Our project

Abstract

Greenhouse gasses play a significant role in climate change, with carbon dioxide contributing to 79.7% of total emissions [1]. However, while CO2 is abundant, it is not the most potent greenhouse gas. Methane has 86 times the warming impact of CO2 over 20 years [2] yet comprising only 11.1% of emissions [1]. Its severe environmental impact contributes to the formation of ground-level ozone, which damages crops and poses significant health risks [3,4].

Thus, reducing methane emissions offers a more effective climate change strategy than focusing solely on CO2. Landfills are major methane sources, contributing to 17.1% of emissions in the United States [1]. Our project aims to address this issue by genetically modifying the soil bacterium Bacillus subtilis to produce enzymes that convert methane into compounds that boost soil fertility. This innovative approach reduces methane emissions while enhancing soil health.

(Image from source [1])

The Issue

There are several greenhouse gasses contributing to climate change, including nitrous oxide, carbon dioxide, and sulfur hexafluoride. Among these, methane plays a significant role, accounting for approximately 11.1% of all greenhouse gasses [1]. Although methane is less abundant than carbon dioxide, it is about 86 times more effective at trapping heat in the atmosphere, making even small reductions in methane emissions impactful [2].

(Image from Source [1])

The U.S. Environmental Protection Agency (EPA) identifies landfills as the third-largest source of human-caused methane emissions in the United States, responsible for about 17.1% of the total [1]. Landfills in the United States have released an estimated 119.8 million metric tons of carbon dioxide equivalent (MMTCO2e) of methane into the atmosphere in 2022 [5].

Methane's impact extends far beyond climate change, as its oxidation drives most tropospheric ozone formation. Ground-level ozone is linked to respiratory illnesses such as asthma and chronic obstructive pulmonary disease (COPD), leading to 365,000 deaths in 2019 alone [6]. Reducing methane emissions by 40% by 2030 could prevent approximately 180,000 deaths, 540,000 asthma-related emergency room visits, and 11,000 hospitalizations of elderly individuals annually [7]. Additionally, the reduction of methane-driven ozone formation could save 18 million tons of crops each year, preventing $5 billion in agricultural losses by 2030 [7].

Current Solutions

There are two types of landfills: open and closed. Open landfills are active sites where new waste is regularly dumped and compacted down to fill the landfill. These sites tend to leak significant amounts of methane because they aren't fully sealed, making containment more difficult. Closed landfills, on the other hand, are covered with dirt and often have pipe systems designed to capture and manage the methane produced. However, not all closed landfills have functional systems, and some lack them entirely.

(Open landfill, artwork by Meilin Hansen)

(Closed landfill, artwork by Meilin Hansen)

Methane is a highly flammable and hazardous gas, making its formation in landfills a major concern. Many modern landfills are required to keep methane concentrations below the lower flammability limit. To manage this, they use technology to capture and burn methane, a process known as flaring. Unfortunately, these systems are often inefficient and prone to leaks, requiring additional energy and surveillance. Moreover, flaring converts methane into carbon dioxide, further contributing to climate change. Additionally, this process can also produce nitrous oxide, another greenhouse gas. Despite the potential to generate energy from methane, many landfills lack the necessary technology due to high costs.

Our Project

At the Khan Lab School iGEM Team, we are developing a solution for methane emissions by using Bacillus subtilis, a naturally occurring soil bacterium that can easily be found in landfills.

Our engineered B. subtilis would act as a biofilter, absorbing and breaking down methane.

We realized that by synthetic biology techniques, we could genetically edit Bacillus subtilis to absorb methane from the surrounding air. While methanotrophic bacteria already exist, we chose Bacillus due to variants of Bacillus making up 95% of gram-positive soil bacteria populations [8].

To allow B. subtilis to metabolize methane, we start by inserting mini-smmo, an enzyme that oxidizes methane to methanol, with the eventual goal of converting methane into formaldehyde. This formaldehyde is converted into fructose via the RuMP cycle, which is naturally present in Bacillus. For this iGEM project, we focus on the first step, converting methane to methanol.

To insert a gene into Bacillus subtilis, we decided to create a construct where the two sides are homologous to the Bacillus genome. We then planned to synthesize these two end fragments from PDR110. We then planned to rely on homologous recombination, a process where the Bacillus automatically integrates DNA similar to its own genome into its genome, to insert our gene. If the transformation is successful, the resulting Bacillus is supposed to have spectinomycin resistance, and be unable to digest starch, due to a knocked out amylase gene.

How we selected our idea

Our original idea for this project was to genetically engineer ornamental tobacco plants via agrobacterium. These edits would allow for the expression of two enzymes, those being methane monooxygenase and nitrous oxide reductase. However, we realized that past teams had already tried to and other individuals in the scientific community have as well [9]. We decided to focus on methane monooxygenase, which many have tried before with little success. However, two articles were published recently that successfully expressed methane monooxygenase. One of them successfully expressed particulate methane monooxygenase(pMMO) in plants, but not successfully getting it to work [10]. Another successfully expressed a synthetically edited version of soluble methane monooxygenase(sMMO) that successfully was expressed and worked in E. coli [11]. We decided to try to see if the successfully expressed enzyme would also function in B. subtilis due to its large presence in soil bacterial populations.

Potential Impacts

Deployment

As previously discussed, our plan for deploying the genetically engineered bacteria involves integrating it into the soil used in landfills. This soil will be layered with waste and also applied on top as part of the biofilter, where the majority of methane is oxidized. Therefore, we consulted with experts to gather their feedback on this approach.

The project aims to significantly reduce methane emissions from landfills, which will not only help mitigate climate change but also reduce health issues caused by ground-level ozone. Methane is a potent greenhouse gas, and by targeting its emissions at landfills, our project directly addresses a major environmental concern. However, the potential applications extend far beyond landfills. Our system can also be implemented at other anaerobic methane-emitting sources such as manure ponds, dairy farms, and gas refineries, which are major contributors to methane emissions.

In comparison to current solutions, our approach has several advantages. The genetically engineered Bacillus subtilis strains expressing methane monooxygenase (MMO) show a higher efficacy in methane oxidation than existing methods. Furthermore, while conventional landfill gas systems still release carbon dioxide into the atmosphere, our project offers the additional benefit of carbon sequestration. This makes our solution not only an efficient methane mitigation strategy but also a carbon-negative approach, as it captures methane and helps store carbon in the ground.

The project could have widespread environmental benefits by applying this technology across various methane-emitting sites, positioning it as a superior alternative to existing methane capture technologies.

Improvements

Our project uses methane monooxygenase to turn methane into methanol. However, methanol is toxic to many bacteria, including those used in our project, which results in them being killed. We would mitigate this issue by adding a way to process methanol into formaldehyde, using the enzyme methanol dehydrogenase, which is processed via the RuMP cycle allowing the bacteria to turn it into food. In addition to this, extra safety measures would need to be added for the bacteria to be implemented into open environments, including a kill switch and environment exclusivity.

References

1. U.S. Environmental Protection Agency. (2024). Inventory of U.S. greenhouse gas emissions and sinks: 1990-2022 (EPA 430-R-24-004). https://www.epa.gov/ghgemissions/inventory-us-greenhouse-gas-emissions-and-sinks-1990-2022
2. CCAC. (n.d.). Methane. Climate and Clean Air Coalition. https://www.ccacoalition.org/short-lived-climate-pollutants/methane
3. Shindell, D. T. (2016), Crop yield changes induced by emissions of individual climate-altering pollutants, Earth’s Future, 4, 373–380, doi:10.1002/2016EF000377.
4. Zhou, X., Sampath, V., & Nadeau, K. C. (2024). Effect of air pollution on asthma. Annals of Allergy, Asthma & Immunology, 132(4), 426-432. https://doi.org/10.1016/j.anai.2024.01.017
5. “Frequent Questions about Landfill Gas | US EPA.” Environmental Protection Agency (EPA), https://www.epa.gov/lmop/frequent-questions-about-landfill-gas. Accessed 22 September 2024
6. Domingo, N. G. G., Fiore, A. M., Lamarque, J.-F., Kinney, P. L., Jiang, L., Gasparrini, A., Breitner, S., Lavigne, E., Madureira, J., Masselot, P., Pereira da Silva, S. N., Ng, C. F. S., Kyselý, J., Guo, Y., Tong, S., Kan, H., Urban, A., Orru, H., Maasikmets, M., ... Chen, K. (2024). Ozone-related acute excess mortality projected to increase in the absence of climate and air quality controls consistent with the Paris Agreement. One Earth, 7(2), 325–335. https://doi.org/10.1016/j.oneear.2023.12.008
7. Climate & Clean Air Coalition. (2021, March 22). Methane’s links to respiratory diseases strengthens the case for its rapid reduction. Climate & Clean Air Coalition. https://www.ccacoalition.org/news/methanes-links-respiratory-diseases-strengthens-case-its-rapid-reduction
8. Prashar P., Kapoor N., Sachdeva S. Rhizosphere: Its structure, bacterial diversity and significance. Rev. Environ. Sci. Biotechnol. 2013;13:63–77. doi: 10.1007/s11157-013-9317-z.
9. Wan S, Johnson AM, Altosaar I. Expression of nitrous oxide reductase from Pseudomonas stutzeri in transgenic tobacco roots using the root-specific rolD promoter from Agrobacterium rhizogenes. Ecol Evol. 2012 Feb;2(2):286-97. doi: 10.1002/ece3.74. PMID: 22423324; PMCID: PMC3298943.
10. Spatola Rossi, T., Tolmie, A.F., Nichol, T. et al. Recombinant expression and subcellular targeting of the particulate methane monooxygenase (pMMO) protein components in plants. Sci Rep 13, 15337 (2023). https://doi.org/10.1038/s41598-023-42224-9
11. Yu Y, Shi Y, Kwon YW, Choi Y, Kim Y, Na JG, Huh J, Lee J. A rationally designed miniature of soluble methane monooxygenase enables rapid and high-yield methanol production in Escherichia coli. Nat Commun. 2024 May 23;15(1):4399. doi: 10.1038/s41467-024-48671-w. PMID: 38782897; PMCID: PMC11116448.