varying soil conditions. These samples will be used in set areas, with microbe and soil health to acquire accurate information regarding effectiveness in different conditions.

After the initial phase of gathering data is completed, the full commercial process will follow a path to ensure investors are delivered with an optimised form of our product. First, the stakeholder’s soil will have to be analysed to understand the necessary application of fertiliser. This will be done through the collection of multiple samples across their growing sites, which will then be delivered to a lab to be tested for multiple factors such as nitrogen capacity, pH levels, and phosphorus. With the results acquired from such tests, we will be able to evaluate the amount of fertiliser required, then deliver it to the stakeholder.

Advice from our interview with Bach Mai Ly suggested that the standards for regularly testing soil samples should be around every 3 - 4 months. Testing will be done following these intervals, allowing us to gain an idea on the health and activity of the nitrogen fixing microbes.

Safety and Challenges to Implementation

To ensure the safety of people and the environment the bacteria will be involved in, numerous measures were made during the development of our project to minimise the development of pathogenic attributes. The K12 strains that make up our bacteria are prevented from independently manufacturing amino acids or vitamin B5. Such features prevent them from thriving in widely variable environments whilst also discouraging the intermixing of DNA with other bacteria. This prevents pathogenic microorganisms from developing features that could enhance the negative effects they already cause. Intermixing DNA is further mitigated through the inclusion of the rec-A minus gene within the strains, eliminating the ability to engage in homologous recombination while also restricting their growth in variable environments. Finally, the T7 promoter used for controlling Green Fluorescent Protein (GFP) fluorescence is a B strain. As such, it is considered safe for use in our applications.

The integrated FNR deactivates the nitrogenase cycle when it detects an unfavourable oxygen level in the environment, allowing them to survive in the midst of a change in their preferred anaerobic environment. This protective measure acts as a killswitch, preventing the nitrogenase from being damaged while also preventing the damage to the surrounding environment if leakage somehow occurs.

Citations