Team:Wageningen UR/Description


iGEM Wageningen 2021

DESCRIPTION

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Cattlelyst

Reducing ammonia and methane emissions from cattle farms

Feeding our ever-growing world population comes with a heavy burden to the environment. The agricultural sector is one of the biggest contributors to ammonia and methane emissions in the Netherlands, mainly originating from livestock farming [1]–[4]. Ammonia emissions cause acid deposition, which changes the soil chemistry and water quality. This results in a loss of biodiversity, weakening ecosystems such as the heather fields in the Netherlands [1], [5]. Methane is a greenhouse gas much more potent than CO2, and methane emissions greatly accelerate global warming [6], [7]. Cattle are the biggest producers of both compounds of all livestock in the Netherlands. Whereas ammonia originates primarily from their manure, methane is both released from manure and produced by cattle during digestion, releasing it directly through their breath [3], [4].

Schematic illustration displaying acid deposition and dlimate warming by ammonia and methane, respectively
Figure 1: Schematic illustration displaying the different ways in which cattle stall emissions ammonia and methane affect the environment: (1) ammonia changes soil chemistry by acid deposition, while (2) methane enhances the global greenhouse effect.

Approximately 40% of protected areas worldwide are at risk of biodiversity loss due to excessive reactive nitrogen [8]. In the Netherlands, this is even higher: 73% of nature reserves are under threat [9]. Within Europe, the Netherlands has the highest nitrogen-emission density. Ammonia makes up 60% of total Dutch nitrogen emissions of which more than 90% originates from the livestock sector [10]. Since 2019, a series of court verdicts and rulings by the Dutch Council of State forced the government to act promptly against the country’s excess ammonia emissions [2], [11]. Many governmental parties proposed measures like halving the Dutch livestock population and decreasing the amount of protein fed to livestock [8]. These measures have caused friction between the government and farmers, as their implementation would cause many farmers to go out of business. As a result, thousands of farmers rallied in the streets, in one instance even flooding the center of the Dutch administrative capital The Hague in protest [12].


Although methane emissions did not receive quite the same amount of media attention as the nitrogen crisis in The Netherlands, they are a significant contributor to global warming. Despite the economic and industrial downturn caused by the global pandemic, the largest annual increase of methane measured since 1983 was in 2020 [13]. The European Commission has proposed a European Climate Law to reduce greenhouse gas (GHG) emissions by at least 55% by 2030 compared to 1990 levels [14]. To achieve this goal, The Netherlands would need to cut its GHG emissions by 40% [15].


It is clear we need a solution. One that reduces both methane and ammonia emissions of the livestock sector.

Read more about our Project Background

Biofiltration

Cattlelyst is an innovative solution that deals with both of the harmful emissions of the cattle industry, which have contributed to complex environmental and political problems. This solution consists of a biofilter containing genetically modified microorganisms, which convert ammonia to innocuous dinitrogen (N2) and methane to carbon-neutral CO2. As such, the Cattlelyst biofilter applies Synthetic Biology. Figure 2 schematically shows what the system surrounding the biofilter would look like. Methane-rich air from the cattle stalls will be captured by the “hood system”, inspired by the cow-friendly hood sampler described by Wu [12]. This airflow is combined with the ammonia-rich air from the manure pits and pushed through the biofilter. This way, the Cattlelyst biofilter provides an elegant solution for both types of harmful gasses emitted by the livestock sector, allowing farmers to continue feeding the world while nature and the climate remain unharmed.

Schematic farm and application of cattlelyst
Figure 2. Scheme displaying a farm in which the Cattlelyst biofilter is applied: exhaust air coming from the stall cubicles and manure pit pass though the biofilter, where genetically modified bacteria convert ammonia and methane into CO2 and N2.

Our biofilter has an environment with unique conditions, so it is unlikely that naturally occurring organisms will realize the optimal conversion of ammonia and methane. To make sure our biofilter is as efficient as possible, we chose to create our own synthetic organisms to fit the unique environment of the biofilter. These microorganisms should possess several key characteristics to fulfill their function. The Cattlelyst microbes should be able to grow on ammonia and methane and convert them to N2 and CO2 under aerobic conditions. As our microorganisms will be genetically modified (GMOs), they should also be unable to escape the confines of the biofilter. Therefore, they should ideally contain intrinsic safety mechanisms that prevent them from surviving in the outside environment. We divided these fundamental traits into three separate pillars that we worked on extensively both computationally and in the lab. We attempted to implement these pillars into a co-culture of two organisms.

Schematic view inside of the Cattlelyst biofilter
Figure 3. Schematic overview of the inside of the Cattlelyst biofilter. Polluted air coming from the cattle stall passes though the biofilter, where genetically modified bacteria convert ammonia and methane into CO2 and N2.
The three pillars of Cattlelyst
Ammonia icon
Ammonia

Recently, microorganisms have been discovered that are able to nitrify and denitrify simultaneously, meaning they can convert ammonia all the way to N2 by themselves. However, little is known about the cellular mechanisms behind this conversion. Therefore, we decided to develop a dynamic metabolic model to increase our conceptual understanding of this phenomenon. We applied this knowledge in the design of our lab experiments, where we worked on engineering the entire nitrification and denitrification pathways in one organism. We tested multiple enzymes originating from different native (de)nitrifying organisms to achieve the maximum conversion rate of ammonia to N2 in Pseudomonas putida, our non-denitrifying chassis organism [16]. One of the intermediates in the introduced pathway is nitrous oxide, which has a global warming potential 300 times that of CO2 over 100 years [7], [17]. It was of paramount importance to prevent any accumulation of this compound. We therefore limited nitrous oxide production by redirecting the electron flux of the denitrification machinery by means of CRISPR interference.

Read more on ammonia removal

Methane icon
Methane

In nature, conversion of methane to CO2 is carried out by methanotrophs, that use methane as their carbon- and energy source. However, these organisms often grow very slowly and are difficult to engineer, making them unsuitable for the Cattlelyst biofilter. Fortunately, Chen et al. [18] recently modified a strain of Escherichia coli to grow on methanol and produce CO2. By adding the conversion step of methane to methanol, we could create a synthetic methanotroph that is perfect for our biofilter. To achieve this, just one type of enzyme is required: methane monooxygenase.

Read more on methane oxidation

Safety icon
Safety

Ultimately, the Cattlelyst biofilter needs to be safe for implementation in real-world situations. Therefore, we designed and modeled a multi-layered safety system based on conditions that are unique to the biofilter. The first safety mechanism relies on the high concentration of methane coming from the cattle stall, and the other on the high density of bacteria inside the biofilter. If our microorganisms should escape, the absence of either of those conditions would trigger a kill-switch, preventing them from surviving outside of the biofilter. A third cross-feeding mechanism was added to prevent individual microbes from the microbial culture of the biofilter to survive when escaping alone.

Read more on safety by design

To summarize, we engineered engineered E. coli to convert methane into CO2 and P. putida to convert ammonia into N2. Our two bacteria are made to grow stably together in a co-culture and our three-layered safety system makes sure that they can grow exclusively in the specific conditions of the biofilter. To learn more about the process of developing Cattlelyst, visit our Engineering page. If you want to know more about the final outcomes of our project, visit the Overview page.

Ammonia icon
Ammonia

Recently, microorganisms have been discovered that are able to nitrify and denitrify simultaneously, meaning they can convert ammonia all the way to N2 by themselves. However, little is known about the cellular mechanisms behind this conversion. Therefore, we decided to develop a dynamic metabolic model to increase our conceptual understanding of this phenomenon. We applied this knowledge in the design of our lab experiments, where we worked on engineering the entire nitrification and denitrification pathways in one organism. We tested multiple enzymes originating from different native (de)nitrifying organisms to achieve the maximum conversion rate of ammonia to N2 in Pseudomonas putida, our non-denitrifying chassis organism [16]. One of the intermediates in the introduced pathway is nitrous oxide, which has a global warming potential 300 times that of CO2 over 100 years [7], [17]. It was of paramount importance to prevent any accumulation of this compound. We therefore limited nitrous oxide production by redirecting the electron flux of the denitrification machinery by means of CRISPR interference.

Read more on ammonia removal

Methane

In nature, conversion of methane to CO2 is carried out by methanotrophs, that use methane as their carbon- and energy source. However, these organisms often grow very slowly and are difficult to engineer, making them unsuitable for the Cattlelyst biofilter. Fortunately, Chen et al. [18] recently modified a strain of Escherichia coli to grow on methanol and produce CO2. By adding the conversion step of methane to methanol, we could create a synthetic methanotroph that is perfect for our biofilter. To achieve this, just one type of enzyme is required: methane monooxygenase.

Read more on methane oxidation

Methane icon
Safety icon
Safety

Ultimately, the Cattlelyst biofilter needs to be safe for implementation in real-world situations. Therefore, we designed and modeled a multi-layered safety system based on conditions that are unique to the biofilter. The first safety mechanism relies on the high concentration of methane coming from the cattle stall, and the other on the high density of bacteria inside the biofilter. If our microorganisms should escape, the absence of either of those conditions would trigger a kill-switch, preventing them from surviving outside of the biofilter. A third cross-feeding mechanism was added to prevent individual microbes from the microbial culture of the biofilter to survive when escaping alone.

Read more on safety by design

To summarize, we engineered engineered E. coli to convert methane into CO2 and P. putida to convert ammonia into N2. Our two bacteria are made to grow stably together in a co-culture and our three-layered safety system makes sure that they can grow exclusively in the specific conditions of the biofilter. To learn more about the process of developing Cattlelyst, visit our Engineering page. If you want to know more about the final outcomes of our project, visit the Overview page.

  • References
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    1. PBL, Zure regen, een analyse van dertig jaar verzuringsproblematiek in Nederland. Bilthoven: PBL publicatie, 2010.
    2. A. Sikkema, “The nitrogen problem in five questions,” Resource, WUR, 2019.
    3. RVO, “De Nederlandse landbouw en het klimaat,” 2016.
    4. CLO, “Ammoniakemissie door de land- en tuinbouw, 1990-2017,” 2019. [Online]. Available: https://www.clo.nl/indicatoren/nl0101-ammoniakemissie-door-de-land--en-tuinbouw. [Accessed: 16-Sep-2020].
    5. RIVM, “Over de uitspraak op 29 mei 2019: Programma Aanpak Stikstof (PAS) & beweiden en bemesten,” 2019. [Online]. Available: https://www.aanpakstikstof.nl/achtergrond/vragen-en-antwoorden/uitspraak-29-mei-2019-programma-aanpak-stikstof-beweiden-en-bemesten. [Accessed: 10-Oct-2020].
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    10. TNO, “Factsheet Emissies en depositie van stikstof in Nederland,” pp. 1–16, 2019.
    11. RIVM, “Nitrogen and PFAS suddenly big societal issues in the Netherlands,” 2020. [Online]. Available: https://www.rivm.nl/en/newsletter/content/2020/issue1/nitrogen-pfas-in-NL. [Accessed: 07-Sep-2020].
    12. A. J. A. Aarnink, W. J. M. Landman, R. W. Melse, Y. Zhao, J. P. M. Ploegaert, and T. T. T. Huynh, “Scrubber capabilities to remove airborne microorganisms and other aerial pollutants from the exhaust air of animal houses,” Trans. ASABE, vol. 54, no. 5, pp. 1921–1930, 2011.
    13. NOAA Research, “Despite pandemic shutdowns, carbon dioxide and methane surged in 2020 ,” 07-Apr-2021. [Online]. Available: https://research.noaa.gov/article/ArtMID/587/ArticleID/2742/Despite-pandemic-shutdowns-carbon-dioxide-and-methane-surged-in-2020. [Accessed: 23-Apr-2021].
    14. European Commission, “Impact Assessment, accompanying Communication ’Stepping up Europe’s 2030 climate ambition - Investing in a climate-neutral future for the benefit of our people - part 1/2,” 2020.
    15. European Parliament and Council of the European Union, “Regulation (EU) 2018/842 of the European Parliamanet and the Council of 30 May 2018,” Off. J. Eur. Union, pp. 26–42, 2018.
    16. M. Martin-Pascual et al., “A navigation guide of synthetic biology tools for Pseudomonas putida,” 2021.
    17. UNFCCC, “Global Warming Potentials (IPCC Second Assessment Report).” [Online]. Available: https://unfccc.int/process/transparency-and-reporting/greenhouse-gas-data/greenhouse-gas-data-unfccc/global-warming-potentials. [Accessed: 08-Sep-2020].
    18. F. Y. H. Chen, H. W. Jung, C. Y. Tsuei, and J. C. Liao, “Converting Escherichia coli to a Synthetic Methylotroph Growing Solely on Methanol,” Cell, vol. 182, no. 4, pp. 933-946.e14, 2020.
About Cattlelyst

Cattlelyst is the name of the iGEM 2021 WUR team. Our name is a mix of 1) our loyal furry friends, cattle, and 2) catalyst, which is something that increases the rate of a reaction. We are developing “the something” that converts the detrimental gaseous emissions of cattle, hence our name Cattlelyst.

Are you curious about our journey? We have written about our adventures in our blog, which you can find here:

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