Introduction
Currently, the preservation of the planet and ecosystems is a topic of discussion. Technological solutions are urgently required to address and potentially reverse the prevailing issue of microplastic pollution. This is where our project comes into action by which we can offer various applications to prevent further accumulation of microplastics in our water bodies, such as rivers, lakes and ponds. To achieve this, we are using bacteria to produce Curli fiber that will capture microplastics from the environment.
Possible real life application
Biofilm on road drain filters
During our project, we discovered that most of the microplastics in natural environments come from tire degradation (rubber microparticles).1 Therefore, we decided to look for an application that could stop these particles directly at their source. In Switzerland, filtration chambers have already been installed in street drains to capture larger plastics, such as bottle caps and packaging. However, these filtration chambers are unable to trap microplastics.
Application
We propose two potential applications: growing the biofilm directly on the inner surfaces of filtration chambers or cultivating it on filters placed at the inlet of pipes leading to the lake. The core concept of this application is that water coming from roads and rich in rubber micro-particles enters these filtration chambers. Then, the microplastics would be captured in the biofilm and the filtered water would reach the lakes and rivers.
End users
The final product can be installed on a street drain. Thus, both the government and small private individuals who are interested in minimizing their environmental impact can benefit through our implementation idea. Furthermore, should the industry producing the filtration chambers be interested, it might be possible to introduce the biofilm at an industrial production level. For example, the filters on which the biofilm grows could be produced directly by these industries, so that there would not be the problem of how to grow biofilm on an existing filter in these facilities, but it would simply be replaced by one already equipped with biofilm. Proper installation requires expertise, as working with road drain filters and filtration chambers isn't a typical task for the average individual.
Biofilm inside waste water treatment plants (WWTP)
Another possible application of our project would be to place biofilm inside water treatment tanks. In fact, one of the steps in water purification facilities is the biological basins. In this stage, microorganisms, including bacteria, are used to degrade organic matter.
Application
The slow circulation of water in these biological basins makes them an ideal environment for the growth of our biofilm, as there is no risk of it being damaged by the current.
Furthermore, the application of biofilm could increase the capture of microplastics within these ponds, as there are currently no specific techniques to capture them in Switzerland.
The application works as follows: the biofilm, generated within the biological tanks in wastewater treatment systems, would begin to capture the microplastics within it.
This process would lead to the progressive formation of larger and larger aggregates consisting of microplastics and biofilm.2 As these aggregates become larger and heavier, they would tend to settle to the bottom of the tanks, similar to what happens with other organic matter digested by bacteria. Subsequently, this denser matter would be removed, following the procedure already in place in this type of plant, by drying and subsequent incineration.
→ Read more: STEP Morges interview
End users
In this scenario, the individuals responsible for interfacing with our product and overseeing its installation are the personnel working at wastewater treatment facilities. However, it's important to emphasize that the advantages stemming from the capture of microplastics would extend far beyond the confines of these facilities, ultimately benefiting the entire community whose wastewater is processed at these purification stations.
Biofilm in washing machine filters
A large amount of microplastics that leak into the environment are coming from our daily self-care products and cosmetics. These microplastics are then released when we shower, brush our teeth, and do our laundry. An estimate of the mass of plastic fibres released through textile washing each year is around 150,000 kg*.3
Our towels and clothes release fibers through wear and tear3, especially during the washing and drying process, including PET fibers.
→ Read more: Mitrano
*Data taken from a study done in Finland.
Application
Our biofilm can be applied inside washing machines or tumble dryers with the aim of capturing microplastics coming from daily self-care, cosmetics, and cleaning products as well as synthetic fibers that are released from clothes. Ideally, the biofilm will be grown on the existing filters of these domestic appliances. Alternatively, an additional filter can be placed at the outlet of the machine, just before the start of the pipes carrying the waste water.
End users
Our product will primarily be used by people who own household appliances and want to reduce their environmental footprint. Using our product might mean that these appliances need to be replaced more frequently for regular maintenance.
Quality concerns
All these application ideas have great potential and would certainly decrease the amount of microplastics that inadvertently end up in various aquatic ecosystems. By implementing these solutions in real life, it would be possible to prevent microplastics from accumulating within the various food chains and causing damage at different levels of these chains.
For these solutions to be as effective as possible, it would be necessary to improve certain aspects of our biofilm. It would be necessary to test the biofilm's adhesion to different types of materials and to check its resistance in the presence of strong water currents, as would happen inside a washing machine or manholes in seasons with heavy rain.
Another very important factor that would need to be tested is, for example, after how long a given biofilm surface would reach saturation, so that users can be informed of the timing of replacement of the various filters.
→ Read more: Challenges and Improvements below
Safety concerns
While our applications are designed with safety in mind we must always remember that we are dealing with genetically modified organisms and before that with bacteria.
To prevent bacterial cells or even genetic material containing antibiotic resistance from being lost in the environment, we could resort to a cell-free system.
→ Read more: cell-free system below
Challenges and improvements
Large-scale biofilm production:
In order to make our implementation ideas possible, especially those in street drains and washing machine filters, it is necessary to increase biofilm production.
In fact, during our months in the lab, we only worked with small volumes of medium that allowed us to grow small quantities of biofilm.
→ Read more: Results - biofilm capturing microparticles
Simultaneous introduction of several peptides:
It could be interesting to introduce several peptides simultaneously.
This would allow the biofilm to target different types of microplastics simultaneously.
Search for a peptide targeting PET:
Together with Prof. Mitrano and DePoly company, we discussed the possibility of further developing the project to capture PET fibers. We can improve our biofilm by introducing a specific peptide to bind PET polymers. This could make the adaptation of washing machine and dryer filters even more customized and potentially improve its effectiveness.
→ Read more: Human Practices - Mitrano & DePoly
Biofilm growth on different surfaces:
In order to prove the feasibility of our implementation, it would be necessary to test the ability of the biofilm to grow on different surfaces, such as existing filters, or in the inner walls of the filter chambers of street drains. We are particularly confident that the biofilm can successfully install itself on plastic surfaces as it has been modified to bind to plastic. Furthermore, Curli fibers naturally attach to hydrophobic materials, which would be ideal for our implementation.
Testing the biofilm's resistance to large flows of water:
Since both in drains and washing machines the flow of water can be very strong, it is necessary to test when our biofilm can manage this.
Stability of the biofilm in other environments:
The stability of the biofilm within culture environments other than Lysogeny Broth (LB) needs to be tested.
This is especially necessary because our implementation ideas require the biofilm to grow on a plastic surface in the presence of other substances or submerged in a waste water. Furthermore, the biofilm needs to be tested whether it can tolerate any chemicals that are frequently found on the roads or the washing products.
Biofilm maximum binding capacity test: It is necessary to test after how long a given amount of biofilm reaches its maximum saturation of microplastics. In this way, it would be possible to estimate the feasibility of our implementation ideas, particularly for capturing microplastics.
Integration with recycling systems:
While currently our project does not include a plan to recycle microplastics, a question would arise; what do we do now with all these microplastics we have captured? Our biofilm is compatible to be coupled with a recycling technique such as the one proposed by DePoly company or with other iGEM projects, such as the Heidelberg 2023 team.
Our project is one step of many, no just to capture, but to also facilitate the next steps towards a sustainable solution for the microplastics issue.
Cell-free system
To prevent E. coli cells or genetic material from our engineered bacterial cells from being dispersed into the environment, potentially causing unforeseen ecological problems, we could turn to a cell-free system. In fact, we could remove, through different techniques, the bacterial cells from the biofilm they created and use only the extracellular matrix rich in fibers, such as our engineered Curli fiber, for our applications. In this way, we would not have to worry about the biological hazard that our biofilm might entail, but at the same time take advantage of its microplastic trapping functionality. To create this cell-free system, we found three possible techniques in the literature. The first strategy would consist in cutting the fibers produced by the bacterium with the help of an enzyme. In Bacillus subtilis, the SipW peptidase can recognize and cleave the signal peptide in the TasA subunit, the main biofilm component.4 In this way the biofilm fibers simply dissociate from the bacterial cells.4 Alternatively, we could use salts, such as SDS and GdmCl, to kill the cells and then filter the engineered biofilm to obtain a 'Curli aquagel' that could be modeled and used in the different situations we propose.5 As a last option, it would be possible to obtain a Curli fiber separate from the bacterial cell by removing the C-terminal of the CsgF subunit, responsible for binding to the CsgB subunit, beforehand (Figure 4). Both genes are part of the operon we worked on during the project.6 In this way, the engineered Curli fibers would self-assemble in the extracellular space, forming the matrix without attaching to the cells.
Interconnection with other projects
During one of our collaborations (collaboration with the Stockholm team) we became aware of another iGEM 2023 team's project, the Heidelberg team, that treats the problem of microplastics in the environment.
It would be very desirable for us to create a connection between the two projects as part of theirs could solve our question: once the microplastics are recovered thanks to our biofilm, where do they go?
Our idea would be to use their 'helper strain' to upcycle the peptides of PP, PS and PE captured by our biofilm into PET. It would then be possible to send the upcycled-PET to the company, De Poly, we have already contacted to recycle this material without the need to generate further waste.
→ Read more: Heidelberg Wiki
→ Read more: Human Practices - DePoly
Conclusions
Throughout our project we have worked to take into account the fact that our biofilm could have an impact on different levels, for example, on industrial water treatment plants, on a state level as in the case of street drains, or in the lives of private individuals such as in household appliances. Our goal is to prevent the accumulation of microplastics in the environment and this can be achieved much more effectively and efficiently by working together with all the stakeholders to tackle the different angles of the environmental microplastics accumulation problem.