Human Practices
A range of new technologies aim at the reduction of greenhouse gas emissions. However, none of these technologies has yet been able to prevail and are subject to further development and are tied to fundamental structural change. With our approach we want to contribute to the development of climate-neutral fuels, and spoke to experts from the industry, to scientists and to the federal environmental agency to discuss the potential our proposal holds. During our human practices activities we mainly evaluated aspects regarding the sustainability and ethics of our idea, while recommended improvements were discussed in the integrated human practices section. Overall, integrated human practices and human practices were very helpful for us and encouraged us to continuously overthink and improve our project.
How the world affects our project and how our project affects the world
The world we live in today, is facing great challenges. Climate change is one of them and already has a great impact on our lives. Recent years impressively demonstrated that human behaviour affects earth’s ecosystems as much as earth’s ecosystems affect us. In the past and in the present humans have developed a series of technologies and processes that allowed us to live the way we do. However, industrialization also led to ever increasing greenhouse gas emissions and an anthropogenic greenhouse effect. Although scientists were alarmed by this development in the past, and predicted negative outcomes, we were not able to stop the dramatic events that followed. Now that the consequences of climate change affect us more than ever, humans finally start to become aware of this problem and their part in it.
We, as students, experience that people in our environments question their personal ecological footprint and behaviour in terms of climate action. We also observe this development in the industrial sector and the products that supermarkets, drug stores and even clothing stores offer us. But will those efforts be sufficient? As this question affects us personally as well as humans all over the world, we want to contribute to a solution for this great problem. Therewith, we believe that our efforts can motivate other people to take part in climate action.
To assess our approach for the generation of carbon-neutral fuels and to capture different perspectives on biotechnological fuel production, we spoke to different experts including scientists, industry representatives and the federal environmental agency. In the section below, you will find our evaluation of those talks and an in detail list of what we learned and why we approached them.
Attitude towards carbon-neutrality
The experts we talked to had different subject areas and perspectives on our project and its impact on the world. We learned from the UBA (German Federal Environmental Agency) and the BASF (chemical company) that the final energy-efficiency of a technology can be an important factor for its sustainability and success. While talking to the UBA and an expert from the steel industry it became clear that there is a structural transformation going on that is directed towards decreased greenhouse gas emissions which has brought many different new technologies onto the scene. However, we heard that none of these technologies has reached its full potential which is why they are still subject to further development.
Sustainable transportation
In Germany, politics focus on the electrification of the transportation sector. The UBA mentioned that a problem of this technology is that it cannot be applied to heavy transport, shipping and aviation in the near future. Here, liquid fuels are required to fill this gap. Another advantage is that our liquid fuel is produced without fossil resources and that there is no competition over land use as it is the case for biofuels from plant material. This also makes our project ethical, as it was mentioned by the UBA that fossil resource extraction often conflicts with local residents or poor working conditions. Additionally, it was stated by the Environmental Agency that the fossil resources that are required for batteries of electric vehicles will currently not suffice to supply the whole world with this technology. Therefore, they believe that our project has the potential to be an advanced alternative to replace harmful fossil fuels until better solutions are found in the long run. Our project could thus be used as an important transitional technology.
Recycled-carbon fuels and Power
Representatives of UBA indicated potential hurdles for carbon dioxide assimilation techniques as they require energy for the conversion of CO2 to CO via hydrogen gas. However, LanzaTech showed before that especially in the steel industry or in municipal solid waste gasification a suitable environment for acetogenic bacteria is created. These industrial processes are even beneficial for our technology from another standpoint. The bacterial fermenters can reach their highest potential when a gas substrate is constantly supplied. The above mentioned techniques fulfil this requirement as they produce a constant output of waste gas. To date the purification of butanol from bacterial fermenters can oppose obstacles as it can make the process cost inefficient. However, as our process only utilizes waste gases, which are usually disposed expensively, as substrate, a great financial advantage is generated. Ultimately, "[...] ABE fermentation is currently the most important method for biobutanol production [...]"[1]. Furthermore, taking ethanol production from gas fermentation as an example, the greenhouse gas reduction is estimated to reach up to 67% (substrate from steel production) and 98% (substrate from gasified biomass) when compared to fossil gasoline[2]. Biobutanol production by utilization of waste gases from steel industry or waste gasification therefore provide a great advantage over other CO/CO2 utilizing techniques and allows a large scale carbon dioxide emission reduction while producing valuable biofuel.
An expert from the steel industry mentioned that it is currently still difficult for steel companies to become carbon-neutral as the development and establishment of new technologies such as direct reduction with hydrogen still needs some more time. Due to that reason, the expert verified that our technology would help steel companies to become climate-friendly. Here, it was much appreciated that the Wood-Ljungdahl Pathway in Clostria allows for carbon fixation from different gas mixtures.
Safety
Talking to non-biologists, especially during our communications activities, we experienced that some people distrust genetic engineering. Therefore, we also wanted to include this topic shortly in our human practices assessment. Our bacteria are classified as S1 organisms and therefore are not harmful for humans. Additionally, LanzaTech verified that strictly anaerobic bacteria (such as ours), oppose a low risk of unintentional spread in the environment, as they die immediately when they come into contact with oxygen.
Vector system
We were happy to talk to various experts in our own field to exchange scientific knowledge and experience. Doing so, we learned a few things that helped us improve our work. A major influence on the initial design of our project was the work of Dr. Saskia Baur. She worked with solventogenic Clostridia in her PhD thesis[3] and used the ClosTron plasmid system for the transformation of her target strain. Similar to us, she also transformed some rather large plasmids (12 kB), so we based our own work on hers. We used the same promoters and E. coli strains as Dr. Baur. In addition, we used mostly the same media and procedures, but still had difficulties with our project. Our E. coli strains did not grow well or modified our plasmids. These changes were mostly in the promoter region, so we concluded that the promoter might be toxic to the cells. However, talking to Dr. Baur we heard that this difficulty has not gone unnoticed. The phenomenon that E. coli cells modify toxic plasmid vectors was also known to Prof. Winzer who is an expert on metabolic engineering of Clostridia. He mentioned that it can be helpful to modify the plasmid step by step in order to deal with its toxicity. We did that by trying to construct our plasmids beginning from the empty vector and trying different promoters, such as the galactose-inducible promoter PbgaL.
Cloning and Regulation
Concerning the size of the DNA we wanted to clone, Prof. Winzer told us that it is important to consider that the transformation efficiency may decrease with plasmid size which encouraged our plan to increase the size of the cloned DNA stepwise and the idea to use two plasmids for the total size instead of one. However, our supervisor Dr. Poehlein also warned us to consider the transcription units which should not be damaged during the stepwise cloning of genetic material. Furthermore, Prof. Winzer drew our attention to regulatory processes and the generation of energy or reduction equivalents that can determine if genes for a metabolic pathway are used or not. As an example he mentioned the Rnf and the Ech-complex which is needed for production of energy by bacteria using the Wood-Ljungdhal pathway.
Strains
Coming to clostridial strains, he listed four strains, including C. beijerinckii and C. saccharoperbutylacetonicum, that are most commonly used and well-suited for genetic engineering and butanol production. On his advice we decided to use C. saccharoperbutylacetonicum as our acceptor organism.
Alternative Methods
Hearing of our difficulties with cloning, Prof. Winzer drew our attention to an alternative method of genetic engineering, namely the CRISPR-Cas9 system. He informed us about the Ribo-Cas system and allelic exchange method which may be helpful for cloning of large DNA fragments. We also heard of CRISPR-Cas when we were talking to Dr. Koepke from LanzaTech where we learned that it is important for strain stability that the transformed genetic material is integrated into the genome. Taking these assessments into account, we started to overthink our approach in terms of switching from ClosTron to CRISPR-Cas, this time directly including transformation into the genome. Unfortunately, we did not have enough time to pursue and implement this idea any further regarding the iGEM competition, but are considering working with CRISPR in the future.
Industrial implementation
Regarding future steps on the road to industrial implementation, we learned that the substrate gas composition is important in terms of product yield, but also application area. We knew that most industrial processes are subject to continuous improvement. From the beginning on we were aware that we have to further optimise our target strain after the end of our iGEM project period. Dr. Koepke, BASF and Dr. Redenius helped us to get an idea of useful optimization targets. We learned that the steel industry can be a target sector for the carbon assimilation technology and that it could be helpful if the target strain was able to use large amounts of CO2. Furthermore, strain resistance to fermentation products and butanol production rate should be assessed and optimised. Another crucial aspect for feasibility and sustainability is the overall energy-efficiency and CO2 footprint.
Dr. Saskia Baur – Research Associate
Dr. Saskia Baur was a PhD student at the university of Ulm where she constructed acid-producing Clostridium saccharoperbutylacetonicum strains by genetic engineering. Now, she is a research associate at the Eberhard Karls University Tübingen in the Environmental Biotechnology Group.
Why did we reach out to Dr. Baur?
The working group of Prof. Daniel has a lot of experience with the cultivation and genetic traits of the genus Clostridia, but mainly focuses on genome sequencing and metagenomics. Therefore, we contacted Dr. Saskia Baur for additional advice on genetic engineering of Clostridia. As Dr. Saskia Baur was working in her dissertation on genetically engineered butanol producers, her expertise was helpful and welcomed by our team. By reaching out to Dr. Baur we hoped to get some insights and advice concerning the genetic engineering of our bacterial strains.
What did we learn?
Dr. Saskia Baur introduced the ClosTron vector system to us as she had successfully used this vector system already. Additionally, our work has been shaped by the promoters that Dr. Baur has used. Furthermore, she advised us in clostridial transformation procedures, and we took her approach as the starting point for our own transformation protocol.
Takeaways:
- A functional vector system is the ClosTron plasmid system which is specifically designed for the genus of clostridia, but works also in other well established organisms like E. coli.
- Even though clostridia are not that well-established in molecular biology, there are already existing methods we can utilise and take these as base for our own approaches.
Prof. Dr. Klaus Winzer - Scientist
Prof. Dr. Klaus Winzer is an associate professor at the university of Nottingham and is part of the synthetic biology research centre. He is an expert on clostridial metabolism and metabolic engineering. His focus is on biofuel production and the metabolism of gas-fermenting species.
Why Prof. Dr. Klaus Winzer?
Prof. Winzer is an expert on the field we are working in. We reached out to him to talk about genetic engineering strategies suitable for Clostridia and his experience with some of these methods. The focus of his working group is on the integration of complex metabolic pathways into hosts. The Wood-Ljungdahl pathway is such a complex metabolic pathway and we had the chance to ask about our intended integration into solventogenic clostridia.
What did we learn?
- Well suited for solvent production and genetic engineering are C. beijerinckii, C. acetobutylicum, C. saccharobutylicum and C. saccharoperbutylacetonicum as they are primary butanol producers. The latter was therefore used from us as the host organism for the integration of the Wood-Ljungdahl pathway.
- It makes sense to work with Clostridia that are well known which is the case for our chosen organisms (C. ljungdahlii, A. woodii and C. saccharoperbutylacetonicum).
- Similar to us, Prof. Winzer also observed that E. coli cells can initiate insertions or point mutations on plasmid vectors if the plasmids are somehow toxic to the organisms.
- In response to the intended integration of bigger sized gene clusters, he proposed alternative methods in case we are struggling with the ClosTron plasmid: the allelic exchange system which was already used for Clostridium acetobutylicum. Additionally, he introduced us to the RiboCas system which uses CRISPR/Cas for gene editing in Clostridia.
- An important aspect for the development of CRISPR-Cas9 systems is the optimal expression of guideRNA and Cas9 and to find the right time point for activation.
- An important factor for functional pathways is the regulation. If the regulation is not optimal, it is possible that the microorganism does not use the pathway although it has all the required genes. Here, reduction equivalents and ATP production are crucial.
- More general: A technique that is also used for industrial purposes is the adaptive laboratory evolution. Here, a selective pressure is exerted in order to evoke specific features or to determine target genes or system vulnerabilities.
Takeaways:
- A well-suited organism for butanol production is C. saccharoperbutylacetonicum. Hence, this organism was chosen for later genetic engineering.
- One has to consider that transformation efficiency can decrease with plasmid size, so we could distribute the genetic material that we want to transform over several plasmid vectors to prevent that issue
- The RiboCas or allelic exchange system could be a promising alternative technology to transform our target organism if we are troubling with
- In vitro-evolution can be useful for improving our engineered strain according to its butanol-resistance and turnover
Dr. Michael Koepke – VP Synthetic Biology at LanzaTech
Dr. Michael Koepke is an expert on carbon fixing microbes and strain engineering at LanzaTech and was involved in establishing a process that generates ethanol from syngas. In contrast to our approach, their organism naturally possesses all the genes for a functional Wood-Ljungdahl Pathway and is able to assimilate syngas with it.
Why LanzaTech?
As LanzaTech uses similar bacteria as we do, we wanted to know how they upscaled their project and how their bacteria behaved in industrial production tanks. Furthermore, we were interested in the overall future and success of their process.
What did we learn?
- Industrial waste gases that would be emitted into the atmosphere can be assimilated by organisms and can be used for the biotechnological production of chemicals (such as LanzaTech is already doing it). This is climate friendly and sustainable and will be demanded increasingly, due to the severe problems we are facing caused by climate change.
- Anaerobic bacteria are robust to cultivate on an industrial level in anaerobic fermentation tanks.
- Anaerobic fermentation processes are easier to control than aerobic fermentation processes
- Due to the fact that bacterial production tanks can be located right at the waste gas source we reflected that CO2 emission by transport processes is prevented.
Takeaways:
- So far, we have been working with plasmid systems to transform our bacteria. To ensure the stability of the strain, we should integrate the DNA into the bacterial genome.
- The composition of syngas from the steel industry is relatively constant, whereas it gets harder to use waste gases from municipal waste. We should use syngas from the steel industry as substrate to start with.
- The product yield depends on the composition of the gas as well as the type of product. We will have to assess these components once we have constructed our bacterial strain.
- We want to use CO2, CO and H2 (Syngas) as substrates. We should learn more about the safe handling of these gases in the industrial environment.
Given the fact that LanzaTech produces ethanol with their bacterium, we think that the new strain that will be constructed by us, may be used to complement the existing technology and specialise in biofuel production. Furthermore, after detailed investigation of the strain, new possibilities for further products may open up that can expand the current repertoire.
Prof. Michael Helmut Kopf - Senior Research Manager at BASF
Prof. Kopf is Director of Alternative Fermentation Platforms and Senior Research Manager at BASF. BASF collaborates with LanzaTech in the development of sustainable processes and products via gas fermentation.
Why BASF?
Due to their collaboration with LanzaTech and their chemical knowledge, we reached out to BASF to learn more about their view on carbon-recycling technologies.
What did we learn?
An important aspect is the purification of the products synthesized by the fermentation process. As an example, a challenge of current fermentative butanol production is that the downstream processing is not as energy efficient yet as desired. Nevertheless, we found that “[...] ABE fermentation is the current main method to produce biobutanol [...]”[4].
- A high concentration of butanol is toxic for microorganisms. This is a main reason why respective fermentations, e.g. ABE and gas fermentation take place in a dilute environment. This however, leads to low product concentrations in the fermentation broth.
- In case of gas fermentation, the composition of the gas that is used as substrate for the fermentation process is an important factor for the efficiency of the production. Here, the stoichiometry of product formation from CO2, CO and H2 and the energy need of the microorganism must be considered.
- To use butanol as biofuel or for chemical applications, a purity of 99% is desired, but high product purity causes high energy consumption.
- Butanol production (fermentation and DSP) currently requires more energy than e.g. Ethanol because of its separation characteristics
In consequence of the above, it would be advantageous to improve downstream processing techniques and to engineer microorganisms to achieve high resistance to toxic fermentation products.
Takeaways:
- Because of given toxicity of the target product (here BuOH), a high space-time-yield (STY) combined with an in-situ product recovery is preferable over high-titer batch fermentation.
- Goals for synthetic biology and strain development are high product tolerance, low side product formation and good STY.
- Gas composition i.e. the amount of hydrogen available determines conversion efficiency in terms of carbon and hydrogen yield.
- Goals for DSP development are minimizing energy need and meeting the set purity specification.
Steel industry
We spoke to the Head of Division Efficiency of Resources and R&D Coordination of a German steel company (expert wanted to stay anonymous).
Why a steel company?
The steel industry is a major player in greenhouse gas emissions which is why it may be a good target for the application of a technology that could be developed with our target microorganism. Furthermore, the gas composition of steel waste gases is suitable to act as a substrate for acetogenic Clostridia. This is why we wanted to know more about the situation of steel companies and their attitude towards a carbon-recycling biotechnology for fuel production.
What did we learn?
So far blast furnaces with coke as reductant were used to produce steel, although the direct reduction method with H2 as reductant is an upcoming technology. Steel companies are retooling accordingly. However,
- Until the technology unfolds its full potential, many years will pass in which a transition solution would be admirable
- Once direct reduction with H2 is running as desired, large amounts of H2 will be needed. Probably energy will have to be imported to cover the need.
- A biotechnological approach such as the one we have in mind is desirable for steel companies if the microorganisms use gases with a high CO2 content
- H2 reduction and biotechnological carbon-recycling are not necessarily mutually exclusive in the future
Takeaways:
- We could optimise our target strain in a way that it uses a maximum amount of CO2 while still being energy-efficient and productive.
Dr. Florian Tiller - Ucaneo
Dr. Florian Tiller is the CEO of Ucaneo Biotech. This start-up has developed a biocatalytic membrane that captures CO2 from the air and which can be used to decrease CO2 levels in the atmosphere.
The direct carbon capture technology has aroused our interest as it is another way how CO2 can be removed from the atmosphere. Furthermore, captured greenhouse gases by the technology of Ucaneo could be used to feed our bacterial target strain.
Besides, Dr. Tiller told us from experience, what requirements have to be fulfilled to create a start-up and how the up-scaling of a process works. He also told us how to get funding and what investors are interested in. For this purpose, he explained to us how a pitch is constructed.
Dr. Karl-Heinz Maurer - CLIB
Initially, we reached out to Dr. Karl-Heinz Maurer to talk to an expert in the practical application of biotechnology. He is the chairman of CLIB, an industrial cluster which connects a huge network of biotechnological companies. He then referred us to other specialists from CLIB, which could help us out during our project.
Why CLIB?
CLIB is a huge organization for in the field where our project could be realized someday. It was important for us to hear out the expertise on how research projects in life sciences are brought to industrial stage. Since CLIB combines many experts from the research world as well as the industry, we hoped to reach the go-between here. Furthermore, some of the members are experienced in the industrial work with C1, which also was accommodating to us.
What did we learn?
To be sure about your basics, before you try to take the step to the industrial level. The experts pointed out to us that it is of great importance to have first checked all the risks that the method could face on an industrial scale. Helpful advises they gave us:
- Make sure, that the construct is integrated into the genome, to receive a stable industrial strain
- Size matters: fact-check how similar organisms behave on a huge industrial scale and talk to other experts
- Slowly step up the size of the production, to exclude any mistake from the start.
- Sort out the process engineering and consult experts before you plan to up-scale your project
To bring your technology to an industrial level takes time and accurate planning, as well as communication. The CLIB-experts really changed our point of view on the industrial industry and again ensured us about the importance of trans-disciplinary work.
Takeaways:
The talks with CLIB members:
- Adapted our planning for the continuation of the project.
- Provided us with important information for our implementation part.
- Helped us to frame our research in the industrial world of biotechnology and, accordingly, to adapt our communication about our project.
Life Science Factory Göttingen
Together with the iGEM Team TU Dresden, we visited the Life Science Factory Göttingen. This facility was founded by Sartorius but acts independently from the company. The purpose of the Life Science Factory is to promote research and development in the field of life sciences and to support start-ups.
What did we learn?
During our trip we learned how to create a start-up, what one has to consider beforehand and how the financing works. Furthermore, we heard how the Life Science Factory can support scientists to create a successful start-up:
- By providing modern laboratories including state of the art interior, offices and working spaces.
- By managing events and mentoring programs.
- By forming a founder community and promoting mutual exchange.
Takeaways:
We have learned a lot about how to successfully create a start-up and how mentoring programs and scientific-/founder-communities are helpful to gain a foothold more easily. This knowledge is important to turn an idea into reality and to make it accessible to others.
Reinhard Herbener - Umweltbundesamt (UBA)
WReinhard Herbener is an expert on fuels and resources at the Umweltbundesamt, the Federal Environmental Agency in Germany. As such, he has been involved in several publications on carbon neutrality and the assessment of decarbonization in the transportation sector.
Why Umweltbundesamt?
As an environmental agency, UBA has a good overview of current technologies and their benefits in terms of the Paris Climate Agreement. We talked to Reinhard Herbener to find out what role our project can play in achieving the climate goals and what factors are crucial to this.
What did we learn?
- It is already being recognized in the industry that something must be done for the environment. However, political regulations are partially lagging behind, especially internationally.
- Per definition there is a difference between biofuels and recycled-carbon fuels. Biofuels are fuels that derive from biomass which is not the case for recycled-carbon fuels.
- Legal regulations for biofuels can be found in the renewable energy directive (RED). However, recycled-carbon fuels are not included so far. In the RED it is defined that a greenhouse gas reduction of about minimum 70% compared to fossil fuels is normally required for biofuels. No calculation requirement for recycled-carbon fuels exists yet.
- Companies that produce large amounts of CO2 from fossil sources are subject to the emission trading system (ETS). This system foresees that a certain amount of CO2 emissions is allocated to such companies. If this amount is exceeded, companies must purchase additional certificates which are costly.
- Some recycled-carbon technologies require electricity and heat which is not sustainable if the electricity derives from fossil energy sources like coal.
- Aviation is regulated by the International Civil Aviation Organisation (ICAO) and also aims for a reduction of greenhouse gas emissions. However, long-distance flights will in the future still rely on liquid fuels that are similar to Kerosin → Biobutanol can be further processed to liquid fuels that can be used for aircrafts
- Aircraft fuels must be approved to guarantee their safety. This costs money and is time consuming. However, if a similar fuel is already approved, the process may be simplified and sped up.
Takeaways:
- We concluded that aviation will still rely on liquid fuels in the future. Similarly, heavy transport and shipping also require sustainable fuel solutions which we think could be achieved with our project.
- An alternative application for biobutanol is the chemical industry. Biobutanol can also be used to produce valuable downstream products in a more sustainable way.
Sources
[1] Lee, H., You, T. & Chen, C., 2022, Energy efficient design of bio-butanol purification process from acetone butanol ethanol fermentation. J Taiwan Inst Chem Eng 130, 104015.
[2] Liew et al., 2016, Gas Fermentation – A Flexible Platform for Commercial Scale Production of Low Carbon Fuels and Chemicals from Waste and Renewable Feedstocks. Frontiers Microbiol 7: 694.
[3] Saskia Tabea Baur, 2021, Construction of acid-producing Clostridium saccharoperbutylacetonicum strains by deletion, overexpression, and interfering with regulation of genes. http://dx.doi.org/10.18725/OPARU-42087
[4] Lee, H., You, T. & Chen, C. Energy efficient design of bio-butanol purification process from acetone butanol ethanol fermentation. J Taiwan Inst Chem Eng 130, 104015 (2022).3https://doi.org/10.1016/j.jtice.2021.08.003