Introduction
iGEM Toronto was heavily focused on ensuring that our project revolved around “human-centered design”. To ensure that our project would solve problems concerning humans, it was critical for our team to involve and consider stakeholders throughout our project’s planning and development. Without the critical advice, feedback, and input from our various stakeholders, it would not have been possible to effectively reflect and consider all the nuances found in our project that we are able to demonstrate today. We ensured that we not only spoke to our stakeholders, but also listened to them, understood their backgrounds and values, in order to properly incorporate their feedback into our proposed solution.
In order to determine if our project would impact Canada’s current methane emissions and climate crisis, a lot of effort was taken into the brainstorming, identification, and outreach of various stakeholders. Furthermore, with the help of our stakeholders and research, our team wanted to ensure that our project would be able to be properly implemented holistically in reducing Canada’s methane emissions. It was important for our team to not just offer a technical solution to stakeholders, but to demonstrate that our project fits in a greater narrative of current ongoing initiatives and movements to reduce methane emissions and landfill gas.
It was important for our team to not just speak with stakeholders, but to understand their feedback and insights to gain a better understanding about how our project fits into the larger picture of the climate crisis and how it can be used to reduce methane emissions in Canada. Our Human Practices Wiki is separated into the following sections:
- Human Practices Frameworks: these frameworks were used to ensure that each stakeholder conversation and interaction we had maximised its potential
- Regulative Politics and ethics: to demonstrate how our project would fit within Canada's landfill site and synthetic biology regulations
- Indigenous communities: Indigenous communities are a key stakeholder group in Canada in regards to the negative impacts of landfill sites and environmental racism within Canada
- Implementation: our team visited a landfill site in Ontario to understand the real-life implementation of our project
- Business: we considered how we would take our project and turn it into a potential start-up company
- Integrated Human Practices: conversations we had with key stakeholders across all subteams to demonstrate how their feedback directed and influenced our project
Frameworks
Our Human Practices team created frameworks that drove our stakeholder interactions at every step along the way. Beginning with the “Stakeholder Management Framework”, our team utilised this guide to consider how each stakeholder fits into the larger scope of our project. Next, our team created a “Feedback Cycle” framework that allowed our stakeholder conversations to be structured, efficient, and informative to ensure that we gained the absolute most out of each and every stakeholder interaction. Lastly, our team used the “Third-Party Feedback Template” to introduce our stakeholders, summarise our conversations, and demonstrate how our stakeholders drove the next steps of our project.
Stakeholder Management Framework
In order to demonstrate to our stakeholders how and in which areas they can help us in our project, we created a stakeholder management framework identifying the four key areas in our project that require feedback. Beginning on the top right on the figure and moving clockwise, the first area that we saw required feedback was on the topic of implementation. Being able to properly ensure that our project has the necessary components in its design and application so that it can realistically be implemented at landfill sites for the conversion of landfill gas was important for our team to know that no component was missing in our project. Furthermore, being able to ensure that our project not only had the necessary design components, but also the correct business model and aims was also important to ensure that our project can successfully be implemented in real life. The next key area was for our stakeholders to help us investigate the sustainability and social impact of our project. We wanted to gain a better understanding of how our project currently fits in current sustainability efforts within Canada and Ontario, as well as understanding current social initiatives when it comes to landfill gas and other topics involved in the climate crisis and landfill sites. Next, we wanted to understand the public perception of our project. Since our project utilises synthetic biology to solve a climate issue, we wanted to understand the public perception of both synthetic biology and the climate crisis. To do so, we asked stakeholders about their own personal knowledge and experience with synthetic biology, and how they feel about its implementation in industry to solve a climate problem. Our last key area was to understand the regulatory and safety space for our project. Landfill sites in Ontario are highly regulated, and each municipality is responsible for their own landfill sites. Therefore, there can be quite a lot of variation regarding how each landfill is run. To understand if our project was following any necessary safety regulations in the field, it was important for us to speak with stakeholders that are familiar with the space and the regulatory environment.
The Feedback Cycle: The Human Practices Approach
To drive conversation and ensure that our team makes the most from each stakeholder interaction, we created a feedback cycle that demonstrates the structure and provided us a framework going into each meeting. The first step in our cycle was to listen to what each stakeholder had to say about their experiences, personal story, or insight. Then, our team would follow-up by asking relevant questions, either to probe more deeply into what our stakeholder shared, or to begin a new topic or direction of conversation. Towards the end of the meeting, we would ask clarifying questions and reiterate some key points from the meeting to ensure that our understanding was correct and that the stakeholder did not want to add anything else. Lastly, we would pursue new directions and new ideas that the stakeholder meeting has catalysed, fostering our team to seek out innovative and novel ideas.
Third-Party Feedback Template
The last framework our team used to drive stakeholder interactions is our “Third-Party Feedback Template”, which we used to introduce individuals to our stakeholders and demonstrate their significance to our project. There are three main sections in this template, beginning with the important question of asking who our stakeholders are, and introducing their backgrounds and why we reached out to them. Next, we summarise the conversations and knowledge exchange that occurred during our interactions. Finally, we share our reflections from our stakeholder conversations and how these interactions drove the nex steps of our project.
There are a variety of regulative policies and policies in place that would impact the implementation and success of iGEM Toronto’s project. There are regulative politics in place that impact landfill sites that govern the maintenance, monitoring, and disposal of landfill gas. These regulatory policies were put in place in an effort to reduce landfill gas emissions in Canada, and govern at what size and capacity would landfill sites be required to monitor their landfill gas emissions and also dispose of the landfill gas. In order to educate individuals who are not familiar with the nuanced landscape of landfill gas regulation, our team created a summary of this framework:
Summary of the Regulatory Framework
Methane is a highly potent greenhouse gas with a warming potential 28 times of carbon dioxide. A source of this methane is from the decomposition of biodegradable waste in landfills, accounting for 24% of national methane emissions in Canada. As such, in facing the dangers posed by methane, there is a regulatory framework in place aimed at reducing emissions from landfills.
Current Strategies
The framework suggests implementing infrastructure and equipment at landfills to recover the gas and either flare it or use it for energy generation. Existing approaches are technically feasible, commercially available, and provide measurable methane emission reductions. Building from these current approaches, emerging strategies involve using specific landfill cover materials and designs to facilitate the biological destruction of methane that will produce less pollution and be more cost effective. iGEM Toronto is interested in being at the forefront of using synthetic biology to reduce the amount of landfill gas emissions in an environmentally safe and efficient way.
Implementation
In implementing the landfill gas strategies discussed above, the regulatory framework proposes several key requirements:
Implementation of a landfill methane control approach: Landfills exceeding a methane generation or emission threshold would be required to implement a landfill methane control approach. This could involve active or passive landfill gas recovery systems, methane destruction devices, engineered biosystems (such as biocovers or biowindows), or landfill design and operational plans to reduce methane emissions.
Methane control monitoring and corrective action plan: Landfills subject to the regulations would need to conduct regular monitoring of methane emissions and have a corrective action plan in place to address any issues or leaks in the methane control systems. Increasing the frequency of monitoring and maintenance has been shown to enhance methane recovery and emission reduction.
Notifications, record keeping, and annual reporting: Landfills would be required to provide notifications to the regulatory authority, keep records of methane control activities and measurements, and submit annual reports on their methane emissions and control efforts.
Applicability Thresholds
These regulations would apply to landfills based on the quantity of municipal solid waste (MSW) disposed of at the site. The applicability thresholds are as follows:
- Closed landfills that accepted any quantity of MSW after January 1, 2009, and have more than 450,000 tonnes of MSW-in-place.
- Open landfills that have more than 100,000 tonnes of MSW-in-place or have accepted more than 10,000 tonnes of MSW for disposal per year after the regulations come into force.
Landfills meeting these criteria would be required to conduct a methane generation assessment using a designated model and report the results to the regulatory authority. If the landfill's annual methane generation exceeds a specified threshold of 664 tonnes per year, the regulations would require the implementation of a landfill methane control approach.
Exemptions may be granted based on measured methane values. For landfills without existing methane control systems, they can be exempted if the methane concentration at or above the landfill surface is below specified thresholds. Closed landfills with existing gas recovery systems may be exempted if methane concentrations in the recovered gas and surface emissions are below specified thresholds.
To determine compliance with thresholds, the regulations propose using drone-based and ground-based methane concentration measurements. Drone-based surveys would be conducted at specified intervals, while ground-based measurements would verify exceedances identified by the drone-based method.
The framework acknowledges that further development and validation of measurement methods and thresholds are necessary. Stakeholders are invited to provide data and information to support this analysis.
Improvements to the Framework
The proposed framework aims to align with Canada's commitment to reducing methane emissions, as outlined in the Global Methane Pledge and the Faster and Further: Canada's Methane Strategy. The regulations seek to achieve the greatest feasible reductions in landfill methane emissions and improve monitoring and mitigation practices. Feedback on the proposed regulatory framework was invited until May 19, 2023, to inform the development of the regulations.
The various requirements for the operation, maintenance, and monitoring of landfill gas recovery systems and methane control approaches are as follows:
Operations and Maintenance
- Landfill gas combustion or processing systems must be operated and maintained according to manufacturer recommendations.
- Flares should be operated at or above the temperature that achieves 99% destruction efficiency, as determined by the most recent source test.
- Valves on gas recovery systems must be closed within one hour of shutting down the equipment.
Methane Monitoring and Corrective Action Plan
- Landfills must develop and implement a methane control monitoring and corrective action plan.
- The plan should include monitoring for landfill methane recovery systems, biosystems (biocovers), and methane leaks.
- Landfill owners have flexibility in setting action thresholds for some parameters, while certain thresholds will be specified in the regulations.
- The plan should define procedures for follow-up actions when thresholds are exceeded and document exceedances and corrective actions taken.
Monitoring Landfill Gas Recovery Systems
- Landfills with active gas recovery systems must conduct monitoring in extraction wells and at the gas recovery plant.
- Monitoring parameters include oxygen, nitrogen, liquid level, and vacuum pressure.
- Monitoring frequency may vary based on methane generation rates, ranging from monthly to continuous measurements.
Monitoring Engineered Biocovers/Biosystems
- Monitoring requirements for biocovers/biosystems may include in situ testing, monitoring media properties, and inspecting the biocover surface.
Monitoring to Identify Methane Leaks
- The methane control plan should include leak detection and repair (LDAR) requirements.
- Leaks are defined based on measured methane concentrations exceeding specified thresholds.
- Monthly visual inspections of the landfill surface and monitoring of gas recovery system components are required.
- Drone-based monitoring of surface methane concentrations is required three times per year.
- Annual monitoring may be allowed in areas where landfill gas is not being recovered and surface methane emissions are below the performance standard.
Alternative Monitoring Methods
- Landfills may propose alternative LDAR approaches that can identify the same number of leaks as the standard requirements.
- Site-specific performance standards may be considered if equivalency can be demonstrated.
Notifications, Record Keeping, and Annual Reporting
- Landfills need to submit methane generation reports, registration of methane destruction devices, and notifications of methane control monitoring and corrective action reports.
- Records should be maintained for five years, including surface emission monitoring results, source testing results, and methane leak detection information.
- Annual reporting of landfill operational status, waste received, and waste-in-place is required.
Climate Crisis Case Study: iGEM Toronto “Methanivore”
Introduction
Pollution is defined as the introduction of harmful substances into the environment and can be categorised as either air pollution, water pollution, or land pollution. In particular, air pollution from greenhouse gases is a major concern due to its association with global warming [1] . Greenhouse gases such as carbon dioxide and methane absorb sunlight reflected from the earth’s surface, trapping heat in the atmosphere via the greenhouse effect. Although this greenhouse effect is a natural process, recent human activities have been increasing the amount of greenhouse gases released into the atmosphere, causing rising global temperatures [1] .
Landfill gas occurs due to the anaerobic decomposition of organic materials such as food and paper deposited at landfill sites [2] . As of today, solid waste landfills are responsible for a staggering 23% of Canada’s total methane emissions. Instead of allowing methane to escape into the atmosphere, methane can be collected within landfill sites using a series of vertical and horizontal pipes. The methane is then collected at a central site where it can undergo treatment and be used to generate electricity or create fuel. Alternatively, the landfill gas can also be burned in a process known as “flaring” to create carbon dioxide, which has a lower impact on the greenhouse effect than methane [3] . According to a survey done by the Government of Canada in 2020, approximately 1,401 kilotonnes (kt) of methane were generated from Canadian landfills [4] . Of this amount, only 418 kt, roughly a third, of it was captured (30%) [4] .
It is evident that new regulations and technologies are necessary to reduce methane emissions in Canada. More landfill sites should begin to capture methane in an effort to reduce the environmental impact of our garbage. Furthermore, greener, more clean solutions for utilising methane gas should be considered in lieu of flaring, which still introduces a greenhouse gas (carbon dioxide) into the environment.
iGEM Toronto’s 2023 project, named “Methanivore”, aims to engineer bacteria capable of consuming methane and methane derivatives, such as methanol, formate, and formaldehyde, and implement this system in landfill sites. To do this, the methane from landfill gas will be isolated and then oxidized to methanol. This methanol will serve as a carbon feedstock into methylotrophic strain bacteria. The bacteria that feed on the methanol will also have to be cultivated on an industrial scale to effectively remove methane from the landfill gas ecosystem. The Toronto iGEM’s hardware team aims to design a separation process for the landfill gas, a process for oxidizing the resultant methane gas, and a fermenter to cultivate the methylotrophic bacteria at scale, while the wet lab and dry lab team are utilizing a variety of experimental techniques to enhance the assimilation of single-carbon compounds in our engineered strains using overexpression, CIRPSR-Cas system knockout, and adaptive evolution.
Regulatory Landscape and Risk Assessment
To the best of iGEM Toronto’s 2023 team’s knowledge, the application of genetically modified organisms (GMOs) to manage landfill gas emissions has not been previously explored. However, current research offers valuable insights into engineered methanotrophs, a field our team is actively expanding upon in our project. Past studies have investigated the utilization of genetic tools to research and optimize methanotroph physiology, aiming to enable metabolic engineering for converting methane into valuable bioproducts [5] [6] .
While the research techniques the team is employing, such as CRISPR-Cas9, are vital for optimizing methane conversion, the true bioethical and regulatory challenges arise not within the laboratory setting, but when considering the real-world implementation of these engineered methanotrophs at landfill sites. Therefore, there are a variety of bioethical considerations and perspectives that our team considered during the creation of our project, which are discussed below.
The use of GMOs in Canada extends into various industrial processes and consumer goods within Canada and are included in a category of approximately 23,000 different substances that are utilized in the country.
To ensure public health and environmental safety, Health Canada and Environment Canada jointly established the Canadian Environmental Protection Act, 1999 [7] . This legislation mandates a thorough risk assessment of environmental and industrial products, including those that are utilized in biotechnology. The product is evaluated for its potential risk against both human health and the environment, and if it is toxic. A substance is considered to be toxic if it enters or may enter an environment where it can pose a risk to human health, the environment (such as wildlife), and environment in which wildlife depend on, such as water, air, or soil.
In order to separate chemicals and other products from biotechnology products involving living organisms, a specialized section of the CEPA 1999 act addresses the threat and risk assessment of these entities. This section requires information from companies or manufacturers regarding the organism’s identification, characteristics, exposure, and any potential health threats to humans or to the environment. While iGEM Toronto believes that our engineered methanotrophs pose no threat to human health, there are some considerations when weighing the potential environmental risk of our organisms to the environment.
Engineered methanotrophs, modified within the confines of a lab, cannot be deemed native to the local environment. Methanotrophs naturally inhibit various ecosystems, such as in rice fields, wetlands, sewage treatment facilities, and landfills [8] . It can be expected that each geographical region may have different predominant and local species of methanotrophs, resulting in each location having its own unique biological footprint. Consequently, the potential introduction of genetically engineered methanotrophs may jeopardize the natural diversity of methanotrophs at a landfill site if they were to escape confinement within our hardware system and outcompete native strains.
The concern about engineered methanotrophs outcompeting local strains underscores the importance of preserving nature’s biodiversity, since it is unlikely that the exposure of engineered methanotrophs would cause long lasting environmental damage that could potentially impact the ecosystem. In fact, previous researchers have contemplated the deliberate introduction of engineered methanotrophs directly into the soil at sites for soil remediation [9] , and waste water treatment [10] , and in coal mines to mitigate high methane emissions [11]
Conclusion
The iGEM Toronto 2023 team, “Methanivore”, worked on an innovative and unique project by introducing genetically modified organisms into a landfill gas collection system—a unique application in the field. Given the stringent regulations surrounding genetically modified organisms and synthetic biology, our team dedicated significant effort to comprehensively research the regulatory and bioethical dimensions of our project, with the aim of demonstrating our project’s viability for real-world implementation beyond the laboratory.
In Canada, the utilization of genetically modified organisms is highly regulated under the 1999 Canadian Environmental Protection Act. This legislation requires companies, manufacturers, or other entities to prove that their chemical, product, or living organisms pose no threat to human health or the environment.
While our project poses no direct harm to human health, we acknowledge valid environmental concerns regarding the potential consequences should our engineered methanotrophs escape into the landfill site. The engineered methanotrophs can potentially interbreed with native local strains, or competitively displace them, thereby affecting the local populations. This concern extends to biodiversity preservation, emphasizing our commitment to safeguarding nature’s intricate ecosystems, regardless of their size and big or small.
While assessing the risks of our hardware system and project, our team has come to the conclusion that the chances of our engineered methanotrophs escaping our hardware system are minimal, reducing the risk of contamination or leakage. Nevertheless, we advocate for a proactive stance by endorsing the importance of well-defined spill cleaning procedures at landfill sites. These procedures encompass the use of materials designed to absorb spills, having biohazard disposal bags, and disinfects, all which serve as a contingency plan to swiftly address and solve any unforeseen incidents.
In summary, the iGEM Toronto team, "Methanivore," recognizes the importance of addressing regulatory and bioethical considerations when exploring novel applications of genetically modified organisms in landfill gas collection systems. While there are potential environmental concerns regarding biodiversity, the team's risk assessment indicates a low likelihood of escape from our system. We emphasize the imperative need for proactive bio spill cleaning procedures, reinforcing our commitment to responsible and secure implementation of our innovative solution in the world.
In order for our team to understand the implementation of our project at landfill sites, and to also understand how current landfill sites are regulated and how landfill gas is collected, our team visited a local landfill site in London, Ontario. The embedded document at the end of this section contains key information and takeaways from our landfill site visit.
Most importantly, the landfill site visit allowed our team to become more informed regarding the next steps of our project and how we wanted to continue our hardware team, entrepreneurship team, and human practices teams’ directions. While this information is found in the embedded document, key points and takeaways are summarised below to highlight the importance the landfill site had on our project’s direction and knowledge.
Hardware
The biggest research question our hardware team pursued was to understand how our synthetic biology solution utilising methanotrophs could be implemented in the field at landfill sites and create models to scale the solution up such that it is capable of processing the landfill gas from a landfill site realistically. To do so, the hardware team asked 3 main questions at the landfill site to better understand the constraints and necessary components required to implement our hardware device, and had 1 key takeaway regarding our hardware device and its effectiveness compared to other methods of getting rid of landfill gas such as flaring.
Questions & Answers
What size does our hardware solution have to be?
Space is largely available at the landfill site, and placing anything at the buffer area will not greatly impact the landfill’s future plans or logistics. Therefore, our hardware solution is not constrained by size, however the diameter of the pipe connecting the landfill gas collection system has to match the pipe we use to our hardware solution.
What is the volume of landfill gas produced and its trend?
The landfill site tour guide informed us what capacity we would have to match if we wanted to propose a system to treat landfill gas.
What is the budget for landfill sites when implementing a new landfill gas solution?
We would have to match the cost or be more cost-effective than a new incinerator for flaring.
Key Takeaways
Our preliminary results of the membrane separation and oxidation model shows that the separation and conversion rates are low, with a small amount of methane leaking out of our device. This could put our solution in a disadvantageous position in comparison to our flaring system, and therefore our team will have to ensure that there are no leakages.
Entrepreneurship
The Entrepreneurship team gained valuable insight and new considerations needed if this project were to become a start-up. These considerations are further researched and investigated and can be found in the Business Plan in the Entrepreneurship Page.
Questions & Answers
What is the competitiveness of our landfill gas solution?
Synthetic biology as a potential solution for landfill gas management is novel and unique in the market. Using bacteria to assimilate methane and carbon dioxide could enable zero amount of methane and carbon dioxide released to the atmosphere. iGEM Toronto must continue to search for ways to improve the costs and efficiencies of our hardware and chassis engineering design. The cost analysis shows that a scaled up version of our solution is more expensive than installing a new incinerator, and so iGEM Toronto must continue to find additional value behind our solution such that the landfill site authorities and/or the accelerator programs can be persuaded.
What is the business model of existing landfills and their value chain propositions?
Publicly owned landfills are not interested in turning a profit, but private ones might, especially if we can add value to their waste products.
What is our target market?
Our target market is not limited to just the smaller landfill sites, but also large landfill sites with existing flaring infrastructure could benefit from our project. Private landfill sites can also potentially be a good target.
Key Takeaways
The Entrepreneurship team discovered key insight regarding the cost and competitiveness of landfill gas solutions at landfills. Our solution is novel in the field, however we may have to consider additional benefits to our solution to persuade landfill sites to implement our solution if a scaled up version of our solution is more expensive than installing a new incinerator. Furthermore, the team discovered potential future opportunities working with landfills in general, such as leachate management or odour management, which is potentially more profitable.
Human Practice
Those working at the landfill site suggested that iGEM Toronto could gain valuable feedback or consultations on the design and implementation of our project from Government Entities:
- iGEM Toronto Human Practice team reached out to the Green Party of Canada.
- iGEM Toronto Entrepreneurship team will visit a government office to inquire about the Environmental Protection Act.
Our team reached out to a variety of industry and academic experts to discuss our projects. As discussed in our framework section above, we followed our “Third-Party Feedback Template” to showcase the conversations we had and highlight how our stakeholders drove our project forward.
Wet Lab
Professor Christoper Lawson
Who is the stakeholder?
Professor Lawson is part of Biozone - Center for Applied Bioscience and Bioengineering at the University of Toronto. His research investigates metabolic regulation of anaerobic microbiome metabolism. He has expertises in implementing synthetic biology solutions to wastewater treatment and solid waste management, which aligns with iGEM Toronto project this year.
What did they say?
In Biozone, there are two devices that can measure metabolites: Gas Chromatograph or HPLC. Professor Lawson recommended HPLC because the training required for GC is much more intensive than HPLC. The preparation of GC includes gas handling. Although GC is known to be better for volatile compounds (Methanol in our case), the safety hazard behind preparing or operating GC is higher than HPLC, specially none of the wet lab members had experience with GC.
Reflection or next steps
Wet lab discarded the GC protocol and developed a protocol for preparing our sample for HPLC. We also reached out to Biozone members who have experience with HPLC to help our team out with the details.
Dr. Nico Claassens
Who is the stakeholder?
Dr. Claassens is a renowned professor and researcher in the field of Reductive Glycine Pathway (rGlyp) pathway and C1 assimilation pathways and is affiliated with the Laboratory of Microbiology, from the University of Wageningen. Our team was interested in acquiring one of the strains that was generated in his lab. He gave advice to our team and gave recommendations on selecting C1-assimilating pathways and strains, as well as giving insight on potential chemostat setup.
What did they say?
Dr. Claassens told us that it would be important to prioritize genomic manipulations rather than plasmid overexpression in metabolic engineering, and that mdh is a bottleneck and important genetic target in the Rump cycle. Lastly, we tried to acquire the P. putida rGlyp strain from his lab.
Reflection or next steps
From our conversation, we listed tpiA gene KO and mdh KI in our plan, and we researched different mdh variants.
Dafni Giannari
Who is the stakeholder?
Dafni is a graduate student at the Department of Chemical Engineering at the University of Toronto, and was one of our academic advisors. We reached out because we needed help determining which tools would be best for modeling and narrowing focus in flux balance analysis to industry standard tools.
What did they say?
Dafni provided recommendations on how to make the in-silico metabolic model simulations more accurate (when modeling the methylotrophic E. coli).
Reflection or next steps
Worked with Danfi iteratively over the course of the project to update the approach to modelling. We continuously updated a list of questions for her and changed the analysis approach using her guidance. For example, our team chose flux balance software based on her suggestion, and made adjustments to media concentrations based on her later feedback.
Ethan Agena
Who is the stakeholder?
Ethan was one of our team’s academic advisor’s. We needed to use the university's lab space in BioZone, and Ethan is a graduate student within the lab who has experience with cloning and common synthetic biology techniques. He also has previous iGEM experience as an undergrad. Due to his experience and personal connection to iGEM we felt as though he would be a great asset to our team as a stakeholder for our project.
What did they say?
Ethan worked with our wet lab team and allocated time and space for our experiments in his lab. He also helped our team establish a protocol for HPLC and aided us in preparing samples for it.
Reflection or next steps
We incorporated his feedback towards our protocols. He oriented us with lab facilities, hence allowing us to do work on the project.
Mohamed Nasr
Who is the stakeholder?
Mohamed was one of our team’s academic advisor’s from the Department of Chemical Engineering at the University of Toronto, and provided advice for the experimental designs for CRISPR-Cas9 knockout of tpiA. He offered insights in designing primers, assembling sgRNA and co-transformation. Mohamed has a lot of experience in CRISPR genetic engineering in different E.Coli strains and yeast. Our team is fresh to CRISPR-Cas9 designs so we consulted him for expert opinions on CRISPR knockouts.
What did they say?
He provided valid information regarding designing primers for inverse PCR to assemble sgRNA.
Reflection or next steps
We successfully assembled sgRNA by conducting inverse PCR using the primers designs Mohamed suggested. We also successfully designed and acquired donor DNA for knockout of tpiA
Gary Hoang
Who is the stakeholder?
Gary is a lab technician at the University of Toronto and was our team’s wet lab advisor for safety training, lab methods, and helping our team develop our protocol. We needed to use the lab space within the Institute of Biomedical Engineering, and Gary was there with us during experimentation to help us solve any problems. He has extensive experience with synthetic biology techniques and troubleshooted our protocols with us. He also provided us with reagents and consumables.
What did they say?
He gave us invaluable advice by helping us troubleshoot all protocols that failed and improving our protocols where necessary.
Reflection or next steps
His feedback was instrumental towards the success of our cloning experiments. He provided us with the space to even begin the project at all.
Dr. Olanrewaju Raji
Who is the stakeholder?
Dr. Raji is a post-doctoral research fellow at the University of Toronto. We reached out to him for help and access to HPLC equipment. He advised the team on the best way to perform HPLC experiments and how to prepare samples.
What did they say?
Information conveyed to us includes the following: describing how the BioZone HPLC equipment functions, describing how to prepare samples containing MeOH, and describing how to schedule HPLC tasks.
Reflection or next steps
Collaboration with Dr. Raijji allowed our team to quantify the MeOH consumption of the T-B18 bacteria in BioZone. His advice informed our decisions about how to properly prepare HPLC samples.
Jenny Baker
Who is the stakeholder?
Jenny was a past member from the University of Wageningen iGEM team that worked with methane assimilation strains, and she is experienced in experiments relevant to what we wanted to conduct in our project. Jenny gave advice and recommendations on selecting C1-assimilating pathways & strains.
What did they say?
She gave details on improving on our experimental approaches.
Reflection or next steps
We have gained insight on how to potentially apply toxic compounds (methanol) into our experiment and ALE.
Michelle Feigis
Who is the stakeholder?
Michelle is a graduate student at the University of Toronto and was one of our academic advisors. No one on the team had previous experience with adaptive evolution. Michelle has the most experience in ALE in the chemical engineering department, thus we reached out to seek her expertise. We were planning to do ALE through serial passaging, but there are not clear protocols in publications (due to its "simplicity"). However, we wanted to make sure the protocol we made from extracting information from publications makes sense, hence we reached out to Michelle for her input.
What did they say?
Michelle clarified and explained many things that's part of a standard ALE protocol, and pointed out potential issues in ours:
- Catabolite repression is likely (glucose consumed first, then will switch to methanol). Usually having two fluxes change at the same time is usually what you would to in ALE.
- Passaging: occasionally make a glycerol stock so we don't lose progress.
- Sterility: we had no choice but to use fume hoods due to methanol. Fume hoods are very dirty (adding antibiotics should prevent contamination, but we should also be very careful during this procedure).
- M9 media: add MOPS buffer into the media (in case of acid byproducts).
- OD600: make sure we have enough volume, also need to confirm OD range (e.g., there might be a need for dilutions).
- Passage transfer volume: use 10%
Reflection or next steps
We took her advice and adjusted our protocol. Meeting with her was very helpful in clarifying things we were confused about, especially about the general guidelines for certain ALE methods. She also gave us confidence in believing our experiments will work.
Dr. Mona Abo-Hashesh
Who is the stakeholder?
Dr. Mona helped us with suggesting changes to our gel extraction protocol to increase concentration and offering advice on our restriction digestion and ligation timeline. We were experiencing some trouble with low DNA concentrations after running a gel purification, and reached out to her for some help.
What did they say?
She suggested that we try several things:
- Increasing initial DNA concentration
- Combining multiple gel excisions into one column
- Incubate for 15 mins at 37 C for the elution step
Reflection or next steps
We changed our gel extraction protocol to apply the suggested modifications.
iGEM Muenster Team
Who is the stakeholder?
As a fellow iGEM team, we contacted iGEM Muenster since our team's software design is based upon the same FSEOF algorithm as the Muenster's team but follows the original research paper more closely.
What did they say?
They confirmed having had the same difficulties implementing FVSEOF, clarified some of the misunderstandings regarding the enforcing of flux bounds in FSEOF implementation, and suggested us to focus less on trying out new nonlinear solvers.
Reflection or next steps
We decided to focus more on re-implementing FSEOF, keep our choice of enforcing flux bounds on both ends and settled on only trying out the MindtPy non-linear solver.
Hardware
Professor Jay Werber
Who is the stakeholder?
Prof. Werber is an assistant professor at the University of Toronto Chemical. Engineering department. His interests are in membrane separation and he runs the Advanced Membranes lab there. The hardware team needed to identify potential solutions to separating methane from landfill gas, and to identify means of modelling membranes to size them and so they reached out to him for guidance.
What did they say?
Prof. Werber provided us with reference material to begin designing a membrane separation, and introduced us to concepts such as stage cut and the solution-diffusion model. He also directed us to finite element models which we implemented.
Reflection or next steps
At the beginning of the project we were debating on using an adsorption column vs membranes for separation. Prof. Werber's input allowed us to converge on membranes as a separation solution. His advice gave us the technical background required to begin looking into research materials for membranes, and presented an approach to membrane modelling despite Aspen lacking a membrane model.
Niloofar Shirali Zadeh
Who is the stakeholder?
Niloofar is a PhD student at the University of Toronto who works under Prof. Jay Werber at the Advanced Membranes Lab. She investigates bipolar membranes but is very knowledeable on gas separation and runs the gas separation setup at that lab. Hardware needed to demonstrate a proof-of-concept for membrane separations on gas mixtures and the gas separation setup in the Advanced Membranes Lab is managed by Niloofar.
What did they say?
Niloofar provided us with a hardware design, including a list of materials required and supplier information, and usage protocols for a single-gas membrane flow setup.
Reflection or next steps
Our team wanted to prototype mixed-gas separations, which is an improvement over what Niloofar manages in the Advanced Membranes lab. We took upon Niloofar's design and modified it for that purpose.
Yuxin (Sindy) Zhang
Who is the stakeholder?
Sindy is a masters student at the University of Toronto who works under Prof. Jay Werber at the Advanced Membranes Lab. She investigates polysulfone membranes and their behaviour at high stress and near failure conditions, and is an expert at polysulfone membrane synthesis. The Hardware team needed to inquire on membrane synthesis techniques and needed a polysulfone membrane to carry out the prototyping.
What did they say?
Sindy provided us with membrane synthesis techniques and a polysulfone membrane sample which was intended for the prototyping.
Reflection or next steps
We gained an insight into membrane separation techniques and allowed us to acquire membranes for the gas separation setup without requiring external sponsors.
Professor Daniela Galatro
Who is the stakeholder?
Professor Daniela Galatro is an assistant professor at the University of Toronto who mainly engages in process simulation and heat transfer pedagogy. She has over two decades of industry working with process simulation software, and her research interests are on physical modelling and machine learning. The Hardware team needed to inquire on the quality of the Aspen Plus simulations performed.
What did they say?
Prof. Galatro provided us with feedback on Aspen model use, including thermodynamics model selection, recycle stream validity, phase separation techniques, and membrane modelling automation with Python.
Reflection or next steps
We obtained tangible next steps for Aspen Plus modelling should the project continue for longer. We gained insight into how recycle streams are not very ideal in processes and should be kept small, and how distillation columns could be used for high performance phase separation.
Human Practices
Ravneet Gill
Who is the stakeholder?
Ravneet Gill works at Waste to Resource Ontario, and is in charge of the stakeholders and collaborations division at the organisation.
What did they say?
Due to her in-depth experience in the field of waste management in Ontario, we asked her questions regarding the implementation of our project and how we can collaborate with landfills and governments. She explained to us that landfill site management is actually a very complicated process, and that each municipality is in charge of its own landfill site with little to no oversight from the federal government. Landfill sites often hire companies to manage their landfills for them, and recommended for us to research these management companies. To her knowledge, there is no other company or current initiatives within Ontario that is using synthetic biology to convert landfill gas to reduce landfill gas emissions. She thinks that our project is unique and that it is a good idea. Furthermore, she recognized that there is currently a lot of talk regarding landfill gas right now within the industry, and that our project is very much so on-trend. Lastly, she emphasised the fact that the landfill industry is very much so dependent on cents and dollars, and that if we wanted to market our product and have it succeed compared to other competitors we need to ensure that our project is budget friendly and realistic.
Reflection or next steps
- How did we use the feedback to change/improve our project? Our conversation with Ravneet changed the way we approached our human practices outreach because without fully understanding how landfill sites are being regulated, we were interested in reaching out to the federal government to gain more insight regarding landfill site regulations. However, after speaking with her and understanding that each landfill site is regulated municipally, we focused on reaching out to city members as well as landfill site management companies that have a more direct role in our project.
- How did this feedback change how we think? This feedback changed our fundamental impression of landfill sites. It is much more nuanced that anyone on the team was originally expecting.
Randi Ramdeen
Who is the stakeholder?
A critical component for the success of our project is to consider the political support for our project. Projects addressing politically visible issues can often receive more resources and receive a stronger push for implementation, while discomfort with synthetic biology or a lack of interest can hinder usage of our project in landfills. We spoke with Randi Ramdeen, a member of the Green Party of Canada who had a common goal in preventing climate change. Furthermore, speaking to a stakeholder involved in Canada's political scene has strong knowledge regarding current climate initiatives and priorities in the country. We were referred to her from the Hamilton Stoney Creek area.
What did they say?
Randi helped us learn more about how to talk about climate change and raise awareness in different communities, as well as provided us an inside look at how the Green Party is structured. Randi explained that currently landfill gas, to her knowledge, is not currently on the radar for many politicians and climate initiatives in Ontario. This demonstrated to our team that landfill gas and its effects on climate change is currently neglected within Ontarios current policy makers and drivers. Therefore, it is crucial for our project to also educate the public when it comes to the significance of landfill gas on global warming.
Reflection or next steps
These insights from someone who worked on the front lines of talking to vulnerable populations, youths and numerous organization have provided novel approaches on how we can identify and persuade new stakeholders in supporting our cause. Furthermore, we are planning to further our relationship with the Green Party to learn more about the legislation, political climate, and potential collaborations. Randi has offered to connect us with Mike Morris, a Member of Parliament from the party, as well as other contacts. We hope that this will become an ongoing relationship that will help our team learn about the rules and regulations and gather the support to implement our project.
Nancy Hurst
Who is the stakeholder?
The implementation of our project cannot be separated from the people making the purchasing decisions. As such, we wanted to gauge the interest and funding for projects like ours, as well as the process of pitching our solution. As such, we spoke with Nancy Hurst who is an environmental advisor from Ward 12 of the City of Hamilton, who was heavily involved in environmental activism against Doug Ford's proposed urban expansion before joining city council. We were interested in her perspective, as she has seen both sides of pushing topics into public consciousness and putting political pressure onto city councillors. We were referred to her from the Green Party representative in the Hamilton Ancaster/Dundas region.
What did they say?
Nancy provided valuable insights into the current priorities and funding of the Hamilton municipality, including the Greenbelt expansion, coal use in steel industry and landfill gases. Specifically, she indicated that the municipality is aware of the landfill gas issue, and some of the landfills are already using methane capturing solutions. As such, she gave two critical ideas: our solution has competitors that are currently implemented, and landfill sites are open to using methane capture technology. This gives us a target to ensure that our solution has an advantage over current solutions. For example, this made it clear that we must produce a useful product downstream rather than simple methane capture that is currently used. Moreover, this conversation gave us reassurance that there is interest in our solutions for landfill gases, not only from the municipality and landfill sites, but also from the public. As she mentioned, the public support for the green initiative is higher than ever, especially among the younger population. Just recently, there was a parade on Yonge street for environmental activism.
Understanding the public's opinion and pitching solutions that match their interest is necessary for gaining the support for enacting change and direct funding to implement our project. To do this, Nancy told us about her experiences of organizing a petition, including how she gathered support over Facebook, how to target certain demographics, the approximate timeline to gain supporters, and how to leverage windows of opportunities.
Reflection or next steps
In our conversations, we were given insight into how to improve both our project, as well as the messaging behind our project. One of the chief complaints of the public regarding landfill sites is the smell, which could be translated into changes in our filtering hardware to also redirect noxious-smelling gases like sulfides and ammonia. This is important information, as she told us a part of ensuring a successful campaign is to have catchy slogans that captures public interest. In fact, she brainstormed some with us, including “Our dumps are farting, put a plug on it,” to evoke the imagery of foul smelling gases and the potential of our solution to stop it. She also told us how to target the different demographic, such as using affordable advertising spaces on Instagram and other social media to get interest from the younger demographic. We also learned about her experience of viewing her initiative of stopping urban boundary expansion from multiple perspectives to cater towards different interests, such as from the perspective of tax increases for people who may be less interested in climate change but are concerned with their finances.
Furthermore, we learned about groups in Hamilton, like McMaster City Lab, with whom we can collaborate, because they have more experience introducing solutions to the community and implementing pilot trials that can become permanent solutions.
Nancy also reshaped our thinking of public perception of synthetic biology. Our collaborators will likely be environmental groups who are in support of reducing climate change and decreasing greenhouse gas emissions. However, given the backlash and fear of the public and certain environmental groups around GMOs, we were worried that the perception of synthetic biology would hinder our messaging to our biggest supporters. However, Nancy stated that people are unlikely to tie the concept of genetically modified crops or food to genetically modified bacteria aimed to reduce emissions. As such, we do not have to fear pushbacks from environmental groups about genetically modifying the bacteria. She also changed our thinking on how complete our project has to be and who to communicate with. For example, because the municipalities regulate the landfills, we should be pitching our solutions to the city councils rather than the landfills themselves. In order to do so, our project needs to reach a level where it is a complete product, ready to be purchased and implemented. In addition to the private pitch, we learned that we could push for public support to enact a bylaw requiring landfills to use some form of methane capture. Overall, speaking with Nancy gave us direction in how to improve the hardware of our project as well as the messaging and promotion of our project.
Entrepreneurship
Rebecca Tam
Who is the stakeholder?
Rebecca was one of our external industry stakeholders who we reached out to in order to find opportunities to talk about our project to other founders, students, and researchers. Rebecca hosts a platform to talk and discuss startup projects with researchers and other startup founders. She served as connector between Methanivore and other department staff.
What did they say?
Rebecca helped our team choose and practice a specific pitching style, encompassing key points and differentiators that make our project idea standout.
Reflection or next steps
We polished our business plan to incorporate sections important. We also implemented her advice on how to pitch, and talk about our project for future presentations.
Brittany Goldhawke
Who is the stakeholder?
Brittany is from Toronto’s Creative Destruction Lab (CDL), a seed-stage program for start-up companies created by the Rotman School of Management at the University of Toronto. We reached out to her to seek mentorship, and to apply to the CDL incubator.
What did they say?
Brittany handled Methanivore’s CDL application, and invited our team to the interview stage of the admission process. She explained that our idea is still too young to enter the CDL incubator, but that our mission has strong potential down the line. They offered avenues for CDL to help us mature our idea further, and invited us to an exclusive agreement to receive mentorship.
Reflection or next steps
We will continue our exclusive mentorship with CDL to help our project grow after the iGEM competition.
Shannon Lee
Who is the stakeholder?
Shannon Lee is the Program Lead at the Faculty of Applied Science and Engineering’s incubator program. We connected with her to acquire advice and admission to the startup incubation program and to receive insight of our company’s current state.
What did they say?
She provided recommendations of alternative incubation programs to pursue appropriate for the current maturity of the company, while providing recommendations of alternative programs for future development.
Reflection or next steps
After receiving the feedback, we were able to further develop our growth potential and exit strategy within our Entrepreneurship business plan.
Introduction
During our team's preliminary research, we found that Indigenous communities are heavily impacted by landfill sites due to environmental racism and a lack of resources [1] [2] . Environmental racism is an incredibly broad and nuanced topic, and includes actions that expose marginalized communities to contamination and pollution, having a lack of voice or representation in decision-making regarding the creation and management of landfill sites near their land, and slower rates of clean-up [1] [2] . While we were originally interested in how our project could potentially impact indigenous communities, after more thorough research it became apparent that indigenous communities are primarily affected by poor solid waste management and a lack of proper landfill site infrastructure and funding to properly collect, treat, and dispose of garbage [1] [2] . Our project is dependent upon utilizing a landfill site's already existing landfill gas capturing infrastructure to collect the landfill gas to our hardware system. Therefore, our hardware could not be implemented at landfill sites by or near indigenous communities that do not have the proper infrastructure already. This demonstrates a lack of resources, funding, and most of all, priority, that the government has given to our indigenous communities. Although our project cannot be directly implemented at their landfill sites, our team has therefore decided to use this opportunity to promote the unfair treatment and lack of resources indigenous communities face for their landfill sites, and to advocate for better landfill waste management on their land.
Inspired by our exploration of these issues and guided by the urgent need for cultural sensitivity and respect in collaborative efforts, we found inspiration in the work of Dr. Justina Ray, a distinguished conservationist and advocate for inclusive conservation projects with Indigenous communities. This revelation motivated us to create a practical resource—a guideline aimed at empowering researchers, organizations, and individuals with the knowledge and tools needed for meaningful and equitable engagement with Indigenous communities.
Tips and Guidelines for Working with First Nation Communities
We acknowledge that all of us in Canada are on the traditional territory of many Indigenous nations. This idea is more than a statement. Engaging with Indigenous communities in research is a vital aspect of conducting studies that involve their lands, resources, and well-being. This document provides guidelines and tips to ensure respectful and effective interactions with Indigenous communities while conducting research. These guidelines and tips have been gathered from the invaluable expertise and insights of Prof. Shelby Riskin and Prof. Justina Ray. Through their extensive experience and dedication to working with Indigenous communities, they have provided invaluable guidance in shaping these recommendations for respectful and effective interactions in research involving First Nation communities.
Build relationships and establish trust
Prioritize building relationships with community members before initiating research. Attend community events, engage in open dialogue, and actively listen to community concerns and priorities. Identify and approach community leaders, elders, and Knowledge Keepers as key partners in the research process. Seek their guidance and respectfully follow their protocols for engagement.
Obtain informed consent and maintain confidentiality
Prioritize obtaining informed consent from individuals and the community as a whole. Provide clear and accessible information about the purpose, risks, benefits, and potential outcomes of the research. Respect community decisions regarding data ownership and confidentiality. Seek consent before sharing or publishing any community-specific information, ensuring anonymity and confidentiality where required.
Respect cultural protocols and local knowledge
Obtain permission before entering Indigenous lands or conducting research on culturally sensitive topics. Respect community boundaries and practices. Recognize and respect the unique knowledge systems and traditional ecological knowledge held by Indigenous communities. Collaborate to integrate indigenous insights into the research design, data interpretation, and analysis.
Communicate clearly and transparently
Use clear and jargon-free language when communicating with Indigenous communities. Ensure that all information, consent forms, and project documentation are accessible, culturally appropriate, and available in both English and Indigenous languages. Provide regular updates to the community, keeping them informed about the progress, findings, and any potential impacts of the research. Share information in community spaces, such as community centers or council meetings, using multiple communication channels to reach a broader audience.
Recognize and accommodate community protocols and timelines
Respect the community's sense of time and recognize that decision-making processes may take longer due to the importance placed on consensus-building. Be patient and flexible, allowing adequate time for community consultations and engagement. Familiarize yourself with the community's protocols and procedures for meetings, ceremonies, and other cultural activities. Seek guidance from community members to ensure that research activities align with cultural customs and traditions.
Consider community research priorities
Engage in active dialogue with community members to identify their research priorities related to landfill gas impacts. Prioritize community concerns and seek to address their questions and interests through the research project. Ensure that research activities are relevant and beneficial to the community. Collaboratively determine the research scope, objectives, and methodologies that align with community needs and interests.
Respect intellectual property and traditional knowledge
Obtain consent and respect protocols when accessing and utilizing Indigenous intellectual property or traditional knowledge. Seek permission from Knowledge Keepers and Elders before documenting or incorporating traditional knowledge into the research. Protect Indigenous intellectual property rights and ensure that any use of traditional knowledge or cultural materials is done with the community's explicit consent, acknowledging their ownership and providing appropriate attribution.
Facilitate knowledge exchange between academic researchers and Indigenous Knowledge Keepers, fostering a reciprocal learning environment that respects and values both Western and Indigenous knowledge systems.
Respect community decision-making processes
Understand that decision-making processes in Indigenous communities often involve community councils, elders, and knowledge holders. Seek guidance from community leaders on how decisions are made and ensure that research activities align with their protocols. Be prepared to adapt the research project based on community feedback and decisions. Collaborate with community members to develop strategies for resolving conflicts or addressing any concerns that may arise during the research process.