MercuLess: How does it work?

Our solution is a transportable photobioreactor in which the cyanobacteria cells modify methylmercury from the contaminated water into less toxic and collectable forms of mercury: ionic and elemental. Cyanobacteria are photosynthetic microbes which means they can utilize energy from light and carbon dioxide making them an affordable, sustainable and scalable choice of host organism.

We engineered a species of cyanobacteria called Synechocystis sp. PCC 6803 to convert methylmercury into elemental mercury by adding non-native merB gene and controlling the native merA gene expression with a operon-based system. The merB gene codes for the MerB protein that cuts the bond between the methyl group and mercury. The merA gene codes for the MerA protein that reduces the ionic form into elemental mercury. You can read more about our construct design in the Design page.

In addition to the biochemical reaction of transforming methylmercury there are technical aspects to consider in our photobioreactor. We contacted Pihla Hasan who is a project manager working with CircLab which is a new Research and Development environment project. We had the chance to meet Pihla and her colleague Kirsi Koivula who is working with the same project. We gained a lot of insights regarding photobioreactors and what to consider when designing and operating them. On this wiki page we examine the different topics that came up in the conversation and how the topics should be implemented in our project. You can read more about our conversation with them on the Human Practices page.

Energy provided by the sun and carbon dioxide provided from CO2 emissions

Transportable and optimized design

When deciding on the design of our photobioreactor, light availability was one of the most crucial things to consider. After discussing different photobioreactors with both our PI Pauli Kallio and our external contributors Pihla Hasan and Kirsi Koivula, we decided that our system would be a tubular photobioreactor. Together we also had a look at current solutions and images of them, which inspired our design decision.

Our solution would be transportable so we can reach the different contaminated areas efficiently and no water would have to be shipped. The photobioreactor and the additional necessary devices would be in the container. The container roof would open during the day time to collect the energy from sunlight and close during the night to eliminate any light pollution therefore maintaining the natural conditions at the site.

Ensuring balanced system and preventing cell clumping

Mixing is a major factor in bioreactors because it keeps the conditions even and prevents cell clumping and the cells attaching to the walls of the reactor. In our photobioreactor the mixing would happen due to the water flow and added CO2 gas bubbles. (Huang et al., 2017)

Collecting and upcycling the mercury for new appliances

The methylmercury entering the system would be transformed into elemental mercury by the cyanobacteria cells before it is captured. Elemental mercury is liquid at room temperature but it evaporates due to its high volatility. The higher the temperature the more vapors are released. (Gaffney & Marley, 2014) The temperature inside the bioreactor would be at least room temperature in order to secure the optimal circumstances for the cells. This means elemental mercury vapors are released inside the bioreactor. These mercury fumes could be recovered through adsorption onto activated carbon. The advantage of transforming the mercury into its elemental form is its high rate of vaporization and low solubility to water. These attributes make it efficient to gather as it easily volatilizes onto the adsorbent. Collecting the elemental mercury in a concentrated form saves the economical and environmental costs concerning the final disposal. (Oehmen et al., 2014)

Mercury has been used for centuries in many daily appliances: electronics, dental amalgams and lighting and is still needed as a raw material for electronic appliances such as observation, surveillance and measurement appliances, and some cosmetics where it cannot be replaced. The final product of our solution, elemental mercury, can thus be upcycled for components of new products. (Häkkinen et al., 2021)

Confirming a closed system and biosafety

Our team is persistent in creating a responsible solution to a global problem. While using genetically modified organisms in our solution, we have to be especially precise with biosafety. In MercuLess, we ensure that neither our strain is contaminated nor our cells are released to the environment.

The mercury-contaminated water goes through a particle filter to ensure that external biomass and various particles do not enter and disturb the system. Biocontamination is a crucial part when working with genetically modified organisms and it is the most common concern of our stakeholders surrounding our project in addition to the overall use of GMOs. There are physical, chemical and biological containment methods. In our system before releasing the clean water to the environment, it goes through a filter to keep our genetically modified cyanobacteria cells in the reactor. In addition to this physical containment other appropriate safety measures would be used such as ultraviolet radiation to chemically kill the cell. Possible biological containment methods that could be used are the kill switches, genetic circuit-based controls of cell viability. (Chan et al., 2016) Given the large operating scale, adding molecules to the bioreactor to control the kill switch is neither cost-effective nor practical. Hence our system would use only physical and chemical containment methods, to ensure that no cells would be released to the environment whilst being economically reasonable.

Monitoring the parameters and optimizing them with the gathered data

To monitor and optimize this system we need information about the current circumstances and changes happening in the reactor. For this we need sensors. It is important to monitor the pH, the temperature, the levels of oxygen and carbon dioxide, the amount of mercury vapor, the pressure and the nutrient levels. The sensors would be located inside the reactor and near the filters. With the data gathered we can analyze the accuracy and the efficiency of the system and improve it further by doing more DBTL Design, Build, Test and Learn cycles. Improvements may be done to the construct as well as to for example the promoter to achieve a stronger and continuously active expression.

Individuals and whole industries as possible end users

Our primary end users are landowners who want to take care of their land and industries producing mercury wastewater. Finland is known for its many lakes and one third of the land area is marshlands. Marshlands have gathered harmful compounds over centuries and when they are ditched these compounds wash away with the runoff water to surrendering lakes and forests. When speaking with Arja Alikoivisto who is a forest owner and bioeconomy agrologist in Silva ry which is the Finnish association for the continuous growth of the forest, we learned that forests are an important livelihood in Finland and many are ready to protect them if it would bring them some kind of a profit. The European Union restoration law 2022 states that at least 20% of land and water area in the EU region should be restored by 2030. This increases the pressure for the governments and individual owners to remediate the bases of water. In the future, as knowledge about mercury and its presence in the environment increases, as does the need for safe water and living habitats, the need to remediate bodies of water that are toxicated by methylmercury may increase even more. Besides the legislation pressure when we talked with Risto Sulkava who is the chairperson and scientific director of Hiilipörssi, a peatland compensation company, we learned that there has not been a shortage of people willing to restore their peatlands. Nevertheless, there are also people willing to restorate their lands without any financial profit but they are scarce. You can read more about our conversation with Arja and Risto in the Human Practice page.

Besides individual landowners, our possible end users would be several industries that cause mercury pollution. For example non-ferrous metals production, cement production and by stationary combustion of coal produce large amounts of mercury emission every year. (Technical Background Report to the Global Mercury Assessment 2018, 2019) Laws against mercury pollution have been introduced over the years globally by the Minamata Convention and locally by the European Union. These new laws create a demand for mercury cleaning methods, which is why we believe our continuous cost-effective solution will interest several parties and different industries. Our solution to the mercury problem is more sustainable than alternative techniques and therefore we believe nature conservationist organizations and ministries would be interested in assisting with the initial infrastructure costs for this system to be spread to wider use.

Adaptable solution for various conditions

Our system can be used both in fresh and saline waters as cyanobacteria thrive in various environments. (Chorus & Bartram, 1999) The water from the natural source is applicable because cyanobacteria need only light and carbon dioxide for their sources of energy and carbon. This means that no expensive sugars are needed and scalability is easily achieved.

Our solution could be transported next to the most mercury contaminated waters. An example site would be Pyhäselkä, a part of the large natural lake Saimaa, in Joensuu, which is mostly contaminated by a pulp mill. (Mononen et al., 2022) Another example site in Finland would be one near a mining site that produces sulfate rich sewage. The sewage can increase the methylation of mercury as a by-product of sulfate-reducing bacteria, therefore causing enrichment of methylmercury in the waters. (Jeremiason et al., 2006)

Some of the mines also use marshlands as a filter for their pollutants. As these marshlands are used for long periods of time, the pollutants do not accumulate anymore but will be washed off and flow to other bodies of water. (Yaraghi et al., 2020)

Besides being used as filters, marshlands naturally gather harmful compounds. When speaking with Postdoctoral Researcher of Marshland Ecology Elisa Männistö from the University of Eastern Finland, we understood that mercury is not such a problem at marshlands in natural state. The mercury is bound into the natural biomass and is not an issue because it holds still and does not enter any food chain. Problems occur when this natural state is disturbed, by for example ditching, because the water flow can dissolve harmful compounds to nearby areas. Also, this risk is involved in restoration of marshlands because the returning water could have the same effect. Hence, one application of our solution is to use it as a part of the restoration process.

Mercury is also a problem on a global scale and the biggest sector causing mercury emissions is small-scale mining. (Technical Background Report to the Global Mercury Assessment 2018, 2019) For example in India, China and the Democratic Republic of Congo, the most mercury polluted areas are connected to artisanal mining sites (Zandt, 2022). Although we have focused our resources to research local problems, we believe our system could be used globally achieving a broader impact.

By monitoring, gathering data and analyzing the data we can modify both our organism and the gene construct to fit the circumstances and needs of various applications even better. Likewise the mechanical parts could be modified to a system that purifies waters from factories that produce heavy metal sewage, for example. This decreases the sewage costs of factories and makes work at water treatment plants more safe, energy efficient and environment-friendly. When used indoors, the system would use artificial lighting that could be produced sustainably by using for example solar panels.

Safety considerations and dual-use

Responsible solutions are safe and well-thought of. To ensure that our project is responsible we considered different safety aspects. Firstly we considered biosafety: our system would be a closed system to keep our cells in control. The European Union legislation controls the use of GMOs and their release but even without the legislation pressure we understand that biosafety and the control of GMOs are a vital aspect of responsible synthetic biology projects. Risk assessment is a critical part of biosafety therefore a report must be written before piloting and starting our operations.

Secondly, to make our project whole we considered the upcycling of mercury. Our system would gather the mercury but its journey does not end there. Mercury can be upcycled for example for new surveillance and measurement appliances. This means that the collection process must be joined with the appropriate downstream technologies for recycling.

Thirdly, because our project aims to clean large bodies of water a mobile purification system is the only reasonable choice. Our transportable truck would be brought to the contaminated site. Because our project is meant to promote nature’s wellbeing the system is to be designed to cause the least amount of disturbance to nature. Light, noise and other pollutants would be taken into consideration.

Although all iGEM projects aim for good, they can be misused. Our intention is to not promote irresponsible mining but to create clean and safe water for us all. Yet, as we live during the time of electrification, mining metals for electronics is vital. With our solution, mining could be more sustainable and reduce the current harmful ecological effect. Additionally, our system promotes the upcycling of mercury and hopefully in the future other metals. Besides mining, the solution we created could decrease the negative effects of marshland ditching, without justifying the increase of these practices. Ditching is known to decrease the carbon sequestration ability of the marshlands, thus enhancing climate change. It also causes a dramatic change to the ecosystem causing habitat loss and threatening biodiversity. MercuLess is meant for cleaning up the mess we have already made rather than making it worse.

The purified water makes the use of fish, also large predator fish, safe. With this we aim to promote hobby fishing and traditions. Nevertheless we do not want to promote loosely regulated professional fishing that causes disturbances in the ecological balances.

Promoting circular bioeconomy in multiple levels

Our solution follows the principles of circular economy. Although our host organism is photoautotrophic, it needs nutrients to reproduce and grow. Cyanobacteria may use eutrophicated waters from agriculture, such as sewage water of greenhouses, while simultaneously purifying them, for example from nitrogen and phosphorus, and thus promoting circular economy. An example of this kind of a solution can be found from Ruissalo, Turku where they purify water from an aquaponic greenhouse of nitrogen and phosphorus by using cyanobacteria. (Salazar et al., 2023)

As our host organism is a photosynthetic microorganism it has the unique ability to use only carbon dioxide and light. These organisms are the greenest and the most sustainable solutions synthetic biology has to offer because there is no need for an external source of carbon or energy. Because cyanobacteria use carbon dioxide as their carbon source, they function also as a carbon sink. Therefore, the carbon dioxide emissions of industrial sites can be bound into biomass by locating our solution near to these point sources.

While the cells are working in our solution they gather energy from light and carbon dioxide into themselves. When they have done their part in our bioreactor the collected energy in the cell biomass can be upcycled for industrial use. Biofuels, fertilizers and secondary metabolites like pigments can be made by using cyanobacteria biomass that has been used for wastewater treatment. (Bolatkhan et al., 2020; Sharma & Sharma, 2017) This promotes a circular economy and makes our solution even more responsible.

The purified water coming out of our system could be used for both agriculture and industry in addition to providing a safe habitat. Clean and safe water will be a scarce resource in the future, as climate change increases the need for irrigation and makes waters more acidic and eutrophicated.

Next steps and future prospects

Our project has immense potential to be continued beyond the iGEM competition by either one of the team members or other students of the Turku region for example in their master’s thesis. For example the Faculty of Technology in the University of Turku offers a master’s programme in Molecular Plant Biology, which would be a perfect programme in which to both continue our iGEM project and gain more knowledge about the use of photoautotrophic organisms.

The University of Turku, Åbo Akademi University and the Turku University of Applied Sciences have various different programs and collaborations with these would be ideal so that the project would continue and grow, for example, into a concrete water treatment system with the help of the Department of Mechanical and Materials Engineering. In addition, our project adds knowledge about the conditions, requirements and considerations that have to be taken into account when doing material or mechanical engineering with bacteria: to make better filters and other parts to our system, mechanical and material engineering are needed to support synthetic biology.

We will share our project results with different foundations that work with our topics and could be interested in continuing our work with cyanobacteria and bioremediation of local on global bodies of water and wetlands. Our work will provide a lot of new data for the field studying Synechocystis sp. PCC 6803, other photoautotrophic organisms and enable development of sustainable alternatives for different processes. With our study, we will get knowledge on how Synechocystis sp. PCC 6803 works with heavy metals, the insertion of different constructs to Synechocystis sp. PCC 6803 and how Synechocystis sp. PCC 6803 can be used to produce secondary metabolites.

With MercuLess we focused on the battle against mercury but our aim is that our system can be enhanced so it could be used in the fight against other heavy metals and harmful compounds in contaminated water. Cyanoremediation is a growing field and therefore we are positive that this will be possible in the near future.

When our system would be ready to use in practice, we hope that the EU has taken more courageous steps towards the use of synthetic biology in commercial use. Despite the current strict legislation we have thought about the opportunities and challenges our solution could face in the markets.

References

We took cues to our project from previous iGEM projects. Teams Minnesota 2014, Cornell 2014, Mit_mahe 2020, UFAM-UEA Brazil 2016 and TU Darmstadt 2020 did their projects concerning bioremediation of mercury or wastewater treatment. We delved into their wiki pages, contacted many of them and took inspiration from their work to come up with a more efficient and sustainable solution of our own.