Traditionally, university education is often theoretical and focussed on fundamental research, in iGEM we saw an opportunity to use our knowledge in science to make an impact on the world. One of the biggest challenges humanity will face in the coming decades is climate change. To have a big impact we sought to reduce the carbon footprint of a product everyone uses and uses a lot: plastic. At the same time we aimed to utilize an underused waste stream.
Two challenges… Cellulosic waste is an underused waste stream. A lot of cellulose is “lost” as it is burned for energy  , because there are currently no alternatives. Sources of cellulosic waste can be in the form of e.g. wood, algae or toilet paper. Current methods, as designed by Avantium for example, use acids to degrade cellulose to glucose. However, global desire for a biobased economy is influencing the industry; therefore the interest in bioplastics is growing. Polylactic acid, polyhydroxurethanes and polyethylene are being produced as bioplastics, but through expert interviews we discovered that these do not meet the current demands of the plastic industry. Conversely Styrene, one of the most used plastic monomers in the world, styrene, is not being produced in the biobased way.
... one solution
Through interdisciplinary brainstorming sessions we aimed to merge these two challenges into an opportunity.: using vast amounts of cellulosic waste for the production of bio based styrene (StyGreen). Due to our enzymatic approach, we bypass the need for acidic degradation
We thoroughly investigated different biomass sources in order to find the source most suitable for our project. After comprehensive research, we found three potential sources; wood, algae and toilet paper.
Our first idea was to process wood (lignocellulosic biomass). We approached Avantium, a company experienced with converting wood chips into glucose. Additionally, we analyzed the financial aspects surrounding wood pulp. We quickly found that the kraft technique, which is used for the conversion, is financially and environmentally expensive. If we wanted to use wood chips as our biomass source, StyGreen would not be environmentally friendly or economically feasible.
The second possible cellulosic biomass source was algae. We interviewed prof. Dr. Klaas Timmermans of NIOZ about their extensive research on algae. It is argued that future use of algae as a biomass source may be highly promising. However, as the industry is still in its infancy, lack of scale puts upward pressure on pricing. Therefore, this is currently not an attractive possible cellulosic biomass source.
Thirdly, we investigated “sludge:” toilet paper waste streams. Toilet paper is a second generation biomass source. It is underused and virtually valueless. This makes it a promising biomass source. KNN cellulose was enthusiastic about our project and provided us with their recycled toilet paper product (Recell): this consists of cellulosic biomass (99% cellulose) filtered out of sewage locally in the Wastewater Treatment Plant. To further investigate the environmental impact of this potential cellulosic source we looked into its production.
KNN Cellulose provided us with monsters of their product, which we tested in our lab. Degrading Recell without any preprocessing proved to be challenging. After literature review and expert interviews we decided to use Ball Milling . We successfully degraded ball milled Recell using one of our cellulases. Read all about that here
Carbon Footprint Analysis
We performed a thorough analysis of the carbon emission of a potential toiletpaper derived StyGreen, and compared these to the emissions of petrochemically produced styrene. Naturally, our primary emissions were caused by the energy which is used during the production process. However, due to the high energy consumption of the production of regular styrene, we are able to potentially improve the carbon emission by 71%! We'll explain the calculations in more detail here. Local availability, a favorably predicted carbon footprint analysis, proven degradation and under utilization of waste toilet paper make this the perfect source of cellulose.
One of the factors which influences the carbon emission of our production process is the mass balance: the amount of biomass required to produce a kilogram of StyGreen. Due to increased efficiency of “economies of scale”, upscaling our process to the bioreactor level is important. With the input of one of our partners (EV Biotech); we designed a bioreactor to make upscaling possible in the future, that enables our yeast to grow under ideal circumstances while optimizing styrene yield. The two main challenges we tackle 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 held together by hydrogen bonds. Apolar stacking interactions also contribute to insolubility. Both the polar and apolar interactions of cellulose strains can be decreased through phosphorylation of the 6 position or methylation of the 2, 3 or 6 position. However, this requires chemicals and preparations steps that greatly reduce profitability and increases the carbon footprint of StyGreen. Hence, we decided to keep these alterations to a minimum. The solubility of cellulose can 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 site where the cellulose binding domain of our enzyme scaffold binds to cellulose.
The second challenge we faced is, that the apolar styrene is likely to localize into the lipid bilayer which will eventually destroy the membrane, limiting the amount of StyGreen that can be produced in a single batch before the cells die. Styrene may get actively transported out of the cell, using the s.cerevisiae native Pleiotropic Multidrug Resistance system as styrene can be toxic to the DNA through intercalation and covalent binding. The PDR5 system is be able to export styrene, although it cannot yet be expected to efficiently export large quantities. Therefore, 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 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. This can simply be introduced by terminating fermenter stirring. The apolar phase contains extracellular, apolar impurities which can be removed through reverse extraction with water: styrene is the most apolar compound in the entire mixture. The styrene is separated from the ethyl acetate through evaporation. The ethyl acetate and the reverse extract are recycled into the bioreactor system: no fresh apolar phase is required.
Through use of our metabolic modelling we discovered that trading culture growth for styrene production was not beneficial. 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 substrate chorismate, thereby increasing the flow to phenylalanine. Continuous fermenting has many advantages over a batch or fed-batch approach, because of the relatively slow growth on cellulose, the tryptophan knockout and most importantly the biphasic medium with easy styrene removal. 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.
Investigating bio-commodity alternatives
In order to consider how well StyGreen addresses the problem versus other potential solutions we investigated bio-commodity alternatives. We contacted Avantium and BioBTX, producers of bio-aromatics and biopolymers. These innovative biotech companies explained their technologies, and gave us a tour of their pilot plants. The pilot plant of Avantium turns wood chips in glucose, which is used to produce PEF, a new alternative to PET. BioBTX has found a novel method to produce aromatics from waste. Avantium and BioBT work with chemicals to, where as we work with microorganisms exclusively. The disadvantage of working with microorganisms, these firms told us, is that biological processing is less robust. However, when it works, it has the chance to be a lot more sustainable than working in a chemical way.
Potential Buyers of StyGreen
To understand the needs of 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 the kind of plastics used in the toy industry. Next to a start-up, we also talked to a multinational in toy producing. You can find that conversation here. We aimed to find producers of durable non-single use plastics for our StyGreen. The price point, as well as the ideology, of the product makes it more suitable for use in durable products. Therefore we work together with toy manufacturers such as Lego and Ludos Imaginem.
Investigating possible downside
No plastic solution can be presented without taking into considering the downside of plastic usage: plastic waste. Our oceans and environment are being filled with the consequence of our plastic addiction. Throughout our project we have had many discussions about this obvious problem, even presenting a Ted talk on the “Cause, Size and Solution to our Plastic Problem” at “Noorderzon Festival.”. Here we educated the public on the negative consequences of plastic use. Feedback from public engagement further motivated us to focus on durable plastic solutions. In the foreseeable future plastics, because of their broad applicability, are still highly in demand for durable applications. These do not only include Lego, but also medical equipment and precision electronic devices. For these purposes, we are determined to provide a durable viable solution: StyGreen.
Humanity’s hunger for styrene has been steadily increasing over the past decades. More and more fossil fuels are needed to create these vast amounts of styrene . By recycling toilet paper, we are able to create StyGreen: a modern and sustainable solution to our “plastic problem.” Due to interest from both buyers and suppliers, we expect this technology to turn the plastics market from black, to green.
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 Tianjiao Qu et al.: Ball Milling for Biomass Fractionation and Pretreatment with Aqueous Hydroxide Solutions. ACS Sustainable Chemistry and Engineering 5, 7733-7742 (2017)
 Mckenna, R., Thompson, B., Pugh, S., & Nielsen, D. R. (2014). Rational and combinatorial approaches to engineering styrene production by Saccharomyces cerevisiae. Microbial Cell Factories,13(1). doi:10.1186/s12934-014-0123-2