Applied Design

For Applied Design, we had a look into the world of Bioplastics. We wanted to know how they are produced now and if there are people looking to get better bioplastics. Next to this, we looked into the different sources of biomass.

Two problems...

The first problem is the underusage of cellulose. A lot of cellulose is lost by burning it. This can be in the form of wood, algae or toilet paper. Recent methods, proposed by Avantium for example, use acids to degrade cellulose to glucose. There is a lot of interest in a biobased economy, and the interest in bioplastics is growing. Polylactic acid, polyhydroxurethanes and polyethylene are being produced as bioplastics, but can be fitted as well in the existing industry. Styrene, one of the most used plastic monomers in the world, is not being produced in the biobased way.

… One solution

By combining these problems, we use the vast amounts of cellulose, and use this to produce a biobased styrene (StyGreen). Due to our enzymatic approach, we do not need big amounts of acids, and will not use energy expensive heating techniques.

The resource

We looked into different biomass sources to use, to find the one that is most applicable to our project. After a lot of searching, we came up with three potential candidates; wood, algae and toilet paper. Out of these three we continued with toilet paper, and found a supplier who was eager to work with us.

Our first idea to work with was wood. We had contact with Avantium, who has experience turning wood chips into glucose. Next to that, we had a look at the prices of wood pulp, which is produced by the kraft technique. We quickly found that the kraft technique is financially and environmentally expensive. If we wanted to use this, StyGreen would not be environmentally better and also too expensive.

The second possibility were algae. We talked with NIOZ about their research on algae. In the future we expect this to be our main source of biomass. However, as the research is not yet very sophisticated, we needed a more direct source of biomass.

The final idea was toilet paper. The recycled toilet paper that is provided by our partner KNN, is filtered out of the sewage. This is done locally in the Wastewater Treatment Plant. We looked into this process to see where our source is coming from, and get a clear idea of our product line. Source

1. Wastewater Treatment Plant. At this place the toilet paper is gathered.
2. Grit Removal. The solids such as sands and grit are removed.
3. Cellulose washer. The organic contaminants are removed.
4. Salsnes Filter. The cellulose particles are filtered by a cloth filter.
5. Cell Press. The cellulose particles are dewatered.
6. Hygienization. The product is hygenized to a EPA class A rating, so it’s safe to use.

In cooperation with KNN Cellulose, we received monsters of their product to test in the lab. To break down the cellulose to glucose, we couldn't get our enzymes working right away. To preprocess the toilet paper, we ran tests with Ball Milling and Phosphorylation. We had very promising results with this.

Upscaling

Together with one of our parters (EV Biotech), we designed a bioreactor to make upscaling possible in the future. To implement our microorganism in a working technology we need to find the best way of upscaling the production. For this we designed a bioreactor that enables our yeast to grow under ideal circumstances while optimizing styrene yield. The two main complications tackled through our design are difficulties of bringing cellulose into solution and extracting styrene from the system. Cellulose consists of many long chains of cellobiose polymers that from many hydrogen bonds. The contribution of apolar stacking interactions to the difficulties when dissolving cellulose should not be underestimated either. Both the polar as well as the apolar interactions of cellulose strains can be decreased through phosphorylation of the 6 position or methylation of the 2, 3 or 6 position. These alterations however require chemical conditions and preparations steps that would greatly reduce the profitability as well as the carbon footprint of our final product. Hence we decided to keep these alterations to a minimum. The solubility of cellulose can already be increased significantly when only 30 % of the 6 positions are phosphorylated. On top of that we aim to employ dry ball milling to reduce particle size and crystallinity of the cellulose while increasing the amount of amorphous regions. This is important as amorphous cellulose doesn’t form strong apolar interactions, which increases the solubility while also being the only sites where the cellulose binding domain of our enzyme scaffold can actually bind to cellulose.

The second problem our strain faces is, that the apolar styrene is likely to localize into the lipid bilayer which will eventually destroy the membrane (link MathijsT MD), limiting the amount of styrene that can be produced in a single batch before the cells die. We considered that styrene might get actively transported out of the cell, using the s.cerevisiae native Pleiotropic Multidrug Resistance system as styrene can also be toxic to the DNA through intercalation and covalent binding. Although the PDR5 system seems to be able to export styrene it cannot be expected to export large quantities of styrene at an efficient rate. Hence we decided to employ a biphasic medium in our bioreactor. In a biphasic medium an insoluble, organic phase is added to the aqueous medium. Ethyl acetate was an apparent choice as it can donate hydrogen bonds while not accepting them, making it immiscible in water while not denaturing extracellular proteins. Styrene has a high preference for the apolar phase (log P = 2,8) and hence will likely localize into the ethyl acetate phase which can then be siphoned off the system after phase separation which can be introduced simply by stopping of the fermenter stirring. The apolar phase will likely contain many extracellular, apolar impurities which can be removed through reverse extraction with water as styrene is the most apolar compound in the entire mixture. The styrene can be separated from the ethyl acetate through evaporation. The ethyl acetate and the reverse extract can be recycled into the bioreactor system so no fresh apolar phase is required.

Our metabolic modeling suggested that trading viability and culture growth for more styrene yield is only possible at a very bad ratio as most knockouts in the ARO5 pathways from shikimate to phenylalanine and eventually to trans-cinnamate and styrene introduce sizable metabolic stress onto the strain. Therefore we aim to limit growth and biomass by introducing tryptophan deficiency thorough a knock out which is beneficial as tryptophan competes with phenylalanine synthesis for the ARO5 substrates chorismate, thereby increasing the flow to phenylalanine. Because of the relatively slow growth on cellulose in general, the tryptophan knockout and most importantly the biphasic medium with easy styrene removal, continuous fermenting has many advantages over a batch or fed-batch approach. Running the bioreactor system therefore requires continuous cooling, stirring, phosphorylated and ball milled cellulose, tryptophan and other essential chemicals for medium. The system produces heat, yeast biomass and styrene.

Alternatives

Other options for creating bio aromatics and bioplastics we found in Avantium and BioBTX. These innovative companies explained their technologies, and showed us around in their pilot plants. Avantium showed us their new pilot plant in Delfzijl, where they turn wood chips in glucose, and after that to PEF, a new alternative to PET. BioBTX has found a new way to create aromatics out of waste. They don't need cellulosic waste however. Due to the insight in these companies, we understood our place in the world better. The big difference between Avantium and BioBTX, and us, is that those companies work with chemicals instead of microorganisms. The disadvantage of working with microorganisms, these firms told us, is that biological processing is less robust. However, when it works, it has chance of being a lot more sustainable than working in a chemical way.

Potential Buyers and plastic waste

To get the best possible link with our customers, we had contact with several potential buyers and discussed the possibilities with them. Ludos Imaginem agreed to share the datasheets of the granulate that they order, giving us new insights on what kind of plastics is used in the toy industry. You can find the ideas and conversations with the customers here. We aimed to find only non-single use users for our StyGreen. Due to the price of the product, it is not possible to use it for single use products. Therefore we aim for toys, such as Lego and Ludos Imaginem. These are used a lot, and it is not a problem if the plastics are a bit more expensive. Also, these products stay in the family for more generations and are not left behind on the street.

Carbon Footprint Analysis

To see if our project has a positive influence on the environment, we analyzed the Carbon emission of StyGreen, and compared this to the emission of regular styreen. Our main emissions were caused by energy which is taken by the process. However, due to the high energy consumption of the production of regular styreen, we are able to improve the carbon emission by 71%! We'll explain the calculations in more detail here.

Conclusion

The styreen consumption has been growing and growing over the last few years. More and more fossil fuels are needed to create these vast amounts of styreen. Be recycling toilet paper, we are able to create StyGreen, a new way of saturating the plastic market. The price will increase, but the CO2 emissions will go down. Due to the interest of both buyers and suppliers, we expect this technology to turn the plastics market from black, to green. Of course there is a very big downside of plastic usage. The waste. The oceans and environment are being filled with the different polymers. We think single use plastics are very bad, and there should be a solution for this. But we also know that we will always need plastics for other, more important purposes. These do not only include Lego, but also medical equipment and precision electrics. For these purposes, we want to create StyGreen.