Porter Analysis

Too analyze our position in the market, and find our opportunities we did a five forces analysis. Here we looked at the strengths of our buyers, suppliers, substitutes, competition and new entrants. We found that we have power over our suppliers as there are a lot of ways we can find cellulose. However, for the buyers the scale is very important, which is why the buyers have a very high switching cost. This can be a threat to our technology. For new entrants, a lot of prior knowledge is needed to enter the market, which is a strength for us. The substitutes produce different kinds of plastics, but as the plastic market is so big, and so specific, this is not much of a threat.

EV Biotech

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 great 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 an idea to create a continuous bioreactor. Over the summer we had ten meeting with them to discuss our progress on the project. Next to technical help, we also had a great deal of help by 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 people in the business.

We also had a great deal of help accessing the big network of EV Biotech

Ludos Imaginem: George van den Nieuwenhuizen (19th September 2018)

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 that they need. This way, we can optimize our StyGreen to the demand of the customers. 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. However, they do not want to compete with the food industry, and therefore encourage our way of working with sludge.

"If you find a sustainable solution for current plastics, you will be bigger than Elon Musk"

KNN Cellulose: Yme Flapper(31st August 2018)

We've visited KNN Cellulose! After doing a lot of research in possibilities in biomass, we found a company which produces Recell® . This is an innovative new product from recycled toilet paper which consists for more than 90% out of cellulose. They asked us whether there is a possibility if we can use their product to create styrene. This way we really use waste streams to create StyGreen! The company develops biomass 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 so we can test, and they are very interested in our results.

KNN provded us with a sample of their product

NRK: Martin van Dord (24th July 2018)

NRK is the Dutch Federation of plastic & 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 the NRK facts and figures 2017 the use of bioplastics is ca 20 kiloton (1%) of the 2.000 kton used (2017). Main problem for the use of bioplastics is the price issue. The price is up twice as high as virgin plastics. In order to contribute to the goals of the Climat Agreement of Paris, the objective is to lift this percentage to 15% in 2030. Mr. van Dord thought our project was very interesting, since we find a new way to produce bioplastics. However, he was wondering why we would focus on styrene that much. Why not create a new bioplastic with even better qualities? He also stated the business case should be a part in the project in order to get a better insight in the potential of genetically manufactured/engineered bioplastics and the scale of economic feasible production facilities. NRK also put an article about us on their website

Only 1% of the plastic use is a bioplastic

Bioclear Earth: Jeroen Tideman (27th July 2018)

We had contact with the employees of Bioclear Earth, who gave us great tips on the financial aspects of our project. Because pure cellulose is more expensive than glucose, we needed to find a waste source 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 duo to its imago. After this, they explained to us how the market for enzymes works, and also brought us into contact with various people in the market. Next to the people from the enzyme field, they told us about various parties who are working on turning cellulose into glucose. Lastly they gave us the tip to use glucose instead of cellulose for our project. However, we thought this was not feasible as we do not want to be regarded as competition to the food industry.

Why don't you use toilet paper?

Avantium: Ronny Pals (31st August 2018)

On 31.08. Rianne, Jens, Benno, Bram and team associate Tjerk Douma visited the Chemistry Park Delfzijl where we had a meeting with Avantium. Avantium is breaking down wood chips chemically to hemicellulose, glucose and lignin. Their technology furthermore allows them to break down cellulose with acid to glucose monomers in a one pot reaction with high yields while recovering the acid. We are trying to do exactly the same but enzymatically, employing our cellulosome. We agreed to test the suitability of the glucose Avantium made from wood chips for growth medium for s.cerevisiae. Beyond that we learned a lot about the process of valorizing innovations in general. They gave us a lot of insight 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 very 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 therefore that we have to consider the expenses and environmental implications of our cellulose preparation (grinding, autoclaving, phosphorylating) as well, rather than just our yeast growing on cellulose.

It was amazing to be in a real biomass pilot plant

BioBTX: Pieter Imhof (25th July 2018)

As suggested by the Science Shop, we got into contact with Pieter Imhof of BioBTX. This company is also making chemical intermediates out of biomass, but this company uses a chemical way of working. They explained to us how they use pyrolysis, and combined this by a catalytic conversion step. This way they were able to reach aromatics yields of approximately 30-70%, with BTX (Benzene, Toluene, Xylene) yields ranging from 5-40%, yielde dependent on feed and process condition used. On our project, Mr. Imhof thought that the process of turning glucose into styrene not have enough yields to be economically feasible. However, he thought the cellulose to glucose step in one pot combined with glucose to styrene could be a interesting improvement. Next to this, he explained that with every chemical step, there is more CO2 emission, the magnitude dependent on reaction conditions. So whereas our method would not be able to have industrial needs, it would likely be greener than the chemical process of refining biomass, and significantly better than fossil based. These steps are bound 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 into deep into this, and gave us great references about their own research.

The biological way is less feasible then the chemical way, but probably more sustainable

Fablab: Winand Slingenbergh

As the iGEM team Groningen aims to produce styrene, a plastic monomer, making actual plastic products from our monomer was an obvious idea. As the quantities of styrene we managed to produce are not large enough for industrial applications 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-gluconase 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/.

We developed a kit of building blocks for ABS polymerization

ZAP: Drs. R.J. van Linschoten (4th September 2018)

One of the first steps when considering upscaling is finding a suitable location for a pilot plant. The province of Groningen is a strong agricultural and industrial area. Therefore, the province of Groningen can support the conversion of waste streams from biomass in high-end products. ZAP stands for Zernike Advanced Processing. ZAP offers an unique test environment for bio-based experiments in the northern region of the Netherlands, they offer the facilities to setup a pilot plant. ZAP tries to act on the signal that we need to lessen our reliance on fossil fuels. ZAP is an innovation cluster which is located on the Zernike Campus of the University of Groningen, this can lead to a symbiotic relation between the knowledge of the university and the industry of the surrounding area. We met with drs. R.J. van Linschoten, director of the Zernike Advanced Processing, on the 4th of September. With him we discussed the prerequisites for setting up a pilot plant.

The province of Groningen supports the conversion of waste streams

NIOZ: Prof. Dr. Klaas Timmermans (27th August 2018)

Right now, a lot of research is being done on culturing green algae. If they succeed in culturing green algae on the open sea, this would mean 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, they are involved and research in the Netherlands and far beyond the Dutch border on topics like biology, physics, chemistry and geography. We spoke with Prof. dr. Timmermans about our idea and the potential usage of cellulose from green algae for our iGEM project. At this point in time green algae are mainly cultured for the proteins and partly for their carbohydrates content. However, the cellulose is a residue at the moment that they have not found a purpose for at NIOZ. If we succeed in our iGEM project, we could close the loop in green algae culturing, in that all fractions are used. Prof. dr. Timmermans told us however, that there is still a long way to go in being able to culture green algae on the open sea. On top of that, the separation of the different fractions (proteins, carbohydrates, etc.) proves to be difficult at this point in time. Conclusively, the potential upside is enormous, once the research on culturing green algae has developed further, we are able to tap into an infinity source of cellulose and use all the fractions of the cultured green algae. A win-win situation.

NIOZ hasn't found a purpose for the cellulose yet

RIVM

The Dutch Governmental Institute for Public Health and Environment (RIVM) wants to stimulate the Dutch iGEM teams to consider the broader effects of their project on the world around us. This includes investigating ethical, societal, and technical aspects surrounding the project. They asked us to adhere to the Safe-by-Design concept, that states that safety should be an integral part of each project, to be considered during every phase and aspect from the early beginnings to the end.

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. Jaco was especially curious how progress on the Safe-by-Design assignment was progressing compared to last meeting, and how we are integrating the associated principles into our project. We described what changes we have 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 talked about 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 got some tips on how to improve our infographic.

These remarks pushed us to make the switch

Tjerk Douma (28th June 2018)

On the 28th of June we met with Tjerk Douma, who is a Master student in Energy and Environmental Sciences. Tjerk explained to us the importance of a Life Cycle Analysis (LCA), and how everything is taken up in that. For us, it might 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 several more meetings after this. This resulted in our Carbon Footprint Analysis

We agreed that Tjerk would help us with the LCA

Drs. Karin Ree (11th July 2018)

Drs. Karin Ree is a member of the Science Shop in Groningen. The Science Shop connects ambitious students to companies who are looking for academic research. As we are looking for the connection to the bioplastic industry, Karin was able to give us great tips on who we should contact. She helped us to find people inside and outside of the university who we could contact. Next to this she has send us a many papers on the sustainability of bioplastics.

Karin was able to give us great contacts

Prof. Dr. Gert Jan Euverink (8th August 2018)

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!

In the CaDOS project, cellulose material is used to drain water from sludge

Prof. Dr. Katja Loos (5th July 2018)

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’s okay to throw it away anywhere because it’s “biodegradable”, 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 also has 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:

1. Nylons
2. Polyurethanes
3. Polyamides

PLA is only adding to the problem!

Prof. Dr. Francesco Picchioni (3rd Oktober 2018)

We went to Francesco Picchioni to ask about his opinion on styrene and our project. Did he see benefit in it, or would he think this was unfeasible? He explained to us that styrene is a very good material for various reasons. The first is that it is transparent, which is why you can color it easily with other chemicals. Also styrene has a aromatic ring and has pi-pi stackings of these rings. This makes that the plastics with styrene have a high TG (Glass Transition Temperature). These connections are way stronger than in PET and PLA, because these have esther connections. No other plastics have these characteristics, and therefore styrene is irreplaceable. Right now, styrene is not recycled very much, as the price is more expensive than making new styrene. However, because styrene is a thermoplastic, it is easily recycled when the market pull would be stronger. Dr. Picchioni was very suprised that styrene was able to be made in a biological way. If this could be created with a high yield, this would be a major discovery and he would be very interested.

"If you can make me a few kilo's, you can come back to me!"

Prof. Dr. A. J. M. Driessen

To gain more insight in optimizing a yeast strain we met with prof. dr. A.J.M. Driessen, head of the molecular microbiology department at the University of Groningen. We discussed how we could best implement and optimize our idea. Prof. dr. Driessen gave us very helpful directions. With the help of his feedback we went from the idea of two separate coexisting yeast strains (one producing glucose from cellulose, and one producing styrene from that glucose), to one yeast strain doing both processes. Also, we discussed multiple knock-outs we could implement to gain higher yields. Finally, prof. dr. Driessen proposed the usage of CRISPR-Cas9 technique to us, to genomically integrate the genes we wanted to introduce, instead of using plasmids. Prof. dr. Driessen brought us in contact important people as well as providing us with additional laboratory space.

His help brought us from two seperate yeast strains, to one yeast strain doing both processes.

Prof. Dr. B. Poolman (1st June 2018)

On the first of June one of our team members met with prof. Bert Poolman of the Enzymology research group to discuss styrene toxicity. We had found a number styrene exporter in literature from the organism Pseudomonas putidaDOT-T1E we wanted to discuss expressing these in Saccharomyces cerevisiae. 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 doing an evolution experiment in S. cerevisiae to decrease the sensitivity of our yeast towards styrene.

He suggested an evolution experiment in S. Cerevisae to decrease sensitivity

Shreyans Chordia (27th September 2018)

On the 27th of September one of our team members had a meeting with Shreyans Chordia a PhD at the Biomolecular Chemistry & Catalisys group. Shreyans works on styrene production in Escherichia coli and provided us with a 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 coculturing our cellulose degrading yeast strain with his styrene producing E. coli to convert cellulose to styrene in once bioreactor. He offered to help with setting up the experiment and conducting it. Furthermore he got us in contact with Balin Fridrich working on the degradation of lignocellulose.

Shreyans was able to produce styrene at quite significant levels

Prof. Dr. Marco Fraaije (20th July 2018)

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 detection of cellulase activity. We had a fruitful discussion about the assay described in the paper. One of the points that were discussed is the feasibility to detect cellulase activity with the assay while using our intact yeast cells instead of purified proteins. Finally, professor M. Fraaije provided us with the possibility for assistance, usage of the lab and supplied the materials required for the experiments.

One of the points was the feasibility of the assay

Dr. W.C. Szymanski (27th September 2018)

W.C. 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 found for the phosphorylation of cellulose at the 6th position. Furthermore, he was of great help executing 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.

Szymanski helped us to optimize the phosphorylation of cellulose

Intellectual Property Office RUG (8th October 2018)

To see if we could patent parts of our project, we had contact with the IP center of the University of Groningen. There were 3 important subjects we discussed: inventorship, newness and inventiveness. The first subject we had to discuss was inventorship. Who contributed substantially to our project? Next to our own members, to what extent were the supervisors part of our idea? After consulting other iGEM teams, we found that the patent is normally shared with the supervisors, and we made this decision as well. For the newness of the project, it was important that all the things we wanted to patent were not published already. We encountered problems with our own disclosure here, as we did a lot of work in outreach and education. The most difficult part of a patent is the inventiveness. Since we were putting several methods of degrading cellulose together this was an important part. Here, we also had to think about the financial side of the patent. Would companies pay to use our technology? We discussed with several investors, and found several companies interested in our project.

The most difficult part was inventiveness