Team:Wageningen UR/Background


iGEM Wageningen 2021

Background information

cow in fog

Our project focuses on reducing emissions of methane (CH4) and ammonia (NH3) from the cattle livestock sector. In this page we will guide you through the relevance of our project, the reasons why we chose it and the impact we expect it to have.


CH4 and NH3 are two gaseous compounds produced in large amounts from the agricultural sector. CH4 is a greenhouse gas that has a worryingly high global warming potential (GWP). In a time span of 100 years, methane is able to affect our planet warming 30 times more than carbon dioxide (CO2) [1]. Globally, CH4 emissions from the livestock sector (27%) are second only to CH4 emissions from the fossil fuel industry (33%), as shown in Figure 1 below from United Nations Economic Commission for Europe (UNECE) [1]. On the other hand NH3, a nitrogen compound, is a contributor to the formation of particulate matter. The emission and consequent deposition of forms of inorganic nitrogen is also responsible for loss of biodiversity and enhancement of greenhouse effect [2].

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Figure 1: Global methane (CH4) emission chart per source sector. Note the large contribution to the increase in emissions from the Fossil fuel and the Livestock sectors[1].

Nitrogen Crisis

Within Europe, the Netherlands is relatively the biggest emitter of nitrogen when considering the total amount of nitrogen produced per area of land (hectares). Quantitative data show that the Netherlands emits four times the average emission of nitrogen per hectares of other European countries, of which 60% is attributable to ammonia (NH3) [3]. NH3 originates primarily from livestock manure exposed to the atmosphere [4]. Within the livestock sector cattle is the biggest NH3 producer, accounting for the 49% of the total NH3 derived from livestock [3].


Given the great impact of nitrogen excess that the Netherlands was facing in the last decades and the need to comply to EU rules on acceptable nitrogen levels, the Dutch government ruled to reduce the nitrogen emission. This was done with a series of measures that were put in place from the 1980s and continue being updated in the current days. This had consequences on the population. Among the most affected are the farmers, who responded with protests.

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Protest of farmers that drove their tractors on the highway to The Hague on October 1st 2019. The picture was taken from this page on the BBC News website. For reading more about this protest event check out their article.

The protests took place also very close to us. The 7th of July 2021 a group of farmers came to Wageningen University and Research (WUR) campus with their tractors to protest against the government’s nitrogen policy. We were there to witness and document the protest as you can see in the image slider below, where buildings of WUR campus are visible.

No farmers, no food, no future!

These were the lines on one of the signs carried by the protesting farmers. This is the most recent event triggered by the so-called Dutch Nitrogen Crisis (DNC). The DNC is the environmental and societal crisis to which our project, Cattlelyst, worked to find a solution with the help of synthetic biology.

For more details on the measures and consequences check out the timeline of events of the Dutch Nitrogen Crisis

Go to the timeline of the Dutch Nitrogen Crisis

Environmental background

Why is nitrogen excess such a threat for the environment? Where does it come from? These were questions we asked ourselves. We discovered that the agricultural sector was the largest contributor to the issue (see Figure 2), thus, the consequences were impacting farmers particularly. The cattle livestock industry is also responsible for CH4 emissions, since the animals innately produce this greenhouse gas. From there the idea behind Cattlelyst was born. The project of iGEM WUR 2021 was then developed to reduce the emissions of both NH3 and CH4. Check below for more information on the sources and consequences of emissions of these two gases.

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Figure 2:Ammonia emission per sector. Adapted from data shared by the European Environmental Agency (EEA) on the contribution of different sectors to ammonia emission in 2011 [5]. Note the strikingly high contribution made by the agricultural sector to emissions of NH3.

Approximately 78% of the air consists of nitrogen, an odorless and harmless gas. However, nitrogen can react with other elements to form reactive compounds. The most important reactive nitrogen forms are ammonia (NH3) and nitrogen oxides (NO and NO2, from now referred as NOx) [6]. Emissions of NH3 and NOx contribute to climate change, biodiversity loss, acid rain, soil acidification and degradation of ecosystems [3, 7].


The planetary nitrogen cycle is distorted due to the excessive production of anthropogenic nitrogen. Primary causes for distortion of the nitrogen cycle are large-scale manufacturing of fertilizer and cultivation of leguminous crops [8]. In intensive livestock production, the use of feed produced using fertilizers with additional oxidized nitrogen compounds disrupts the natural nitrogen cycle. To grasp the impact of these sectors, think that these human-driven processes have generated more reactive nitrogen species than all Earth terrestrial processes combined [8]. A major part of the excess nitrogen species from fertilizers and legumes is released in the atmosphere in the form of ammonia produced by livestock’s manure [9].

Methane (CH4) emission is a pressing environmental issue. Levels of greenhouse gasses (GHGs) have increased since the industrial revolution, resulting in an enhanced greenhouse effect [10]. CH4 is one of the most important GHGs next to CO2 and nitrous oxide [10, 11]. However, CH4 has a greater global warming potential (GWP) than carbon dioxide. CH4 traps 84 times more heat than CO2 over the first two decades after it is released into the air [12]. As we introduced earlier, the GWP of CH4 over 100 years is approximately 30 times larger than the GWP of CO2 over the same time span [1, 10, 13].


Although the livestock sector has experienced growth in the last decade in the Netherlands, its GHG emissions have decreased in the last 30 years. This decrease is mainly due to an increased efficiency in livestock farming. Nevertheless, more than two thirds of all methane emissions in the Netherlands still originate from the agricultural sector today [14-16]. Within the livestock sector, cattle are the biggest producer of CH4: 44% of the livestock CH4 emissions can be attributed to cattle. Approximately, 90% of GHG emissions in livestock farming comes from the digestion of food by livestock and from storage of manure due to the activity of certain microorganisms, the methanogens. Methanogens are archaea producing methane that among other places, inhabit the cows digestive system [10, 15].

As mentioned earlier, both gasses have negative environmental effects, some of which are summarized in Figure 3.

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Figure 3: 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.

The ammonia from the manure partly evaporates and is released into the atmosphere. Through precipitation, the ammonia is then deposited in the soil over large areas and ends up in groundwater. Excessive nitrogen is a global environmental problem that exceeds the national borders. The Netherlands, having extensive agricultural and livestock sectors, faces the environmental consequences of ammonia excess more heavily [17]. In the Netherlands, dramatic issues are emerging: the soil becomes enriched with nutrients, causing plants that normally thrive in nutrient-poor soil to disappear. This in turn leads to a loss of biodiversity, which is especially a problem in natural reserves such as the dunes and heather fields. Out of a total of 161 Dutch nature reserves protected under EU guidelines, 118 have nitrogen deposition levels that exceed the critical deposition value [18].


Furthermore, ammonia also plays a significant role in acid deposition. ‘Acid rain’ has been recognized as an issue in Europe and North America since the late 1960s and in Asia since the 1980s [19, 20]. In the Netherlands, the components of acid rain (ammonia and sulfur dioxide) have been known since the 1960s [17, 21]. Now, however, more is known about the negative effects that ammonia has on the environment. The ammonia deposited in the soil is converted into nitric acid (HNO3). When this acid leaches into deeper soil layers it takes along cations like calcium and potassium. As a consequence, the acid neutralizing capacity of the soil decreases and the soil slowly acidifies. This is not only detrimental for plants, but also animals can get calcium deficiencies due to the demineralization of the soil. There have been reports of birds born with broken legs due to calcium deficiencies [22].


As of methane, CH4 from the cow’s breath is one of the largest and hardest to control source of methane. It constitutes 32% of the total non-CO2 emissions from agriculture in 2005 [23]. Cattle accounts for about 2.5 Gt CO2 equivalents of enteric methane annually [12].

Worldwide emissions and consequences

The nitrogen problem is a cause for worldwide concern. We will introduce you to some countries in which this concern is being raised with particular emphasis. In this page we will also have a look at how policy is dealing with the excess of nitrogen outside the case of the Netherlands.

Click here to read more

Similarly, methane emissions from livestock sector are a great source of concern in many countries. The challenges surrounding methane did not get the title of a “societal crisis” as the nitrogen problems did in the Netherlands, but how is the situation in other countries? Here you will find information about worldwide emissions of this GHG.

Click here to read more

Existing knowledge

We, iGEM WUR 2021, chose Cattlelyst as our project because we want to contribute to the reduction of the emission of two gasses that are threatening the environment: methane (CH4) and ammonia (NH3). By doing so, we also aim at providing a technology that could facilitate the farmers in complying to measures aiming at the safety of the environment. Cattlelyst, a biofiltration system for both CH4 and NH3, is a relevant technology in the Netherlands as well as worldwide. Technological development is a process and therefore, as with all innovations, Cattlelyst is also built on giants’ shoulders. We rely on existing knowledge: existing biofiltration systems and microorganisms that provide the biological systems we harnessed with the help of synthetic biology.

Filters

Because the necessity of reducing NH3 and CH4 emissions has been recognized for many years, several possible solutions have been developed, mostly in the form of air filtration systems.

Ammonia filters

Currently, chemical air treatment and biofiltration are the most often used solutions for reducing NH3 in the air from livestock farms. Although these systems can reach high efficiencies depending on the type of equipment, they have evident disadvantages.

Chemical scrubbers rely on dissolving the NH3 in liquid and precipitating it to a salt that can be discharged. To do so, the pH is lowered with the help of sulfuric acid (H2SO4) [1]. This means such systems should be handled carefully, as this strong acid can be dangerous [2]. The discharge water that is produced presents two additional disadvantages. The first is that the produced ammonium sulphate salt ((NH4)2SO4) can present hazards, as toxic hydrogen sulphide (H2S) is produced upon contact with manure. Second, this also means that the produced waste stream needs to be taken care of afterwards, which provides extra work and will cost energy [1].

Another type of solution is biofiltration, where microbes are employed to perform the air treatment. These exist in three types of reactors (bio-scrubbers, biofilters and biotrickling filters) that all take up NH3 from an air or water phase [3,4]. Nitrifying bacteria then oxidize NH3 to nitrate (NO3-) via nitrification, or even to nitrogen gas (N2) by subsequent denitrification performed by another bacterium [1,5]. Flaws of these systems are that nitrification-systems often also produce nitrogen-rich waste streams that need to be processed and both run the risk of producing the toxic nitrite (NO2-) or the very potent greenhouse gas nitrous oxide (N2O), which is undesirable [6,7]. Moreover, denitrification in particular requires additional expensive substrates to supply electrons, methanol, acetic acid and ethanol [8].

Methane filters

For CH4, chemical cleaning or energy recovery technologies are generally insufficient for its abatement. Emissions from the rumen and manure of livestock diffuse in the air in too low concentrations to be efficiently treated and removed [9]. Methanotrophic-based biofilters could be an environmentally friendly solution. Immobilized methanotrophic bacteria (methanotrophs) perform CH4 oxidation, which is more efficient in gas-phase reactions compared to aqueous phase reactions [10]. Even though methanotrophic-based biofilters are already used to combat CH4 emissions from landfills [11,12], they are not yet implemented in livestock farms. CH4 biofilters need a relatively large size to be able to work with existing methanotrophs and CH4 concentrations. Alternatively, better methanotrophs need to be found or designed, or CH4 concentrations in airflows need to increase to decrease the reactor size [13,14].

Combining CH4 and NH3 emission reduction into one system has not been successfully done yet. Efforts that have been made to this end in the form of biofiltration reactors either show low reduction of either of the two gases, or also produce undesirable waste products such as nitrous oxide [5, 15, 16]. More recently, microorganisms capable of heterotrophic nitrification-aerobic denitrification (HNAD) were discovered [17]. With this mechanism, bacteria can convert NH3 into the more desired N2. This lead to the idea of generating microbial communities where methanotrophs and nitrifiers using HNAD to overcome issues in tackling the emission of both CH4 and NH3 at the same time. These cocultures might also work effectively in biofilters and therefore are a promising new, but unused, approach for reducing both NH3 and CH4 emissions. This is where Cattlelyst comes in!

Microorganisms

For Cattlelyst, we want to realize the biological conversion of both methane (CH4) and ammonia (NH3) to less harmful forms in our biofilter. We considered our biofilter to be filled with either a bacterial mono- or coculture (see our Engineering page for details on the two approaches). As there is no accessible organism known that is able to convert both gasses simultaneously, we start by exploring two different types of bacteria: methanotrophs and ammoniatrophs.

Ammoniatrophs
Ammoniatrophs
Methanotroph
Methanotrophs

“Methanotrophs” is a scientifically accepted term to indicate microorganisms that consume methane as the carbon source for energy. How about the term “ammoniatroph”? The Cattlelyst team came up with this name to indicate microorganisms that convert ammonia into dinitrogen gas. You will encounter this unofficial term multiple times in our wiki and, to make it clearer, below you can find more information on how this conversion is performed by bacteria in nature. This is followed by background information about methanotrophs and methane oxidating pathways.

Ammoniatrophs

Conventional microbial ammonia (NH3) removal consists of two steps [18]. The first step is nitrification, which is the conversion of NH3 to nitrate (NO3-) or nitrite (NO2-) by bacteria under oxygen-rich (aerobic) conditions. Nitrification is followed by denitrification, the conversion of NO3- or NO2- to unreactive dinitrogen (N2), by bacteria under anaerobic conditions (without oxygen). The overall procedure is very complex, both due (1) to the discrepancy between conversion rates of nitrification and denitrification, and (2) the necessity for different modes of operation for nitrifiers and denitrifiers. To elaborate (2), nitrifiers require an aerobic process while denitrifiers require an anaerobic process and carbon supply [18], [19]. Because of this, Simultaneous Nitrification and Denitrification (SND) has gained interest over the past couple of decades. SND implies that nitrification and denitrification occur concurrently in one reaction vessel under aerobic conditions [20]. This is of interest for Cattlelyst since we want to convert NH3 into N2 in stalls were oxygen is present.

The exact mechanisms of SND are not completely understood at the moment, although it is suggested that conventional nitrification is actually linked to an oxygen-proof denitrification machinery. Several bacteria capable of coupling nitrification and denitrification in aerobic conditions have been isolated in the past decades, such as Pseudomonas stutzeri or Paracoccus denitrificans. This coupled process is referred to as heterotrophic nitrification aerobic denitrification (HNAD) [18].

HNAD starts with the oxidation of NH3 to hydroxylamine (NH2OH) facilitated by ammonia monooxygenase (AMO) (Figure 3). NH2OH is subsequently oxidized to nitric oxide (NO) by hydroxylamine oxidoreductase (HAO). These two enzymatic steps overlap with the classical nitrification pathway employed by autotrophic nitrifiers, which get their energy out of the ammonia oxidation. ] It his hypothesized that under aerobic conditions NO is oxidized to NO2- and NO3- [21]. The aforementioned oxidation steps are followed by the reduction of NO3- to NO2- by nitrate reductase (NAP) and NO2- to NO by nitrite reductase (NIR). NO is then quickly reduced to nitrous oxide (N2O) by enzyme nitric oxide reductase (NOR). Lastly, N2O is reduced to nitrogen gas (N2) by nitrous oxide reductase (N2OR). The three step nitrite reduction to nitrogen gas overlaps with the classical denitrification pathway utilized by, e.g. P. denitrificans [22-24].

More information about this pathway can be found here.

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Figure 4: Illustration of the pathway for the conversion of ammonia. The orange boxes indicate the enzymes. AMO = ammonia monooxygenase, HAO = Hydroxylamine oxidoreductase, NAP = periplasmic nitrate reductase, NIR = nitrite reductase, NOR = nitric oxide reductase, NOS = nitrous oxide reductase.

Methanotrophs

Methyloccocus capsulatus is a well-studied natural type I methanotroph [25]. We researched it for this project because of its obligate methanotrophy and its nitrogen metabolism [26, 27]. It grows on gaseous methane by converting it into soluble methanol, and subsequently converting this into formaldehyde, formate and finally biomass or carbon dioxide. The pathway is represented in Figure 4. M. capsulatus strain Bath has two enzymes converting methane to methanol [28]. A soluble methane monooxygenase (sMMO) and a membrane-bound, particulate monooxygenase (pMMO), that widely differ in sequence and cofactors but can perform the same conversion. pMMO is more common and has a higher affinity for methane and oxygen than sMMO, but needs relatively high copper concentrations to function [29]. sMMO enzyme has a relatively low efficiency compared to the pMMO. As pMMO is membrane bound, it is more efficient at lower methane concentrations[30]. Both have been expressed heterologously in Escherichia coli [31-33] and by iGEM Braunschweig 2014, that expressed sMMO. As shown in Figure 4 the second step of the pathway is the conversion of methanol into formaldehyde, and this in M. capsulatus is performed by methanol dehydrogenase (MDH, not represented in Figure 4. Then formate is produced and this can follow an assimilatory pathway for biomass production (i.e. growth) or follows further oxidative steps leading to production of CO2. In natural type I methanotrophs (to whom M. capsulatus belong) the conversion of formaldehyde/formate into biomass works via the ribulose monophosphate pathway [29]. information about this pathway can be found here.

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Figure 5: Methane conversion pathway. Methane is converted to methanol via soluble or particulate methane monooxygenase (sMMO/pMMO). Methanol is subsequently converted to formaldehyde via methanoldehydrogenase (MDH). Formaldehyde/formate can be incorporated into biomass or converted into CO2.

If you’ve come so fare you now have all the background information on our project. Check out the Engineering page for details on how we incorporated this knowledge to design Cattlelyst and make sure to go to the Overview page for getting to know our results!

  • References
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    References on the societal and environmental background:
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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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