iGEM Wageningen 2021

Implementation

The iGEM 2021 team of Wageningen University and Research (WUR) developed Cattlelyst, a biofilter for reduction of both methane and ammonia emissions from cattle stalls. This air filtration systems provides a solution to two worldwide needs:

the reduction of nitrogen emission and deposition,
the minimization of greenhouse gas (e.g. methane) release into the atmosphere.

The solution would be used directly by farmers, especially cattle farmers, since cattle are the largest contributor of these harmful emissions [1–4]. Our biofilter will provide a way of reducing the environmental footprint of this large industry. Moreover, the usage of Cattlelyst is likely going to be favourably accepted by the Dutch government. The Dutch government has taken strict measurements in the past 30 years in order to preserve the environment. From talking to Dr.Ir Karin Groenestein, coordinator of the section of the project “Klimaatenveloppe“ (Climate Envelope) on methane emission from the livestock sector, and by getting in touch with the Groene Groeiers network, we had direct confirmation that the effort put in the development of solutions is highly appreciated and promoted.

Experimental efforts to show a proof-of-principle for our proposed biofilter lead to promising results. Our research on the co-culture approach for example, showed that re-routing of the nitrogen pathway for the conversion of ammonia into nitrogen gas was successful and did not lead to the accumulation of toxic compounds.

But, to take Cattlelyst from the lab all the way to the farm, some questions were still left: How will these engineered microorganisms be employed in our biofilter? What will Cattlelyst look like?

In this page you’ll find information on how we envision our SynBio technology to be applied. This implementation strategy is based on investigations on several levels:

Biofilter design:

we designed and modelled our biofilter after consulting experts and we estimated the annual costs associated with installing and using it;

Compliance to regulation

we investigated the current Dutch and European policies on the usage of solutions involving engineered microorganisms;

Alternative applications

we explored in which other contexts our biofilter could be applied in addition to cattle sheds

Cattlelyst Biofilter Design

                Allow us to guide you through our biofilter design. Figure 1 is a schematic representation of our solution. It shows the stable with a cow standing in what is called a cubicle. The air coming from the cow’s breath while ruminating is collected by existing systems such as a hood system (see Figure 2 for details on the hood system developed at WUR [5]).  The collection of this air flow is key to our design since most of cattle methane emissions come from enteric fermentation [6]. Without a collection system the methane-rich air would escape from the stall and add to the green-house effect. Globally, CH4 from enteric fermentation constitutes 32% of the total non-CO2 emissions from agriculture in 2005 [7]. This sector produces about 3.3 Gt CO2 equivalents of enteric methane annually and cattle account for 77% of these emissions [8]. 
            
                You will also notice a second airflow from underneath the floor, where the manure pit is. This is where cow manure gets collected from within the stable. Methane can also be released from the activity of methanogenic bacteria present in the faeces. Therefore, this air flow will channel the methane coming from the manure to our biofilter, although it only contributes to 4% of the CH4 emissions from cattle [9]. More importantly, the air flow from underneath the floor is essential to tackle the second objective of our project: reducing ammonia emissions! Ammonia is, in fact, produced when urine and faecal enzymes in the manure come in contact [10]. This happens often in stables, which leads to high ammonia concentrations just above the pit. For these reasons our biofilter will be placed where the two gas flows converge, allowing it to capture the two pollutants simultaneously.
            
            Figure  1: Schematic of Cattlelyst’s biofilter applied to a cattle stable. The airflows are indicated for the collection of enteric methane from above and ammonia (and methane) from the underneath the stable floor (the manure pit). The biofilter is indicated in orange. 
      
            Figure  1: Schematic of Cattlelyst’s biofilter applied to a cattle stable. The airflows are indicated for the collection of enteric methane from above and ammonia (and methane) from the underneath the stable floor (the manure pit). The biofilter is indicated in orange.  
      
            Figure  1: Schematic of Cattlelyst’s biofilter applied to a cattle stable. The airflows are indicated for the collection of enteric methane from above and ammonia (and methane) from the underneath the stable floor (the manure pit). The biofilter is indicated in orange.

Pollutants entering the biofilter in the gas phase are not immediately available to the microorganisms in the biofilter, as they need transport steps over several phases for this to occur [11]. Biofilters essentially are microbial reactors containing a filter bed medium. This medium can be described as a layer made of many solid particles covered by microbial biofilm. This biofilm is covered in a thin layer of water [12]. Therefore, methane and ammonia need to be transported from the gas, into the liquid and biofilm phases subsequently. The biofilter and transport steps were combined in a model for a conventional biofilter. Our assumptions and design of the model can be found at the link below.

Click here for reading more about the biofilter model and the case study

What did we learn from modelling our Biofilter?

Case study

This model helped us understand the impact of the Cattlelyst biofilter by estimating the performance, size and costs of the reactor. As a case study, we used a cattle farm with 100 milking cows, average herd size in the Netherlands [13], comparing a system with and without a hood system.

The study showed that relatively large reactor sizes (10³ - 10⁴ m³) are needed to accommodate the low pollutant concentrations and high gas flow rates needed in the cattle stall. Nevertheless, the size of the biofilter with hood system is at least five times smaller than without. The model also revealed that the biofilter bottleneck lays in the transport of methane into the biofilter. Most performance measures are highly or only influenced by the initial concentration of methane, which is therefore the most limiting compound for most aspects of the biofilter.

As the model showed a clear benefit from capturing methane-rich air produced by enteric emissions by gas collection systems, we explored the various options currently available. At the WUR, the department of Farm Technology is working on the development of the hood system (Figure 2). PhD student Cécile Levrault showed us how the system is used for monitoring enteric methane emissions by collecting the air from the cubicles, using a sensor to detect and activate the hood when the cow is laying in the cubicle.

Figure  2: Hood system developed by WUR Farm Technology department. On the left, a schematic representation of the system. The hood and the air pumping system are in grey. On the right, a picture we took from the experimental farm where WUR is using the system for measurements. The metal structure delimits the cubicle. The plastic barriers are what makes the hood. The pipes through which the methane-rich air is pumped are the ones visible at the ceiling.

Figure  2: Hood system developed by WUR Farm Technology department. Top: a schematic representation of the system. The hood and the air pumping system are in grey. Bottom: a picture we took from the experimental farm where WUR is using the system for measurements. The metal structure delimits the cubicle. The plastic barriers are what makes the hood. The pipes through which the methane-rich air is pumped are the ones visible at the ceiling.

Click here to read more about our human practices efforts

Cécile and colleagues told us that cows are not bothered by the hood and ventilation. In fact, they observed that cows even seem to prefer cubicles with the hood over the ones without. Our project can nicely make use of this system, giving it an applied function. 
The current costs of installing a hood system are quite high due to the pricing for sensors, installation and stall restructuring. It is additionally laborious to build and requires tailor-made designs for each stall. These aspects need to be improved if the design of the hood is adapted for its practical application, rather than using it exclusively for monitoring.  
According to Cécile there is also room to improve the system for the implementation of methane reduction by increasing methane uptake. 

Talking to stakeholders, we realized that other collection systems could also be coupled to our biofilter. Cattlelyst could for instance make use of manure bags [14]: containers in which the manure is stored. In these, digestion from microorganisms leads to methane accumulation until it could be redirected to the biofilter. However, this has the disadvantage of missing the vast majority of methane coming from the breath of cows. The biofilter could also be built directly in the manure storage, which is usually a silo separated from the stall (and is different from a manure pit, which is built below the stall). In here, high concentrations of ammonia and methane are reached. However, this storage type is usually open to avoid the risk of explosion due to the high concentrations, therefore these storages will need to be adapted for the usage of the biofilter, without running the risk of reaching critical concentrations. Additionally, enteric emission would be missed if Cattlelyst was applied in this location.

The usage of the hood system is the way of implementing Cattlelyst that would give the most environmental benefits as it removes the largest portion of methane emissions. Therefore, we did the cost analysis of our system while considering the conditions (in term of concentration and volumes) that are expected when combining Cattlelyst with the hood system.

With an average biofilter size of 104 m³, as proposed by the previously mentioned model, the total annualized costs for a stall of 100 cows are approximately $1.2M or €1,0M per year. This amounts to $12K or €11K per cow per year. The total annualized costs can be divided in the annualized investment costs (59%), annualized operating costs (35%) and medium replacement costs (6%). In spite of the installation of the hood system, the investment costs are still large and the main contributor to the total costs. The total costs are clearly better with the hood system, as with natural ventilation the total annualized costs would be approximately $2.9M or €2.4M per year for a stall of 100 cows.

Policies and Regulations

Of course, to implement Cattlelyst on a farm, we need to comply to the current laws and regulations. These legislations impact different aspects of our biofilter, like broader environmental laws, food security laws regarding the cows, or labour laws for the farmers, but arguably most important for us is legislation regarding GMO safety.

In the Netherlands, both national and European laws regulate this subject. The European Union (EU) has established a legal framework for all its member states to ensure that the development of modern biotechnology, and more specifically of GMOs, takes place in safe conditions [15]. This framework employs a “precautionary principle”, meaning that usage of GMO’s is not allowed, unless their safety is demonstrated. This can be achieved via an elaborate risk assessment, based on strict criteria. On top of that, a post-market monitoring of the environment is required to identify any unanticipated side-effects [16].

Dutch GMO laws are embedded in this European framework. Following the “risk assessment” directive, the safety for human and animal health and the environment is to be demonstrated before a GMO is allowed on the market. We talked to Dr. Cécile van der Vlugt, who is involved in risk assessment of GMOs at the Dutch National Institute for Public Health and Environment (RIVM). She explained us that risk assessments of GMOs in the Netherlands fall into two categories: “contained use” and “introduction into the environment”, which can happen both at large and small scale. Here we elaborate of which category Cattlelyst fit in and what was our approach to make it pass the risk assessment.

According to the experience of Dr. van der Vlugt, our project could possibly fall within both categories. However, the “contained use” category would likely result in an extra burden for farmers, because they would need their cow stalls to comply to specific conditions and regulations. This would likely cost them more time and money. We reasoned that this was therefore not a preferred option. Therefore, we concluded that in practise, Cattlelyst should fall in the “introduction into the environment category” [17].
An open question is therefore, what needs happen in order to enable Cattelyst to pass the risk assessment and open the door to real-life implementation on a farm? Environment risk assessment looks at the system and its direct surroundings. The first step is scenario development: in what way could the bacteria get out of the system and into the environment? Then, the next question of course is, what are the possible hazards or adverse effects that can arise as a consequence? These hazards can fall into different categories, that are specified in the regulation.
Within our iGEM project, Cattlelyst, we have made important first steps to  be able to reach this stage of implementation in the future and answer these questions thoroughly. As detailed here, we have incorporated safety into the design of Cattlelyst from the very start and kept on improving throughout. We implemented three layers of safety to make sure that the survival of our bacteria is solely dependent on the specific conditions inside the biofilter, and refined these systems based on feedback from experts and the general public. 

Click here to go to our Safety page

On top of that, the concept of safety-by-design can be assured in two different ways. Next to safety of the organisms, it can also be implemented in the design of the biofilter. For instance, Cattlelyst is not a closed system, because liquid must be added to the biofilter, and gases pass through. Therefore, risk minimization strategies should be included in the design. For instance, all air should pass through a filter after Cattlelyst and before being released into the atmosphere; and water can be recirculated to our biofilter. In these ways, risks are minimized and safety can be guaranteed. These things may help Cattlelyst pass the risk assessment and aid its implementation.

It was a true eye-opener for us that, contrary to popular belief, admittance of GMOs on the market is actually allowed, if regulations are met and risks are mitigated adequately. Although regulations in the EU are strict, according to Dr. Van der Vlugt, it’s mostly society’s acceptance that is the limiting factor in admittance of GMOs, and politics that respond accordingly. That is why we believe communication and education are also indispensable in the implementation of Cattlelyst, and any GMO-based solution, in the future!

Alternative applications of Cattlelyst

The majority of ammonia emissions in the Netherlands can be attributed to the cattle industry. But ammonia is also a key component used in fertilizers, which are used worldwide to sustain food production. The extensive use of fertilizer has caused nitrogen pollutants, such as ammonia, to contaminate water bodies.

Icons made by Pixelmeetup from www.flaticon.com

            
            Figure   3: Schematic of a waste water treatment plant. 
      
            Figure   3:  Schematic of a waste water treatment plant.  
      
            Figure   3:  Schematic of a waste water treatment plant  
      
                To remove excess nitrogen compounds form water, wastewater treatment plants (WWTPs) depend on microbial processes. To date, nitrification/denitrification-based biological systems are most widely implemented. In these systems, nitrification and denitrification still occur separately [17,18]. To sustain continuous nitrogen removal, specific and costly operational conditions are required. Especially the addition of a carbon source to sustain denitrification and extensive aeration to complete nitrification add costs to the overall process [20].
            
                Approximately 25 years ago, the anaerobic ammonia oxidation (anammox) process has gained interest for its application in WWTPs. With anammox bacteria on site, one could save on cost both for aeration and supply of an external carbon source.  Thus, switching from conventional nitrification/denitrification to anammox has both an ecological and financial incentive. Since its discovery, approximately 200 WWTPs now depend on anammox bacteria, but at this moment it is not applied ubiquitously in these facilities [20]. Given its superior properties, what is the reason for not being applied in every WWTP?

Implementation of the anammox process in WWTPs still faces many challenges: For instance, the growth rate of anammox bacteria ranges from one to several weeks in doubling time [20]. This makes it especially difficult to use them for continuous ammonia removal. Moreover, numerous external factors affect anammox growth and thus ammonia removal greatly. This makes controlling the conditions in a WWTP very complex.

Cattlelyst for wastewater treatment facilities?

As an alternative, we could apply our Cattlelyst co-culture in WWTPs. The synthetic heterotrophic nitrifier – aerobic denitrifier P. putida has a doubling time within the range of hours. Moreover, P. putida is inherently robust [21]. These features make the application of our bacterium less ‘fragile’ than implementation of natural anammox bacteria. Moreover, our ammoniatroph would improve upon the existing nitrification/denitrification system as the need for two operational modes to remove nitrogen (aerobic/anaerobic) is not required. This is because the conversion from ammonia to nitrogen gas can be achieved in a single bacterium.

But why do we need our methanotroph, E. coli? Wastewater treatment often needs the addition of a carbon source, as the water can be depleted of most of its carbon sources at the (de)nitrification stages [22]. To prevent accumulation of intermediates, these reduction steps need to be fuelled with electrons sufficiently. Methane, originating from anaerobic digesters, has been suggested as a cheap alternative carbon source for denitrification in wastewater treatments [17,18]. Our fast-dividing methanotrophic E. coli could convert methane and provide P. putida with sufficient carbon source. Although the overall system needs to be adapted to an extent, the Cattlelyst co-culture could be a promising alternative to current WWTPs.

Team:Wageningen UR/Implementation