Template:Groningen/Human Practices
To get a good overview, we invite you to have a look at our tree of thoughts. This tree catches ideas as new light in its leaves, and by making the right choices, it grew to great height. We furthermore had great conversations on how to let our tree grow further after iGEM has ended; how to scale up our product StyGreen, and which safety procedures we have to keep in mind. For the StyGreen project, Human Practices was not a box that needed to be ticked. Moreover, it was a tool to integrate our project into the real world. As the production of plastics can be a sensitive subject, Human Practices was important from the start of our project. By reaching out to numerous stakeholders, from suppliers to buyers and start-ups to multinationals, we have gained insights into how the plastic world works, and how StyGreen fits in this picture. As Human Practices is not a binary subject but a continuous and constant process, we here provide a summary of the biggest influences on the design of our project. Additionally, many insights were also gained by talking to our friends, family and complete strangers. One of the first questions always was: “why more plastics?”. We have discussed this subject with several experts and investigated the advantages and disadvantages of different plastics. We looked into ‘biodegradable’ plastics, as well as chemically created bio-plastics.
To investigate our position and opportunities on the market, we performed a Five Forces analysis. Here, we looked at the strengths of our buyers, suppliers, substitutes of our product, competition and new entrants. The results indicate that we have beneficial position towards our suppliers as there are multiple sources of cellulose. However, the buyers have a high switching cost, which is a potential threat to our novel technology. For new entrants, a large amount of prior knowledge is needed to enter the market, which is one of our strengths. The substitutes of our product are different kinds of plastics, but as the plastic market is large and specific, this is not a great threat.
The young, Groningen based biotech startup EV Biotech offered to collaborate with us in many aspects. Represented by Linda Dijkshoorn, Agnieszka Wegryzn and Sergey Lunev, EVBiotech was present at multiple meetings with our subgroups. Linda had fantastic tips about structure and organisation, and helped us to set up a SCRUM way of working. Agnieszka is an expert on modelling and helped a great deal with the flux balance analysis. Sergey helped us to set up the concept of a continuous bioreactor. Over the summer we had ten meetings to discuss our progress on the project. Next to technical help, we also had a great deal of help accessing the big network of EV Biotech. As a special honor, we were invited to the official opening of the new EV Biotech office. Here we had the possibility to pitch our project to several experienced business people.
Creating a bioplastic that is able to compete with conventional plastic means tapping into a market with unlimited potential. For example, 19 billion Lego elements are produced every year. This equals 2.16 million elements every hour, or 36.000 per minute. This premium toy-giant with an annual revenue of 4.8 billion USD (2017) is highly interested to swap their current ABS feedstock to a more sustainable non-crude oil derived source. The Vice-President Materials of a Toy Multinational was able to have a sit down with us, despite a very full agenda, to discuss what drives companies when making transitioning decisions, what toy companies look for in materials and what to take into account throughout development.
While diving into the many applications of styrene, what most interested us was an application of styrene as raw material for a non-single use plastic. This was one of the drivers which pointed us towards Lego bricks: durable playing bricks for playful development. It turns out our visions align. During the course of our project, Lego has published various articles and press releases about their ambition to produce plant-based plastic playing bricks. The mentioned Vice-President Materials was happy to sit down with us to share views on the future of biobased plastics, as well as discussing some opportunities and limitations with regards to their production. Our discussion was highly insightful. Some highlights which will shape the future of our project:
According to the VP Materials we are operating at the right time. Speaking of an ever-increasing visible framework and the fact that we are currently operating in a more circular economy which will have large consequences.
Ludos Imaginem is a new company which creates toys with which you are able to create something out of your own imagination. Because they recently started, they are really interested in making their product as sustainable as possible. Ludos is willing to invest in us when we have the first results, and has shared the data about the styrene they need for their product. This way, we can optimize our StyGreen to the demand of the customer. We keep in contact with Ludos to update them on the developments in our process. Ludos is very interested in a biobased way to create plastics. They do not want to compete with the food industry, and therefore encourage our way of working with sludge.
We visited KNN Cellulose! After extensive research into possibilities of biomass, we found the company that produces Recell®. This is an innovative new product made from recycled toilet paper which consists of over 90% cellulose. KNN asked us whether there is a possibility to create styrene from their product. In this manner, we can really use waste streams to create StyGreen! The company develops biomass-derived chemicals and is looking for new innovative and sustainable ways of production. GMO technology fits this profile. KNN provided us with a sample of their product for our experiments and they are very interested in our results.
NRK is the Dutch Federation of plastic and rubber converters, with 20 different sub associations and 400 member companies. We talked to Martin van Dord, innovation consultant at NRK and Topsector Chemie. According to the NRK facts and figures (2017) circa 2000 kilotonnes of plastics were used in 2017, of which circa 20 kilotonnes (1%) were bioplastics. The main problem associated with the use of bioplastics is the price, which can be up to twice as high as that of virgin plastics. In order to contribute to the goals of the Paris Agreement, the objective is to lift the market share of bioplastics to 15% in 2030. Mr. van Dord thought our project was very interesting, since we came up with a new way to produce bioplastics. However, he was wondering why we would focus so much on styrene. Why not create a new bioplastic with even better qualities? He also stated that the business case should be a part in the project in order to get a better insight in the potential of genetically manufactured or engineered bioplastics and the scale of economic feasible production facilities. NRK also published an article about us on their website
We have been in touch with employees of Bioclear Earth, who gave us great suggestions on the financial aspects of our project. Because pure cellulose is more expensive than glucose, we needed to find a cellulose-rich waste stream which we could use in our process. They came up with the idea to use recycled toilet paper, which can not be used for other purposes due to its public perception. Subsequently, they explained to us how the market for enzymes works and also connected us with various people in the market. Additionally, they told us about various parties that are working on the conversion of cellulose into glucose. Lastly they gave us the suggestion to use glucose instead of cellulose for our project. However, we decided this was not feasible as we do not want to be competing with the the food industry.
Rianne, Jens, Benno, Bram and team associate Tjerk Douma visited Chemical Park Delfzijl where we had a meeting with Avantium. Avantium is chemically breaking down wood chips to hemicellulose, glucose and lignin. Their technology allows them to break down cellulose with acid to glucose monomers in one reactor with high yields while recovering the acid. We are trying to do exactly the same, but enzymatically by employing our cellulosome. We agreed to test the suitability of Avantiums glucose for growth medium for our cells. Beyond that we learned a lot about the process of valorizing innovations in general. They gave us many insights regarding the financial and technical bottlenecks that stand between a promising idea and a large scale profitable industrial process. We were impressed by Avantiums technology as it is robust, works with almost any type of wood and requires only very little material preparation, especially in comparison to our enzymatic approach. An important take-away for us was that we have to consider the expenses and environmental implications of our cellulose pretreatment (grinding, autoclaving, phosphorylating) as well.
As suggested by the Science Shop, we got into contact with Pieter Imhof of BioBTX. This company produces chemical intermediates out of biomass, however they use a chemical process. They explained to us how they use pyrolysis and combine this with a catalytic conversion step. In this way they are able to reach aromatics yields of approximately 30-70%, with BTX (Benzene, Toluene, Xylene) yields ranging from 5-40%, with yields dependent on the feedstock and used process conditions.
Regarding our project, Mr. Imhof thought that the process of turning glucose into styrene does not have high enough yields to be economically feasible. However, he thought that the conversion of cellulose to glucose in one reactor, combined with glucose to styrene conversion could be an interesting improvement. Additionally, he explained that CO2 is released at every chemical reaction step, the magnitude depending on reaction conditions. So, whereas our method would not be able to meet industrial needs, it would likely be greener than the chemical process of refining biomass, and significantly better than a fossil based process. These steps come together in the Life Cycle Analysis, which can be found on the wiki and in given references. Mr. Imhof explained to us that we should not go too deep into this and provided us with helpful references about their own research.
Photanol is a platform renewable chemicals company that utilises proprietary engineered cyanobacteria to process carbon dioxide (CO2) and sunlight into valuable chemical products. They have performed a lot of research in the laboratory, and are scaling up now. They aim to demonstrate that their product works on an industrial scale, and Wilmar elaborated on the process from their concept to where they are now. The concept of Photanol started at university. Together with the university, they went to the University Holding, to get an initial investment. From this point on a biorefinery plan, business plan and LCA were made to attract more investors. He told us that it is important to have a good business plan and LCA when you look for investors, because these are the things that people will look at. Another good way to get income is using different subsidies. Photanol now has an industrial partner with whom they work together to scale their project up further. Wilmar adviced us to contact the GGO bureau and to keep close contact with them, as they know everything about safety and regulations. Especially in the phase where we are in now, it is good to contact them so we can put the safety into the design of our project. One example is that a GMO can never outcompete its wild-type. Besides that, there are two important permits you have to keep in mind; the GMO permit and the environment permit. These take in total about 6 months to get!
Unipol is an European producer of EPS (expanded polystyrene, also known as styrofoam). They produce 90.000 tonnes of high quality EPS annually. Styrene is their main raw material. They are a member of EUMEPS, PlasticsEurope and OIK and are ISO 14001 certified. Unipol is interested in StyGreen, as styrene is their main raw material. Therefore, StyGreen would allow them to significantly increase the sustainability of their EPS production. We have presented our research to Unipol and they are highly interested and enthusiastic about our project. Hence we have acquired a substantial investment from them. Their financial investment as well as interest in our technology as a large industrial player is highly valuable to the entrepreneurial success of our project. We plan to continue our quest to produce biobased styrene in a scaled-up industrial setting.
As we aim to produce the plastic monomer styrene, making actual plastic products was an exciting idea. As the quantities of styrene we managed to produce are not large enough for industrial applications yet, we found an interesting partner in Fablab, a 3D printing venture. Fablab is an open-source, global network that originated from an MIT course titled ‘How to make almost anything'. They have stayed true to this ideal and offer a wide variety of plastic and wood working techniques in their laboratories.
3D printing with ABS plastic is possible, but it has some drawbacks, hence we decided to collaborate with Fablab Groningen without actually using StyGreen for 3D printing. We quickly realized that 3D printed biological structures can be of great educational value. Therefore, we made prints of the most important enzymes in our project: the cellulose binding domain, the endogluconase, the beta-glucosidase and the Phenylalanine Ammonia Lyase. We also printed some of their ligands and matched them size wise to showcase where the pocket with the active site in the enzyme is and which chemical alteration is happening.
We also developed a kit of building blocks for styrene, butadiene, acrylnitril and divinylbenzene that can showcase the process of copolymerization through magnets. On top of that our mascot Styrene Steve was 3D printed multiple times and given as present to some of our sponsors as a nice gesture and to keep iGEM in people’s minds. All structures we designed with FabLab are open source and can be found on their website https://www.thingiverse.com/.
One of the first steps when considering upscaling of a process is finding a suitable location for a pilot plant. The province of Groningen is a strong agricultural and industrial area. Therefore, the province can support the conversion of waste streams from biomass into high-end products. ZAP, the Zernike Advanced Processing innovation cluster, is located on the Zernike campus, along with the University of Groningen, the Hanze University of Applied Sciences and a number of companies. This unique location offers a great opportunity to share and increase knowledge. ZAP offers a test environment for bio-based experiments in the northern region of the Netherlands, where they provide the facilities to set up a pilot plant. ZAP is interested in acting upon the need to lessen our reliance on fossil fuels. We met with drs. R.J. van Linschoten, director of the Zernike Advanced Processing and discussed the prerequisites for setting up a pilot plant.
At present, extensive research is carried out on the culturing of green algae. If green algae can be successfully cultured on the open sea, this provides a virtually unlimited source of cellulose. We arranged a skype meeting with Prof. dr. Klaas Timmermans, Senior scientist ecophysiology of seaweeds, head of Department Estuarine and Delta Systems (EDS) at NIOZ and Honorary Professor at the University of Groningen. NIOZ is the Royal Dutch Institute for Sea Research, and is involved in research in the Netherlands as well as far beyond the Dutch border on topics such as biology, physics, chemistry and geography. We spoke with Prof. dr. Timmermans about our design and the potential usage of cellulose from green algae for our iGEM project. Currently, green algae are mainly cultured for their proteins and partly for their carbohydrate content. However, cellulose is a residual product at the moment, for which no suitable purpose has been found at NIOZ. In case we can show a proof-of-concept in our iGEM project, we could close the loop in green algae culturing, so all fractions are used. Prof. dr. Timmermans informed us that there is still a long way to go before culturing green algae on the open sea is reality. On top of that, the separation of the different fractions (proteins, carbohydrates, etc.) proves to be difficult. All in all, the potential upside is enormous, once the research on culturing green algae has developed further. We are then able to tap into an infinite source of cellulose and use all fractions of the cultured green algae. A win-win situation.
The Dutch Governmental Institute for Public Health and Environment (RIVM) wants to stimulate Dutch iGEM teams to consider the broader effects of their project on the world surrounding us. This includes investigating ethical, societal, and technical aspects concerning the project. They asked us to adhere to the Safe-by-Design concept, which states that safety should be an integral part of each project, to be considered during every phase and aspect from the early beginnings all the way to the end.
The first meeting was with Korienke Smit, a policy advisor, and Niek Savelkoul, a trainee and member of the 2017 iGEM Wageningen team. This meeting took place on june 20th, 2018.
During this meeting we discussed our initial ideas about how we are planning to implement the Safety-by-Design concept into our project. We got some valuable tips and input, especially about the upscaling of our process, which brings a whole set of new challenges with it, something we had not considered until then. We were planning to use antibiotics, which might be difficult to upscale safely regarding antibiotic resistance issues. Styrene is toxic, and having massive bioreactors filled with styrene-producing yeast strains could be a danger to public health. We were already considering to use CRISPR-Cas9 to remove the need for antibiotics, but these remarks contributed to our decision to make the switch. Another factor was our plan to run evolutionary experiments to improve yields. Korienke had suggestions for this as well, and suggested looking into the 2017 Heidelberg iGEM team, that came up with a clever way to accelerate evolution using quickly-mutating phages.
At the end of the conversation we received some advice on how to pitch our idea: our main advantage is the reduction reliance on fossil fuels over traditional methods of styrene production, and the reduction of CO2 emissions.
The second meeting was with Jaco Westra, a coordinator of synthetic biology, and an expert on safety and GMO regulations. This meeting took place on august 14th, 2018.
We talked about our progress in the lab, which was going slower than expected at that time. Jaco was especially curious how progress on the Safe-by-Design assignment was progressing compared to the last meeting, and how we are integrating the associated principles into our project. We described the changes we had made, for example the consideration of using recycled toilet paper as cellulose source, or the plan to focus on toy makers as customers for our product. We discussed the best ways to market our product, and agreed that the focus should be on the reduction of CO2 emissions. Our plan is to do a Carbon Footprint Analysis to come to an exact figure, to make a better comparison. At the end of the conversation we also received advice on how to improve our infographic.
We sat down with Tjerk Douma, a Master student in Energy and Environmental Sciences. Tjerk explained the importance of a Life Cycle Analysis (LCA) and what factors are taken into account. We concluded that it may be interesting to look at the difference in the LCA of StyGreen and oil based Styrene. We agreed that Tjerk would help us with the LCA, and had regular meetings afterwards. This resulted in our Carbon Footprint Analysis
Drs. Karin Ree is a member of the Science Shop in Groningen. The Science Shop connects ambitious students to companies or academic research outside their field. As we were looking for connections to the bioplastic industry, Karin was able to give us great advice on who we should contact. She helped us to find contacts inside and outside of the university. Furthermore, she has sent us a various useful papers on the sustainability of bioplastics.
Gert Jan Euverink is the University of Groningen representative in the CaDOS project. Toilet paper in sewage material contains roughly 80% cellulose. In the CaDOS project, this cellulose material is used to drain water from the sludge, which improves the purification process. Furthermore, Euverink advises companies on the implementation of their technical ideas. His expertise has been helpful to previous iGEM teams, since he was a supervisor of the winning team of Groningen in 2012!
Biodegradable plastics, like PLA, are technically biodegradable but only under controlled
conditions. In nature they still take a long time to degrade on their own, only a bit faster than
for example polystyrene. However, PLA being “biodegradable” sends a message that it is
okay to throw it away anywhere because if its “biodegradability”, only adding to the problem.
Just recently, the EU has moved to ban single use plastics. Therefore, what we should do is
look into non-single use plastics. While polystyrene has numerous non-single use applications, the
stigma of it being used as disposable packaging material is not easily erased. Some
polymers that are nearly always single use include:
We went to Francesco Picchioni, an expert in the field of polymers, to ask his view on styrene and our project design. Did he see the benefits, or would he think it was an unrealistic design? He explained to us that styrene is a very good material for various reasons. The first is its transparency, which is why it is easily colored with other chemicals. Styrene furthermore contains an aromatic ring and pi-pi stackings of these rings. This leads to the fact that styrene-based plastics have a high TG (Glass Transition Temperature). These connections are way stronger compared to the ester bonds in PET and PLA. No other plastics have these special characteristics, and therefore styrene is irreplaceable. Right now, styrene is not recycled to a high extent, as the price of newly produced styrene is lower. However, because styrene is a thermoplastic, it is easily recycled in case of a stronger market pull. Picchioni was suprised that biological styrene production was feasible. If a high yield can be engineered, this would be a major discovery and he would be very interested.
In order to gain more insight into strain optimization, we met with professor Driessen, head of the molecular microbiology department at the University of Groningen. We discussed the best ways to implement and optimize our design. Driessen provided us with many helpful suggestions. For example, we went from the concept of two separate coexisting yeast strains (one cellulolytic, one producing styrene), to one yeast strain doing both processes simultaneously. Furthermore, we discussed metabolic engineering to gain higher yields, for example multiple knock-outs which we could implement. Finally, Driessen proposed to use the CRISPR-Cas9 technique to genomically integrate our genes of interest, instead of using several plasmids. Moreover, Driessen connected us to important contacts as well as providing us with additional laboratory space.
One of our team members visited professor Poolman of the Enzymology research group to discuss the possible effects of styrene toxicity in our design. During literature study we discovered the existence of styrene exporters and we wanted to discuss whether expression of such an exporter in S. cerevisiae would be beneficial for our design and styrene yield. Poolman pointed out that expressing prokaryotic proteins in eukaryotes is extremely difficult, but pointed towards the Pdr5 export protein and ABC transporters. Furthermore he suggested performing an evolution experiment in S. cerevisiae to decrease the sensitivity of our yeast towards styrene.
Shreyans Chordia, a PhD student at the Biomolecular Chemistry & Catalysis group, works on styrene production in E. coli and provided us with an E. coli codon optimized version of the PAL2 gene. Shreyans has been able to produce styrene in E. coli at quite significant levels. He suggested the possibility of coculturing our cellulose degrading yeast strain with his styrene producing E. coli to convert cellulose to styrene in one bioreactor. He offered to help with the experimental setup and conducting the experiments. Furthermore, he got us in contact with Balin Fridrich, who works on the degradation of lignocellulose.
Professor Marco Fraaije is an expert in the fields of biology, biochemistry, biotechnology and in particular enzyme engineering. His group published an extremely useful article for our project describing a fast and sensitive method for the detection of cellulase activity. We had a fruitful discussion about the assay described in the paper. One of the subjects that we discussed was the feasibility to detect cellulase activity with the assay while using our intact yeast cells instead of purified proteins. Finally, professor Fraaije provided us with the possibility for assistance, usage of the lab and supplied the materials required for the experiments.
Szymanski is an assistant professor, at the department of radiology and imaging, at the UMCG (University Medical Center Groningen). His fields of interests are molecular medical imaging and photopharmacology. Wiktor Szymanski was willing to help us optimize the protocol for the phosphorylation of cellulose at the 6th position. Furthermore, he was of great help performing the experiment and provided us with a lab and equipment for the experiment. The phosphorylation of cellulose was performed to increase the solubility of the polymer. The improved solubility resulted in an improved accessibility of the cellulosome complex towards the cellulose polymer. The cellulosome complex chops the cellulose polymer into glucose molecules. These glucose molecules are obtained by the yeast cells, as carbon source, and converted into styrene molecules.
To see if we could patent parts of our project, we contacted the IP center of the University of Groningen. We discussed three important subjects: inventorship, novelty and inventiveness. The first subject we had to discuss was inventorship. Who contributed substantially to our project? Besides our team members, to what extent were the supervisors part of our idea? After consulting other iGEM teams, we decided that the patent should be shared with the supervisors. For the novelty of our project, it was important that the to be patented subject was not published already. We encountered problems with our own disclosure here due to our outreach and education activities. The most difficult part of a patent is the inventiveness. Since we combine several methods of degrading cellulose together, this was an important part. Here, we also had to think about the financial aspects of the patent. Would companies pay to use our technology? We discussed this factor with several investors and several companies are highly interested in our project. 
Stakeholder Analysis
Porter Analysis
For buyers, production scale is very important
Meetings with companies
EV Biotech
We also had a great deal of help accessing the big network of EV Biotech
Interviewing the Vice-President Materials of a Toy Multinational (16th October 2018)
Your choice for a 2G feedstock highly excites me
Optimizing scale and reliable production are of utmost importance when producing a successful bioplastic
Ludos Imaginem: George van den Nieuwenhuizen (19th September 2018)
"If you find a sustainable solution for current plastics, you will be bigger than Elon Musk"
KNN Cellulose: Yme Flapper (31st August 2018)
KNN provided us with a sample of their product
NRK: Martin van Dord (24th July 2018)
Only 1% of the plastic usage consists of bioplastics
Bioclear Earth: Jeroen Tideman (27th July 2018)
Why don't you use toilet paper?
Avantium: Ronny Pals (31st August 2018)
It was an amazing experience to be in a real biomass pilot plant
BioBTX: Pieter Imhof (25th July 2018)
The biological process is less feasible than the chemical, but probably more sustainable
Photanol: Wilmar van Grondelle (12th September 2018)
It is important to have a good businessplan and LCA
Unipol
StyGreen would significantly increase the sustainability of our EPS production
Fablab: Winand Slingenbergh
We developed a kit of building blocks for ABS polymerization
ZAP: Drs. R.J. van Linschoten (4th September 2018)
The province of Groningen supports the conversion of waste streams into high-end products
NIOZ: Prof. Dr. Klaas Timmermans (27th August 2018)
NIOZ has not found a purpose for their cellulose yet
RIVM
These remarks pushed us to make the switch
Meetings with Experts
Tjerk Douma (28th June 2018)
We agreed that Tjerk would help us with the LCA
Drs. Karin Ree (11th July 2018)
Karin was able to provide meaningful contacts
Prof. Dr. Gert Jan Euverink (8th August 2018)
In the CaDOS project, cellulose material is used to drain water from sludge
Prof. Dr. Katja Loos (5th July 2018)
PLA is only adding to the problem!
Prof. Dr. Francesco Picchioni (3rd Oktober 2018)
"If you can make me a few kilo's, you can come back to me!"
Prof. Dr. A. J. M. Driessen
His advice brought us from two seperate yeast strains to one yeast strain performing both processes.
Prof. Dr. B. Poolman (1st June 2018)
An evolution experiment in S. cerevisae to decrease toxic effects from styrene
Shreyans Chordia (27th September 2018)
Shreyans was able to produce styrene at quite significant levels
Prof. Dr. Marco Fraaije (20th July 2018)
How to detect cellulase activity?
Dr. W.C. Szymanski (27th September 2018)
Szymanski helped us to optimize the phosphorylation of cellulose
Intellectual Property Office RUG (8th October 2018)
The most difficult part was inventiveness
