iGEM aims to solve real-world problems using Synthetic Biology and this is only complete with a proposed implementation plan along with the solution, as a process of applying scientific discoveries to the real world to improve our communities and ecosystems, considering various aspects such as end-users, safety, and engineering.

The construction of a project implementation must be aligned with the socio-environmental context of the region of interest, since ideologies, technologies, and opportunities differ. It is therefore understandable that implementation is not a worldwide rule, as it must be made flexible for each country, varying according to the public rules that dominate the region and according to what is already applied. In addition, in order for the application to be feasible, the implementation must contain the information that will appeal to its target audience, justifying its use and guaranteeing its execution and security. To help in this process, we have constructed a sequence of questions that we believe a project should answer:

• What is your project's implementation and action plan?
• Who will benefit from the application and how?
• What is the demand for the topic?
• What problems are expected with the application and how can we avoid them?
• How can it be developed in a way that guarantees safety?

This process of building a project and its applications is not simple and requires a lot of communication with stakeholders and people in the field in order to have a consolidated theoretical basis.

Following these principles, the UNILA-LatAm team had an implementation construction process that required many touches until it was consolidated. That said, at the start of the process, the team identified the lack of monitoring regulations regarding microplastics in water treatment plants, making it possible to apply various projects using this justification. However, from the meeting that the UNILA-LatAm members had with the employees and managers of SANEPAR, the largest sanitation company in the state of Paraná and which was awarded the best sewage treatment plant in the country (check our Integrated Human Practices page here.), it was possible to identify concerns about applying biological methods in water treatment plants and they suggested targeting the application in sewage treatment, considering that biological methods are already used for this, in which microorganisms decompose the organic matter present, and where regulations for the use of organisms are not as strict as in water treatment, which is limited to physicochemical methods.

Therefore, our implementation aims to apply our genetically modified microalgae as an approach to mitigate plastic contamination in wastewater treatment through stabilization ponds.

• What are stabilization ponds?

Stabilization ponds are very similar to open bioreactors, except from the fact that they are used to eliminate contaminants, and not to optimize cultivation. They are sized within technical criteria that, when receiving raw sewage, subject it to biological degradation, mineralizing the maximum possible organic load. Biological treatment can occur under anaerobic, facultative or aerobic conditions, according to the availability of dissolved oxygen, the predominant biological activity, the influent organic load and the physical characteristics of each unit.

• Socioeconomic context

Wastewater stabilization ponds are widely used in developing countries, particularly in tropical regions, due to several socioeconomic and environmental factors. They are generally simpler and more cost-effective to build and operate compared to more advanced sewage treatment systems, such as wastewater treatment plants with sophisticated technologies. This makes them an attractive option in areas with limited financial resources.

The tropical climate often features higher temperatures and greater sunlight exposure, which can be favorable for the operation of stabilization ponds. Elevated temperatures accelerate the biological degradation of pollutants in sewage, while sunlight aids in the photosynthesis process of algae that may be present in the ponds, contributing to water oxygenation. Compared to mechanical systems that require pumps and aerators, stabilization ponds consume much less energy, making them more affordable in areas where electricity supply may be inconsistent or costly.

• Types of stabilization ponds
  • Anaerobic

  Anaerobic ponds are designed to receive such a high organic load that they are completely free of dissolved oxygen. They are used with great advantages as pre-treatment for waters with high solid content. The solids settle to the bottom of the pond, where they are anaerobically digested, and the supernatant liquid, partially clarified, is released into an facultative pond for further treatment (Australian Pond System).

  

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  • Facultative

  Facultative ponds are those that represent both aerobic and anaerobic conditions. Aerobic conditions are maintained in the upper layers close to the surface, while anaerobic conditions predominate in layers deeper in the pond. Although part of the oxygen necessary to maintain the aerobic condition in the upper part is supplied by atmospheric aeration, the majority is supplied by the photosynthetic activity of algae/microalgae, which grow in waters where there are large amounts of nutrients and incident solar energy. The decomposition of organic matter in facultative ponds is based on two fundamental biological principles: respiration and photosynthesis. A cycle is established in the pond in which photosynthetic organisms produce oxygen in the environment, and heterotrophic organisms use oxygen to degrade organic matter, which has carbon dioxide as a byproduct of this activity, necessary for photosynthesis. In the anaerobic zones of the pond, organic matter is degraded by anaerobic bacteria.

  

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  • Maturation

  Maturation ponds are used as a second stage of treatment after facultative ponds. Its main function is the destruction of pathogenic organisms. They are shallower, allowing the effective action of ultraviolet rays on microorganisms present throughout the water column. The factors that influence the process of removing bacteria, viruses and other microorganisms present in its liquid mass are: shallower depths, high penetration of solar radiation, high pH and high concentration of dissolved oxygen.

  

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• Stabilization ponds in Brazil

According to data from the Brazilian Institute of Geography and Statistics (IBGE), until 2017, biological sewage treatment processes in Brazilian municipalities totaled two thousand and thirteen, and of this total, 475 are anaerobic ponds, 817 are facultative ponds and 356 are maturation ponds.



• Advantages and disadvantages

  It is undeniable that the application of any project has advantages and disadvantages, so we bring some points to be considered in relation to stabilization ponds.

  • Advantages
    • Low Construction and Maintenance Costs: Stabilization ponds are generally cheaper to build and maintain compared to more advanced wastewater treatment systems;
    • Operational Simplicity: They are relatively simple to operate, which can be an advantage in areas with limited resources or less technically skilled personnel;
    • Low Energy Requirements: They do not require large amounts of energy for operation as the treatment processes primarily rely on natural biological processes;
    • Efficient Biological Treatment: Stabilization ponds are effective in removing organic matter and nutrients (nitrogen and phosphorus) through natural biological decomposition processes.
  • Disadvantages
    • Long Residence Time: Treatment in stabilization ponds can be slow due to the long residence time required for effective biological degradation;
    • Vulnerability to Weather Conditions: Extreme weather conditions, such as very low or high temperatures, can affect treatment efficiency;
    • Odors: Stabilization ponds can produce unpleasant odors;
    • Space Requirements: The need for extensive land areas can be a disadvantage in densely populated regions.

Considering the points discussed above, it's important to note that while stabilization ponds are a viable option in many contexts, they are not the ideal solution for every situation. In urban areas, more advanced and effective sewage treatment systems may be necessary. It's crucial to ensure that any sewage treatment system is properly designed, constructed, and maintained based on where it will be implemented.

We chose to use a stabilization pond as our implementation after carefully analyzing the potential of incorporating microalgae into existing wastewater treatment processes. This approach allows us to take advantage of the resources of sunlight and the country's tropical climate to enhance biological treatment. Additionally, we view it as an initial step towards addressing the issue of microplastics in sewage, especially since there are still many existing stabilization ponds in the country. Our goal is to gradually transition this concept to more advanced treatment facilities in the future.

• Technical aspects and biosafety
  • It is common to use stabilization ponds in systems. After preliminary sewage treatments, our idea would be to use a system containing 3 ponds, starting with the anaerobic one, followed by the facultative, where the microalgae would be applied, followed by the maturation pond;
  • An important aspect of our choice to work with stabilization ponds, in addition to all the points discussed above, is also due to the fact that the PET degrading enzymes would take a few days to degrade all the microplastic present in the wastewater, being similar to the way stabilization ponds operate, that is, over a period of days. Do you want to know how long it takes for our microalgae to degrade the microplastic present in the stabilization pond? Check out our kinetic modeling here.

  

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  • An extremely important point when we are working with genetically modified organisms is the form of biocontainment, which, in our environmental application, will be used so that we do not run the risk of letting our modified microalgae escape from the system and bring unexpected consequences.
    • Finding a way of biocontainment in a stabilization pond for Chlamydomonas reinhardtii was a challenge, no suitable killswitches were mentioned in the literature for this context. Although the stabilization pond is already a controlled environment, we thought about implementing a physical filter between the facultative pond and the maturation pond, making it harder for microalgae to pass through. Another option to ensure a lower probability of GMO release outside the controlled tank would be to remove the surface biomass, as the strain would be reinserted in the next treatment.
    • We also saw the possibility of a salt-induced killswitch, in which, upon detecting salt, the expressed endonuclease would act in degrading the genetic material of our microalgae. This was the best killswitch we found, and, even though it was the best, it would not be the ideal option in functional and environmental aspects, after all, for the size and volume of a stabilization pond, it would be necessary to add a lot of salt to achieve a minimum of detection, which, in addition to being inefficient, would be harmful to the environment. Therefore, future studies on more suitable forms of killswitch for microalgae applied in stabilization ponds should be developed to ensure better biosafety of our application. Check out our killswitch design here.
  • Often, when applying microalgae, researchers tend to collect and reuse the biomass for other purposes, such as producing feed, biofertilizers or biofuels. However, as our microalgae would be a genetically modified organism, we could not reuse it for any other purpose.
  • Despite our efforts regarding the possible application of Chlamydomonas reinhardtii in sewage treatments, we are aware of its sensitivity to variations in the environment in which it grows. During chassis selection, we prioritized presence in the region, characterization and availability of transformation protocols. In principle, Chlamydomonas reinhardtii, being a model for experiments, is an initial step so that we can later analyze the feasibility of adapting the genetic design in other microalgae with greater resistance capacity.
• Applicability and entrepreneurship

Applying a project in real life and getting away from the theoretical part can be challenging due to the many different aspects of the subject, such as existing regulations, the scaling process, creating a product at a working level, selling the material, funding and studying the stakeholders niche. One way that the UNILA-LatAm team found to develop the research in a more entrepreneurial way was through participation in scientific dissemination events and business meetings, since, in addition to product creation, the team makes it possible to create a brand image.

The implementation of the study is multilateral, requiring the participation of several areas together and transdisciplinary activities to move and encourage the need for attention that environmental factors require in order to contribute to a more sustainable future. The IGEM UNILA-LatAm team also became a non-profit organization (NPO) in 2023, with the aim of building an associative and corporate image in order to establish the scientific contribution to the community more actively in the entrepreneurial field.

In this way, it is possible to establish contacts with the legal and legislative sectors in order to come up with technical and administrative mechanisms that can help build a greener world. For this reason, encouraging research, participating in incubators, publicizing community events, attending conferences and plenary sessions, and meeting with public institutions are extremely important for organizing a broad ecological and entrepreneurial front.

• References

Environmental audit manual for domestic sewage treatment plants. Editora Qualitymark. 2002. Emilio Lebre La Rovere et al.

Sanitary Sewage: collection, transportation, treatment and agricultural reuse. Editora Edgard Blucher. 2003. Ariovaldo Nuvolari et al.

Septic tank and septic tank sludge: guidelines for defining management and disposal alternatives. Editora Abes. 2015. Eraldo Henriques de Carvalho et al.

SILVA FILHO, Pedro Alves da. Operational Diagnosis of Stabilization Ponds. Natal, 2007. Universidade Federal do Rio Grande do Norte, Centro de Tecnologia, Departamento de Engenharia Civil, Programa de Pós-Graduação em Engenharia Sanitária.

ARAÚJO, André Luis Calado et al. Operational and Efficiency Assessment of Stabilization Ponds in the State of Rio Grande do Norte. Natal, Novembro de 2011. Programa de Pesquisa em Saúde e Saneamento. FUNASA-IFRN-FUNCERN.

IBGE - Instituto Brasileiro de Geografia e Estatística. Pesquisa Nacional de Saneamento Básico - PNSB: Data from municipalities with sewage services through a collection network in operation and with sewage treatment plants in operation, by types and sewage treatment processes. 2017.