It was a long way to the idea of the current project of the Unicamp Brazil team. Even though it was not our university's first time in the competition, we were students without any experience in this world and, to add, we were all isolated at home due to the Sars-CoV-2 pandemic. But despite the odds, we were determined to make it happen.
Beyond simply working with synthetic biology unaware of what our project could give back to the world, we wanted our work to have a real impact; to be a solid alternative to the challenge we chose to address. So we tried to think alongside our ideas for the project how they could be translated into real-world action. We later found out that this has a name within the competition: Human practices.
The first idea came together with the first formation of the team. Since our first project ideation sessions, we were clear that addressing and reinforcing environmental and sustainability values should be integral parts of our project. Within this agenda, the topic "recycling and solid waste management" was one of the first to emerge, and it could not be otherwise: the global production of plastics, the main urban solid waste, has increased at a worrying pace in recent decades, jumping from 200 million metric tons in 2002 to 367 in 2020. Of this amount, only a small slice of 9% is properly recycled, while the other 91% is either incinerated, dumped in landfills, or simply discarded into the environment. There is therefore a serious problem regarding solid waste management on the planet, and out of a desire to offer a plausible solution to increase the percentage of waste that can be recycled, we decided to work with waste and biomolecules management.
In the background research to narrow down our object of study, we discovered that packaging contaminated by organic waste, such as greasy pizza boxes, dirty napkins, and the like, is hardly recycled due to the difficulty of cleaning such waste before the recycling process. This being a problem very close to everyone and with the potential to be explored through the lens of synthetic biology, our first idea took shape: use the tools of synthetic biology to clean discards of organic molecules that hinder the recycling of these materials.
Along these lines, our starting point was to look for stakeholders who deal with this waste and could provide us with valuable insights on how to address the problem and where we could intervene with a solution. Furthermore, familiarizing ourselves with the problem and how it is seen through the lens of those who deal with it on a daily basis is fundamental to developing any impactful product, regardless of the area worked on. So we looked for recycling centers, researchers, sanitation companies and other entities committed to a sustainable agenda.
The company Corpus Saneamento e Obras gave us the first opportunity to visit their recycling centers and learn how the workflow for reworking the waste works. The visit to the waste management laboratory of Professor Miriam Gonçalves Miguel, a Unicamp researcher in solid waste management, was also important in this sense. Their availability was fundamental to building the first understanding of what we were dealing with and what are the factors that add complexity to the problem of inappropriate management of these materials.
Another important contact with the theme was the participation in the "Waste Expo 2021" event, a fair held annually in São Paulo that brings together a variety of companies from the sanitation and solid urban waste management sector. Just as companies take the opportunity to probe new technologies and opportunities, we also took the chance to network with companies in the sector and understand the market dimension of our project.
This first couple of feedback gave us valuable insights on the way forward. We found out that organic "contaminants", especially grease, hinder the recycling process, and a pre-treatment of solid waste is still costly and little implemented in recycling centers. We also found that for some types of waste the biggest obstacle is not recycling itself, but the logistical difficulty of transporting large volumes of the material in front of its low density, as is the case of expanded polystyrene (EPS). Besides being a material difficult to reuse, large amounts of polystyrene require a costly logistics operation for companies in the sector since 95% of the volume of this plastic is air, which makes it more expensive and unprofitable to recycle.
At this point, we were already familiar with the topic and the gaps in which we could act. The Styrofoam problem in particular caught our attention. Expanded polystyrene is one of the most produced synthetic polymers annually and also one with the lowest recycling rates: only 12%. The large non-recycled surplus contributes to the pollution of water bodies or is accumulated in landfills. Incineration, the destination of most discarded Styrofoam, is also not a good alternative given the release of possibly carcinogenic gasses such as polycyclic aromatic hydrocarbons.
Virginia's 2019 team sought alternatives to polystyrene. Most innovatively, their Transfoam project has developed strains of Escherichia coli that can transform styrene monomers into PHB, a bioplastic with several potential applications. Although brilliant, there is a gap in the project: there is no known enzyme capable of depolymerizing polystyrene into its forming monomers. Without this a priori step, the whole process of reworking polystyrene into PHB simply does not happen. In meetings, we then decided to change our project to another type of waste and continue the work of the Virginia team in 2019, looking for the piece that would complete the puzzle: an enzyme capable of depolymerizing polystyrene.
Our first step was to search the existing literature for candidate enzymes. In fact, such enzymes exist but have not yet been well characterized; only possible families of proteins related to this function have been described.
We also found very interesting works describing the use of polystyrene as a carbon source by some specific genera of larvae, in particular, Tenebrio molitor: when raised with polystyrene as the only carbon source, the larvae gradually adapt their gut microbiome with bacteria able to digest the polymer. We even put this to the test by breeding our own larvae with polystyrene!
In further research, it was possible to identify some candidate bacteria responsible for the depolymerization of Styrofoam and therefore potential chassis for our idea, with emphasis on bacteria of the genus Streptomyces. In the Institute of Biology of our university, we quickly obtained strains of this bacterium (special thanks to Professor Domingos da Silva Leite), and in conversation with Professor Danielle Biscaro Pedrolli, we also discovered difficulties in working with this bacterium. Streptomyces is a species whose genetic manipulation is a little more complicated, with quite variable gene expression and poorly reproducible colonies.
Thinking about the solubilization of the Styrofoam in culture medium and which solvents to use was another key step, also with a great contribution from Dr. Danielle. Solvents such as acetone and limonene are commonly employed for this purpose, but the toxicity of these materials for potential chassis such as Streptomyces and Bacillus subtilis is a variable to be thought of and tested.
We already had information about potential chassis and solvents for the solubilization of polystyrene, but the lack of knowledge of the enzyme was still a problem. Contacting the Virginia team responsible for the Transfoam project, we found out that not knowing the enzyme responsible for depolymerization was indeed a bottleneck in the project, but they were excited about our idea of continuing the work and willing to help in any way possible.
In contact with Dr. André Damasio, an enzymology specialist from Unicamp, we talked about strategies for prospecting for this enzyme from the bacteria components of the T. molitor microbiota. Although very productive, the meetings were not very encouraging. Despite being technically possible, it would be unfeasible to perform complete bioprospecting work to isolate the enzymes capable of depolymerizing polystyrene with the time and resources we had available for competition. Added to this are the logistical difficulties of large volumes of polystyrene, one of the impediments to Styrofoam recycling that we became aware of with the first visits to the recycling centers.
Finally, as we were aligned with the idea of a truly applicable project, we sought to check the market viability of our idea. We had the unique opportunity to have a very prolific meeting with Colgate's innovation and sustainability sector. With them, we could understand more deeply the need to do surveys such as, for example, what would be the origin of polystyrene to be reworked into PHB, strengthening cooperatives with communities to be benefited from our work with the removal of this polymer, and also with job generation. Aligned with the ideas of responsible innovation, the prospect of an alliance with communities and collectors of recyclable materials brings not only positive impacts for these parties but also speaks directly to the ideas of a circular economy: where economic development is associated with better use of natural resources, through the optimization of manufacturing processes and prioritization of more durable, recyclable and renewable inputs.
Fortunately, there was excitement about the idea and a good acceptance of our project. It was very motivating to receive feedback that with financial and logistical planning in place, our project holds great potential for transformation that is marketable and feasible.
Adding up all the feedback from both experts in the field of waste management and enzymology and professionals in the marketplace, we have again reached a turning point. We realized that although technically feasible, we do not have the resources and time required to perform the full bioprospecting of a new enzyme. However, we remained firm in our desire to work with waste management in some way that was no less innovative than previous attempts. With new research on what was being developed in Brazil within this area, we casually got in touch with Dr. Hernane da Silva Barud, responsible for the Laboratory of Biopolymers and Biomaterials (BioPolMat) of the University of Araraquara - UNIARA, in which one of its research lines focuses on the development of scaffolds based on biopolymers for use in tissue engineering, whose flagship is bacterial cellulose.
Our first contact with bacterial cellulose was in a meeting with Dr. Hernane Barud and his students, where we talked about the cellulose-producing strains, their difficulties on a large scale, and how synthetic biology could act to solve these problems, such as the high cost of production due to a large amount of glucose needed. In our laboratory we cultivated Komagataeibacter strains in conventional HS medium, however also became acquainted with Dr. Barud´s work which involves agroindustrial processing chain waste to optimize bacterial cellulose production in static culture (technology proprietary of the consortium BioPolMat, Biosmart, and, HB in collaboration with JBT Corporation). This technology, in combination with the engineering of Komagataeibacter, has the potential to significantly reduce BC production costs making it more accessible to the population. This was the spark that ignited the idea of starting the Cellulopolis project.
After some time, we made a visit to BioPolMat to learn in practice from the preparation of inocula to the cultivation of cellulose. Moreover, we got to know the headquarters of the startup BioSmart Nanotechnology LTD, making it possible for us to discuss several points about the challenges faced in its commercialization, applicability, and adhesion of bacterial cellulose products in the market.
But the application that really stood out was its use in hospitals to treat burns. Recently in Brazil, there has been a worrying increase in the number of accidents with fire thanks to the increase in the price of cooking gas. With prices unaffordable for people in vulnerable situations, many of them found themselves with no options but to use a wood stove for cooking, a method much more prone to the risk of severe burns. Talking directly to this social problem, we saw in our project the possibility of adding an alternative to the treatment arsenal of hospitals that deal with burn cases daily. Bacterial cellulose has already been evaluated as having curative actions in the treatment of burns, and with the correct conditioning there is nothing to prevent its use in hospital routine. We even tried to contact researchers and hospitals working in the burn treatment area to evaluate the applicability of our project, but unfortunately we did not get any response.
The Celullopolis project was challenging both due to the inherent complexities of the project itself and the logistical difficulties faced by the team. However, it was also a period of intense learning about the potential of this biomaterial. In recent meetings about the project, other ideas emerged about what could be improved in the cultivation of cellulose blankets for the treatment of burns. If the adjustable synthesis of cellulose could be combined with the concomitant synthesis of molecules components of the extracellular matrix of the dermis, such as collagens of various types, the healing of burns could be even more efficient with the treatment by the blankets without increasing costs for patients or hospital units.