Integrated Human Practices
As MSP’s 2021 iGEM team we aim to consider and analyze the potential impact that our project could have on our community, both within the Netherlands and worldwide. This is why we have completed several different projects aimed at acquiring a more in-depth understanding of the ethics and societal opinions regarding synthetic biology. This page begins with a description and information about the different Human Practices projects that our team completed. This is followed by an explanation about how we consolidate this information to improve our project as Integrated Human Practice.
Due to our project affecting a broad range of people, we decided to conduct two separate surveys: one for the general public, and one for those most affected: farmers. For the general public we were interested in their opinions on GM food and the usage of our product. In the farmers survey, we wanted to hear what they thought of cattle and climate, about the role farmers had to play, as well as their opinion about our product. Both surveys were conducted in collaborations with other iGEM teams worldwide! From these surveys we have learned more about how to communicate the methane problem, and how we can show the value of our product for a sustainable future.
To find help in gathering ideas, we reached out and reacted to many teams that have projects of a certain similarity to ours. We wanted to find teams interested in biosafety, ethics, general opinions about GMOs etc. On our search we got in contact with many teams from around the world, with all of which we had some kind of interaction. These interactive exchanges have helped us to share work with others, have common projects fulfill either of our team's needs, and led to a great international exchange. Thanks to all of our collaboration partners for the great work that we have achieved together!
After an initial paper search and information gathering, we encountered certain names over and over again. This is a good indicator that these researchers are interested in similar areas that are related to our project. We therefore have contacted several experts on various topics, such as methanogenesis inhibition and rumen microenvironment as well as ethics experts and business experts. Their valuable input has shaped various aspects of our project, and has allowed us to take more aspects into account.
GMOs are a very polarizing topic. However as a team we have wanted to have a more in depth understanding of this statement. Which ages liked (or disliked) the idea of GMOs more? Who is more knowledgeable about the topic? Does the level or field of education affect the perception of GMOs and if so how? And on top of that we were curious about what people would think when they would hear our research ideas. Would they support them or not?
Therefore we have reached out to the iGEM teams we have previously contacted for collaboration and partnership opportunities to gather our forces to develop and analyze a survey to answer our questions and shape our research accordingly.
Overview - GMO
The alteration of the cow’s microbial community is a step that is not easily accepted by the general population, and the usage of GMOs is often viewed negatively. In a survey in 2004, Hallman et al. found that around 48% of US consumers know very little of GMOs, 16% reported to have no knowledge of GMOs, 30% reporting to know a fair amount and 5% reporting to know a great deal about GMOs. Wunderlich and Gatto (2015) suggested a correlation between lack of information and distrust towards GMOs. Around 20% of US consumers that had a negative attitude towards GM products changed their attitude towards them after given the information that around 50% of food in grocery stores have GM ingredients. Furthermore, those with more scientific knowledge of bioengineering seem to view GMOs as less negative than those with less scientific background, which may mean that deeper scientific understanding of genetic modification may reduce the fear of GMOs (Wunderlich & Gatto, 2015). To further understand public knowledge and perception of GMOs, we are conducting an international survey in collaboration with three other iGEM teams, namely IISER Tirupati, IISER Pune and team Aachen. The aim of the survey is to find attitudes towards the use of GMOs. The survey is designed so that it can be quantitatively assessed. It will be translated into multiple languages and distributed over the summer to a wide range of participants in terms of geography and demography.
- Hallman, W. K., Hebden, W. C., Cuite, C.L., Aquino, H. L., & Lang, J.T. (2004). Americans and GM food: Knowledge, opinion and interest in 2004 (No. 1327-2016-103629).
- Wunderlich, S., & Gatto, K. A. (2015). Consumer Perception of Genetically Modified Organisms and Sources of Information. Advances in nutrition, 6(6), 842-851.
As the iGEM teams contributing to this project we have all set a goal for everyone to get 10 people to fill this survey. As we were more than 60 people collaborating in the project, we have thought to receive 600 results from this strategy.
To reach a higher number of people we have mailed the iGEM team contacts we had using an automated mailing script programmed by our team. We have mailed many iGEM teams using this software to collect more responses.
As a result we have collected 275 responses.
The average is a 5.52/10 for the opinion on GMOs, meaning that they are seen in a slightly positive light in the population. GMO is a term that is more widely known, and has also been stigmatized, with food companies advertising their products as being non-GMO, and fear that it could be used to generate things such as ‘designer babies’. The link between synthetic biology and GMOs is clearly there, and as such this discrepancy needs to be addressed. With appropriate legislation and assurances that GMOs can and will only be used for essential purposes, such as the fight against climate change, this approval may be improved. Yet there is also a cultural and religious aspect to this statistic, meaning that, for some, the negative light on GMOs cannot be turned. Yet this slight trend for GMOs to be viewed positively is a good basis for future education to explain the appropriate usage of GMOs.
Synthetic Biology Opinion
When using adjectives to describe emotions and opinions towards the field of synthetic biology, the trend changes towards more ‘positive’ terms even more drastically. As mentioned before, to many, synthetic biology may be an entirely new term, somewhat fascinating, yet frightening at the same time. It shows that many people are open to hearing what the opportunities of synthetic biology’s applications are, and others have already hopes for the positive aspects of its application. It is important to note that, even though a minority, also those against the use of synthetic biology must be addressed. It is, in the end, OUR planet, and therefore we must include, hear and appreciate their concerns, as well as include them in future undertakings.
Synthetic Biology Opinion
With an average of 7.37, there is a great increase in the approval of using GMOs when used to fight climate change. This, once again, shows that fears about GMOs stem from their misuse mostly, not from their general existence.This can be seen when compared to the graph below. Of course we were very excited to see such graphs, as it seems that, were it a democratic vote, our GMO was approved. Yet once again, it is also the worries that count, and must be addressed. May it be due to lack of knowledge of the urgency to halt climate change, the misunderstanding of what it means to use such a GMO (e.g. is it cows that get genetically modified?), or general disapproval due to perceived threats, these questions need to be addressed. Educational programmes, brochures, open days, legislative campaigning, all those are measures that may lead to more approval, yet also to more input as to what needs to be done to achieve higher approval rates.
As mentioned, it seems that the misuse of GMOs is what sparks fear and distrust towards them.
Overview - Survey
During our search for potential collaborators we got contacted by the iGEM team
Uppsala from Sweden. Their project is about modifying bacteria to produce growth
factors, which can then be used in cellular agriculture growth liquids. After initial
meetings for project idea exchanges and introductions, we found out that we both have
projects that would change the future of farming, in one way or another.
One of our joint team meetings, where we discussed the farmers survey as well as the
webinar (for more, click here).
As we both want to include important stakeholders in our projects, we both came into this collaboration with the same idea: ask farmers what they think of the future and whether they see our project being a part of it. With this promising parallel of ideas, we decided to work on this together. For this, we created a survey to be sent to farmers worldwide.
Various meetings took place, during which we worked on the survey questions, which can be found here. The survey was translated into seven languages, and was sent to over 90 farmers globally. There were multiple choice questions accompanied with optional open answer spaces, allowing for a more individual survey rather than a statistical one.
We have received ten responses in four languages, stemming from six countries: Sweden, Germany, the Netherlands, India, the US and the UK.
Farm Size and Product
The responses we received were mostly from small to medium sized farms, which had a variety of products, as can be seen below.
The average farmer seems to care about having a sustainable business, and views efforts towards sustainability as given.
When asked about the potential of expanding their farm in the future to enhance production or further sustainability, most farmers agreed that their business will expand to adapt to either of these two.
One statement accompanying this question from a farmer reads:
"Higher efficiency is sustainable production, as less input is needed for the same output, it is not 'this or that'."
We agree with this statement, and also argue in this way, since our feed additive (which is a one time administration) would reduce energy lost through methanogenesis, and therefore increase the weight gained since the biomass can be used at a higher efficacy. The two terms ‘efficiency’ and ‘sustainability’ are often viewed as mutually exclusive, even though they are not.
We can integrate this statement, by arguing that through our feed additive, cattle and dairy not only becomes more sustainable, but also more efficient. To see our discussion with Dr. Ungerfeld about increasing efficiency through phloroglucinol, click here.
Furthermore, we see this as proof that a marketing campaign which focuses on the efficiency benefits could further the amount of farmers that could be interested in our product.
Other comments on this question included: "Improve Sustainability"
"Adapt to new Technologies"
Which both indicate that farmers understand that their work is extremely dynamic and is prone to changes due to new technology.
When asked about whether they have witnessed major changes during their work as a farmer, all ten respondents responded with 'Yes', and left us some statements:
"There are now more animals on fewer farms."
"Documentation and animal well-being."
"Thinking more about raw materials and their re-usage."
"Thinking more about biodiversity, such as thinking about natural enemies of pests and fungi."
This shows us that major change has happened in the past, and is currently also in progress. The comments that were added have a lot to do with sustainability and general adaptation to current scientific and climate knowledge. This is a very positive development that will hopefully continue.
This leads us to the next question, which asks whether the farmers see any major changes in the future for their business.
Change in Agriculture
A large portion answered yes, which shows us that change happened in the past, is happening currently, and is anticipated in the future. Some anticipated changes are listed below.
"Livestock farming will be less supported."
"More efficient and sustainable farming."
"Make farming as bio as possible and make it sustainable."
The first statement indicates that more and more people will reduce their red meat consumption, and therefore livestock farming is less profitable and less supported. This is true, but can be countered by giving livestock farming a better GHG footprint, by using our product. It can help to maintain consumption rates (although optimally they would go down eventually) by marketing a greener type of meat.
The other statements both indicate anticipated changes in sustainability and efficiency.
Adaptability of Farms
New technologies may be scary, or just are not defined enough. Yet the trend leans more towards optimism for new technologies and their use.
The statistics are clear here, as there are still tight regulations for the usage of GMOs.
The statistics are clear here, as there are still tight regulations for the usage of GMOs.
Cow's Climate Impact
It seems like less than half the farmers that took part in this survey are aware of the climate impact stemming from cows. This may be due to lacking awareness of what methane is, its effects and where it stems from, or may be due to the belief that there are larger emitters in the world, and that the cow's emissions are not enough to be of matter.
Technology Usage Motivation
Interestingly, many farmers chose the sustainability option as a motivator to adapt to new technologies, which is an interesting sign. Yet sustainability always goes hand in hand with new climate policies, which are rules that need to be followed mandatorily. As has been mentioned before, in our project, efficiency and sustainability go hand in hand, and therefore is an interesting point to make when arguing for the benefits of this product.
Usage of Microbes in Farming
The potential usage of our microbial feed is not very high. Some of the statements that were left for us about this topic included:br
"My cows do not add to the problem."
"It does not fit our way of farming."
"The consequences of GMOs on the ecosystem is not known."
"It depends on the cost."
If a farmer is pushed away from this product by the idea of having to add bacteria to their animal's feed (which may not fit their way of farming), then our product must be advertised using the advantage of one-time administration. We understand that GMOs are a novel subject, especially when they are applied to livestock. Yet we do want to avoid any major changes to the animal or the ecosystem, by having a multitude of kill switches. These should be discussed, tested and explained to the end-consumers (the farmers), to ensure trust in the product.
Throughout the year we had many puzzleing point about our research both in terms of lab work, and human practices such as ethical conciderations. To have better understanding about such aspects about our research we have contacted academicians to guide us.
Assistant Professor Vinayak 'Vinny' Agarwal
Image Caption: GeorgiaTech. (2021). Profile | Georgia Tech Chemistry & Biochemistry. Retrieved 30 July 2021, from https://chemistry.gatech.edu /people/agarwal/vinayak
From the start, our project was mainly focussed around a paper (Thapa et al. 2020), wherein Vinny was the corresponding author.
We had many questions for him, so we had to choose carefully which ones were the most essential one
In their paper (Thapa et al. 2020), E.coli were transformed with algal genes (VHPOs/VBPOs), and made to produce bromoform using different substrates and cofactors. As our E.coli would have to work in a natural environment that lies mostly beyond our control, we had to ask more holistic questions.
Question Answer Integration From our understanding, Vanadium-Dependant Bromoperoxidases / Haloperoxidases (VBPOs/ VHPOs) use pentane-2,4-dione and/or heptane-2,4,6-trione as substrates in his papers (Thapa et al. 2020; Thapa & Agarwal 2021), which resemble thiotemplated beta-keto fatty acid biosynthesis intermediates. According to Thapa & Agarwal (2021), these are present in all life forms, and therefore also in E.coli. With this reasoning, we wanted to know whether it were possible to simply transform the bacteria with the VHPO/ VBPO genes to have them produce bromoform. Vinny reminded us of a crucial part that we have missed in his work. For VHPOs to work, they need hydrogen peroxide (H2O2), which E.coli does not produce a lot of. On the other hand, seaweed produces a lot of H2O2 (as most photosynthetic organisms do), which is why it also produces bromoform. Therefore VHPOs/ VBPOs alone transformed into E.coli would not lead to the production of bromoform. Our supervisor, Erik, encouraged us to re-read Thapa et al. 2020 in order to find out how red seaweed produces H2O2. This revealed to us that we had the (potential) answer right in front of us. The gene mbb2 and its respective protein Mbb2 are involved in the production of reactive oxygen species, amongst them H2O2. Therefore, due to Vinnys reply, we had a major shift in our genetic constructs, as our VHPO genes were now always transformed into E.coli in combination with mbb2, which hopefully would lead to the production of
- Thapa, H. R., Lin, Z., Yi, D., Smith, J. E., Schmidt, E. W., & Agarwal, V. (2020). Genetic and biochemical reconstitution of bromoform biosynthesis in asparagopsis lends insights into seaweed reactive oxygen species enzymology. ACS Chemical Biology, 15(6), 1662-1670.
- Thapa, H. R., & Agarwal, V. (2021). Obligate brominating enzymes underlie bromoform production by marine cyanobacteria. Journal of Phycology.
Dr. Emilio UngerfeldINIA. (2021). INIA » Personal » Emilio Mauricio Ungerfeld Moran. Retrieved 30 July 2021, from https://web.inia.cl/personal/ungerfeld-moran-emilio-mauricio/
Our project has been tremendously shaped with the help of Dr. Emilio Ungerfeld, a Uruguayan scientist living in Chile. Right from first contact, Emilio was very helpful and went above and beyond to answer our questions. Below, a summary of our main email exchange talking points is provided, as well as our integration of his comments and tips.
Zoom Call Summary
question_answerFirst Question - General Tips
Question Answer Integration After hearing of our project idea, do you have general tips that you would like to give us? Examining and experimenting with a food additive for the first time may be challenging. Therefore Emilio proposed that we use rumen mixed batch cultures as our proof of concept in the first instance, due to it being the cheapest and fastest way. We will conduct our ruminal experiments with batches of food added, as well as one initial provision of our product. This can be seen in the simulated rumen experiment.
question_answerSecond Question - Protecting the Rumen
Question Answer Integration How may we avoid drastic changes to the rumen microbiome? How could we ensure the health of the cow, as well as continuous fermentation in the rumen? Our first thought should be the confirmation that our product can inhibit methanogenesis in vitro. If the effectiveness is confirmed, we may observe the effects on fermentation by examining the production and the profile of volatile fatty acids (VFAs), as well as the digestibility of the food substrate. If there is no change in fermentation patterns, we may also check the reaction of the system to continuous and semicontinuous cultures. Only after all of these parameters are taken into account we can focus on the theoretical in vivo consequences, such as the health of the cow, and traces of bromoform in the milk and meat products, as well as traces of bromoform in feces/urine. The inhibition of methanogenesis became our first priority that we want to measure, as without this, the whole rest has no purpose. Additionally we need to find out whether the patterns of the VFAs changes. Although planned, the measurement of VFAs could not be implemented, but plans exist to continue this project with experiments where we do measure VFAs. This can be seen in the simulated rumen experiment.
question_answerThird Question - Alternative H22 sinks
Question Answer Integration When methanogenesis is inhibited, excess H2 may need to be redirected. How do you think this could be achieved? Propionate, reductive acetogenesis as well as microbial biomass are all alternative sinks for excess H2. For our project, it may be favourable if we add an external electron acceptor as a microbial additive. We have given this some thought, and have come up with the idea of adding a gene producing phloroglucinol. This compound, when reduced using H2, can be used as an energy source by the animal. This would both ensure continuous fermentation (unhindered by high H2 concentrations) and give the animal an energy gain. The usage of phloroglucinol is discussed further with Dr. Ungerfeld (see below), as well as with other experts.
question_answerFourth Question - Additional Considerations
Question Answer Integration Emilio wanted us to think about additional subjects. What advantages would our feed additive pose compared to just feeding cows Asparagopsis taxiformis or a less volatile form of bromoform? For example, maybe the E. coli intended to use have less harmful minerals within them than the algae would? In our background summary, we intend to describe how the worldwide scaling of A. taxiformis is not possible, and that the addition of E.coli would be a one-time application instead of a continuous feed additive, due to the chance that it may colonize the rumen indefinitely.
question_answerFifth Question - H2 Mitigation
Question Answer Integration Can cows belch out any excess H2 without additional intervention needed when methanogenesis is reduced? H2 accumulation is a consequence of inhibiting methanogenesis in vitro, yet in vivo this excess H2 can be expelled into the atmosphere. The interesting part is that, stoichiometrically, not all H2 that would reduce CO2 to CH4 remains as H2. When H2 is expelled, this poses a relatively small energy loss compared to the energy loss when methane is expelled. What can be observed is a general increase in dissolved H2 concentrations in the ruminal fluid. As the consequence of increased H2 concentrations in the ruminal fluid is still debated, we did decide to try and find a way to mitigate it. This may or may not be needed for the in vivo application of our product. In future experiments, we would like to measure both the H2 dissolved in ruminal fluid, as well as the gaseous amounts found to be found. In our ruminal simulation, we are restricted to measuring the amounts of methane produced only, yet future experiments will try and establish the consequences on H2 concentrations, and the implications thereof.
question_answerSixth Question - Volatile Fatty Acids (VFAs)
Question Answer Integration As we understood from our research, VFAs are competitors for H2 together with methanogens. It was therefore perplexing to us that, when methanogenesis is reduced, VFA production can also be reduced. Not all VFAs are H2 sinks. Propionate is one, as is reductive acetogenesis. Yet acetate and butyrate formation are associated with a net release of H2. In vitro experiments are more reliable to determine VFA production, as in vivo studies often only measure the VFA concentrations. This does not take into account the absorption and dissolution of VFAs. A decrease in VFA production is the only true indicator of inhibited fermentation, that may be due to increased levels of H2. As mentioned before, these inputs will only be applicable for future experiments that will be conducted later in this project, but they are great advice to give relevant results for our product in the future, and will definitely shape our project.
question_answerSeventh Question - Phloroglucinol
Question Answer Integration Is it possible to use phloroglucinol as an electron acceptor by, for example, adding engineered E.coli which overproduces it? The challenge in this would be microbial ecology. As the rumen is anaerobic, the ATP production is slow, and therefore also the production of phloroglucinol may be low. An efficient feed for these E.coli would be needed, as glucose levels are low in the rumen. Additionally, the E.coli would have to compete with the native microbial populations. Therefore it may be a good idea to give these phloroglucinol-producing E.coli a growth advantage. Due to a lack of certainty about the efficiency of phloroglucinol producing bacteria, we have decided that, in future experiments, we would first establish whether there is an actual need of an H2 absorbing agent or not.
question_answerEight Question - Increased VFAs
Question Answer Integration Would increased levels of VFAs be beneficial for the cows? Theoretically yes. For example, propionate is the main glucose precursor in ruminants, and could therefore be seen as an energy gain. Due to there being stoichiometric differences between the H2 eradicated and used in methanogenesis, we see that an energy gain for the animal would be possible, with or without mitigation. Yet the animals health would be our primary concern, and therefore our experiment strategy will adapt to accomodate for that.
question_answerNineth Question - Consequences
Question Answer Integration What could happen to the cow if there was a CO2 and H2 accumulation as a consequence of methanogenesis being decreased? Ideally you would want to incorporate as much H2 as possible into energy sources, thus combining methanogenesis inhibition with hydrogen harnessing. Although a low rate of methanogenesis inhibition (say 20%) would be more safe for the animals, there would be less noticable change. On the other hand, at 100% inhibition, the levels of bromoform might be toxic. Rumen acetogens may be affected by bromoform, as their role as H2 sinks may increase. Additional concerns would be the accumulation of the bromoform in milk and meat, as well as the rumen epithelium getting damaged by the bromoform (although this may not be linked directly).
He encourages us to look at a more holistic perspective, such as the multitude of interactions bromoform would have with all the microorganisms in the rumen, and the consequences thereof. This could be done by breaking the project up into multiple stages, wherein the safety is assessed individually.
Finally, we will also never be able to fully simulate the true in vivo circumstances, as the in vitro assays cannot consider all variables.
Our project has truly evolved from only stopping methanogenesis into a two step project, which also tries to harness the energy otherwise lost in H2. We therefore try to aim at the highest possible reduction of methanogenesis, while also using as much H2 as possible in other reductive pathways.
Even though this research has already been looked at by our team, we want to determine non-toxic levels of bromoform that can be allowed in the meat and milk products of cows, as well as the direct damage that could be caused by bromoform to the cows.
Zoom Call Screenshot
Dr. Kieran O'Doherty
Image source: University of Guelph. (2021). Kieran O'Doherty | Department of Psychology. Uoguelph.ca. Retrieved 9 September 2021, from https://www.uoguelph.ca/psychology/users/kieran-odoherty.
At the faculty of Psychology of the University of Guelph, Dr. O'Doherty focuses on the ethical implications of science and technology, amongst other focal points. Another major interest of his is finding methods of engaging and involving the broader public in research. These topics were of particular interest to us, as we wanted to find out how we can involve the broader public and present our project to it. Furthermore we wanted to get an outside opinion on the ethical considerations of our project, and whether there are ways to convince the public of its uses. Dr. O'Doherty was very helpful, and agreed to a zoom meeting, where we could ask him questions freely. Below are some of his inputs for our project.
Zoom Call Summary
question_answerFirst Question - Safety
Question Answer Integration We were wondering if he thinks that, taking kill switches into account, the project is safe for the environment, and therefore less unethical. If we always try to find the best solution to a problem, yet the perfect solution does not exist. Even if our kill switches work, and the bacteria is used in ruminants worldwide, we have no control over natural changes. What if, over time, the quality of water changes in certain areas? Maybe our bacteria have become a dominant and needed part of the ruminal microbiome over that time. This change may cause a wide activation of the kill switch, which could have dire consequences to the animals. We were actually surprised to hear this new way of arguing. It was not 'what if your kill switch does not work', but it was 'what if it works really well'. We did not consider this so far.
Our team wanted to make biosafety one of its main priorities, yet biosafety aspects take a lot of planning and time. It is for Dr. O'Doherty's comments that we tried to seek other teams to collaborate with, teams that focus on biosafety.
A good biosafety system should think ahead and anticipate changes in both gene structure, but also environmental input. With the help of constitutional promoters and inducible promoters, we had exchanges with many teams to talk about this topic (see Tirupati, Leiden and MSU)
question_answerSecond Question - Playing God
Question Answer Integration We argued that humanity has been breeding animals and shaping the environment as we please for thousands of years. For this reason we wanted to ask him whether these kinds of projects are inevitable advancements of humanity, or whether they go too far. He doesn't think that using GMOs is an inevitable progress, and it is also fundamentally different from directed breeding. Indigenous populations are living very harmoniously with nature. We, with our western society and lifestyle, have, in a few hundred years, gotten so many species extinct and destroyed so many habitats. To say that this progress with GMOs is inevitable is strange. He is not saying that technology is bad. We use that as an excuse for curiosity and profit. Profit leads to social injustice. Not everything should be done just because it can be done. As this may be more of a philosophical discussion, we could not incorporate these comments, yet we can comment on them. Our curiosity does lead us to such undertakings, but profit is not the end goal. This project wants to help with the fight against climate change, although in a more radical way. 'The way is the goal' yet here the goal is the goal. Climate change will affect our lives and the lives of our children. If we do not act now, in extreme ways, we will face problems manifold higher in magnitude than social injustice. It is often poorer nations that are affected by climate change, thus finding extreme ways to fight climate change are also extreme ways of fighting social injustice.
question_answerThird Question - Risk Assessment
Question Answer Integration How much risk would be deemed reasonable, and who gets to make these decisions? Opinions may diverge on this, we all make decisions individually about personal risks, e.g. driving motorcycles, smoking etc.. But when it comes to things like GMOs, it removes it from the individual decision making process. You will find that different individuals and societies have different standards. It is a situated social decision making. The idea of democratic states is that every voice should be heard before a decision is made. We don't get to vote whether we allow GM foods. There is a lot of expert led decision making. Science and government say some things are ok, but often for very grey area subjects. They are often not technical decisions but established according to the value of an open market decision. In Europe there are more precautions than in the US, where it is more money focussed. Application of different values leads to different decisions. Whose values should lead this decision? Experts have no right to impose their value on society, e.g. scientists, government, lobbyists etc.. Who should be involved? What are the values of the population? Our project would need global values as part of our public engagement to understand the issues from all kinds of societies. What risk is acceptable? Who gets to decide? It is a good question, but what are the alternative solutions that have been proposed? The risk is also relative to the other proposed mechanism. Yet these decisions are made by experts, and not just politicians. Lobbying is often seen as a bad term, yet that also includes ethics experts, scientists and other opinions. Therefore the decision of whether or not to harvest the power of GMOs is influenced by experts on the topic. In countries with direct democracy, such as Switzerland, you actually do get to vote on an issue like this. But letting people vote on every matter can slow progress, and also allows for demagogues to influence the population. We agree that this is a grey area subject, yet this decision would not be made blindly, but under expert advice and with the inclusion of the general public. For this reason we have decided to conduct a global survey on the opinion on GMOs together with three other teams (see more here). Expert led decision making is the only decision making that is somewhat sound. The danger of climate change has already crossed the line of balance with the ethical problems of this project. We therefore believe that the ethical considerations are important, but survival is more important.
question_answerFourth Question - Our Idea
Question Answer Integration Would you think it would be easier to reduce consumption of beef and dairy products worldwide or to apply this product worldwide? There is also synthetic meat, a real possibility. In some parts of India they are very strongly vegetarian. Cultural practices show that it would be possible to stop beef consumption. How many trees would you have to plant to halt global warming? It seemed like a huge project, but it did not seem impossible. Technological solutions seem attractive, but there are also alternatives to look at. He thinks what we are doing is a good thing, but does not know if it is the right thing. It just adds to the things that you can draw from. We agree that there is no more sustainable meat industry than no meat industry. Yet the reduced meat consumption would be a momentous attack on freedom of food choice, which would affect people more directly than if a GMO is inside of a cow's rumen. We also agree that this may not be the first solution, but in times of peril, such radical solutions may be what is needed.
Zoom Call Screenshot
Professor Roderick Mackie
Image source: Yates, D. (2012). Patterns of antibiotic-resistant bacteria seen in Galpagos reptiles. News.illinois.edu. Retrieved 17 October 2021, from https://news.illinois.edu/view/6367/205155.
We were directed towards Prof. Mackie through reading Greening et al. (2019). We immediately received a positive response from him and received very good answers to our questions, which helped us further develop our project.
question_answerFirst Question - The H2 problem.
Question Answer Integration Can cows simply belch out H2 without any further mitigation needed? There is a dose dependent mechanism that defines H2 levels and volatile fatty acid (VFA) levels when it comes to inhibiting methanogenesis. A big problem in this particular research field is the way of measurement of functioning fermentation. Oftentimes, volatile fatty acid production (which can be inhibited by higher H2 concentrations), is only measured in concentrations or molar proportions, whereas more information could be gained if their production rates were measured.
Since H2 has a low solubility in the rumen, more H2
Since the VFA levels and H2 levels are dose-dependant, we want to find out how high our bacterium reduces methanogenesis. If the reduction is at a low level, it may be that no mitigation is needed. To find out, we performed gas measurements in our rumen simulation. For cases where the inhibition is high, we have plans to use phloroglucinol as a compound to be reduced by H2, retaining energy for the animal.
question_answerSecond Question - Volatile Fatty Acids
Question Answer Integration Can phloroglucinol be used as a compound to trap H2 as acetate, in order to retain energy for the animal? In Prof. Mackies opinion, methanogenesis can be inhibited by 20-40%. In this way, the animal feed intake and growth performance should still be maintained. In his opinion, reductive acetogenesis induced by phloroglucinol is not a good competitor for the remaining H2. The opinions of the efficiency of phloroglucinol useage is split. We will keep the idea alive, yet only hypothetically, as we did not have time to test it in vitro.
question_answerThird Question - Phloroglucinol
Question Answer Integration In some papers, propionate is increased upon methanogenesis inhibition. Would this VFA be a good candidate to trap excess H2 to avoid energy loss? Would this be advantageous for the animal? Yes indeed, propionate is a more reduced end product, and can therefore use more H2. Additionally, propionate is the main glucose precursor for ruminants, meaning that it would bring more energy to the animal. To stimulate higher propionate production, high concentrate feeds such as grain can be used. Yet this is also a direct feed for humans, raising the question whether this is efficient. A minute shift in diet may already be enough to avoid energy loss and induce higher propionate production. Different diets may be tested in further rumen simulations, where VFAs are also measured.
question_answerFourth Question - Propionate
Question Answer In a paper by Greening et al. (2019), acetyl CoA synthases from Blautia schinkii were mentioned to be potential H2 sink if methanogenesis were to be inhibited, doing hydrogenotrophic acetogenesis. Furthermore, fumarate reduction and nitrate ammonification with fumarate reductases and nitrate reductases as well as ammonia-forming nitrite reductases could be further H2 sinks. Do you see any of these as a good starting point to mitigate excess H2 in the cow rumen after stopping methanogenesis? Studies are needed for better quantitative measurements of VFA fluxes during fermentation. Nitrate reduction may lead to toxicity if it accumulates, and fumarate reduction most probably makes no difference to the H2 balance.
Dr. Peter H. JanssenImage source: Humboldt.org.nz. (2021). Dr. Peter H. Janssen | New Zealand Association of von Humboldt Fellows. Retrieved 30 July 2021, from https://www.humboldt.org.nz/person/dr-peter-h-janssen/
After reading Greening et al. (2019) and contacting the lead author, we were pointed towards Dr. Peter H. Janssen. He is a researcher at AgResearch in New Zealand and an expert in our field of interest, being one of the leaders of the Global Rumen Consensus. Dr. Janssen answered our questions we had thoroughly and helped us to further understand the impact our product might have on the rumen itself.
Zoom Call Summary
question_answerFirst Question - The H2 Problem
Question Answer Integration Can cows simply belch out excess H2 without any further mitigation needed? Per mol CH4 mitigated, approximately 1mol of H2 is released (the majority of CH4 is formed by 4H2 + CO2 → CH4 + 2H2O). Therefore, fermentation may be redirected to form other reduced products, such as propionate and butyrate, instead of H2. H2, propionate and butyrate are alternative electron sinks in rumen fermentation processes, and H2 follows a gradient towards the gaseous phase, when it is belched out (as a non GHG gas). This expelled H2 still represents an energy loss. As we assumed, the excess H2 may be directly belched out or is formed into VFAs. Yet, as Dr. Janssen explains, the H2 energy loss is still a problem that we decide to tackle.
question_answerSecond Question - VFAs as H2 sinks.
Question Answer Integration From some papers there can be confusion about whether VFA are increased or reduced after methanogenesis inhibition. From our understanding, in a normal rumen, VFAs compete for H2 with methanogens. Is there a consensus? The concentrations of VFAs are point measurements. There is a constant balance between VFA formation by microorganisms and absorption by the cow. This balance may be tipped depending on time, feed and amount of feed. In general, a higher propionate ratio is energetically more favourable to the animal. Increased H2 rates may reduce the rate of VFA formation and/or extend the time needed for feed degradation. Yet this does not mean that the total amount of VFA is reduced. For our simulated rumen experiment, we would take various samples, such as gas and fluids, in order to detect the excess H2 as well as VFA concentrations. Unfortunately, because of time constraints, we were unable to also measure VFA concentrations in our ruminal experiment.
question_answerThird Question - Phloroglucinol
Question Answer Integration Do you think that using phloroglucinol would be an efficient electron acceptor to be added as another transformed bacteria to colonize the rumen? In general, the amounts of electron acceptors that would need to be added are too large to be practical.
A cow makes 320g of CH4 a day (20mol), that is 80mol of H2. If that is reduced by only 25%, then 20mol of H2 is freed up. Therefore, also 20mol of phloroglucinol would be needed, which, at a molecular weight of 126,11 g/mol would result in 2,5kg/day of phloroglucinol needed. This would not be practical.
Keeping the idea still in mind, we have decided to exclude it for the project for the time being.
question_answerFourth Question - Is H2 Mitigation Needed?
Question Answer Integration Would the addition of reductive acetogens or propionate producers to the feed or directly inside the cows rumen be needed? As homoacetogens and propionate producers are already present inside of the rumen, this may not be needed. These producers are already well adapted and their niches might expand upon inhibition of methanogenesis. In some cases for example, the amount of propionate increased when methanogenesis was inhibited. If this holds true, then measurements taken to detect VFAs could show us that. Yet as we had no time for this experiment, we will have to find out at a later point in our research.
question_answerFifth Question - Increased VFAs
Question Answer Integration Are increased VFAs beneficial? They are to the cow, yes. But it could also lead to altered fatty acid compositions within the final products (meat and milk), which need to be analysed. Additional literature is recommended. This is a big part of our project that can sadly only be explored in vivo studies.
- Greening, C., Geier, R., Wang, C., Woods, L. C., Morales, S. E., McDonald, M. J., ... & Mackie, R. I. (2019). Diverse hydrogen production and consumption pathways influence methane production in ruminants. The ISME journal, 13(10), 2617-2632.
In order to create an exemplary project, integrating the information we have required is essential. In this section, we shall illustrate how we have accomplished this in different aspects of the project such as its usage of VHBO, creating gene constructs and kill switches in the lab, and providing a basis for our documentary.
Thanks to an important input by Prof. Agarwal, namely that bromoform is only produced in presence of reactive oxygen species co-factors, we re-read his team’s paper about the genes responsible for bromoform production in seaweed. Thanks to another input by our very own Prof. Redeker, we began to understand that the algae are able to produce bromoform by themselves, and therefore must be able to produce reactive oxygen species. This went on to define how we constructed our plasmids for the rest of the experiment.
Farmers are one of the pillars of any society, providing food for the entire world. Yet oftentimes, they are put under scrutiny by their own government for such things as greenhouse gas emissions. This is especially true here in the Netherlands. Yet the farmers are resisting, and have protested multiple times in the past few years. To understand the frustrations that farmers experience, an informative documentary was produced that aimed to explain the conflict of interest, the opinions of the participants, as well as introducing our product as a solution.
As the release of GMOs into nature are an ethical grey area, we wanted to make sure that our product implements biosafety standards. For this, we thought of, created, and collaborated on a variety of kill switches, amongst other biosafety aspects. This is aimed at a minimal escape rate both within, and without the target space of our product.