Team:Stockholm/Applied Design

iGEM Stockholm 2018 Wiki

Product Design

By utilizing effectively extensive feedback from experts and after numerous iterative circles, our team developed a versatile, innovative solution to detoxify wastewater from active pharmaceutical substances, Biotic Blue. Aquatic pharmaceutical pollution is a looming problem which accelerates antibiotic resistance and causes adverse ecotoxic effects on marine ecosystems. The main source of pharmaceuticals ending up in the water is human consumption, an inescapable activity for our well-being. The key to limit or even prevent pharmaceutical pollution is to detoxify wastewater from pharmaceuticals before released to rivers or the sea. Solutions for treating wastewater from pharmaceuticals exist, but their adoption is either still posing threats to the environment or it’s too costly. To come over these barriers, we developed Biotic Blue, an enzyme-based solution for degrading pharmaceuticals. What makes Biotic Blue unique, is the fact that it can be implemented before the biological treatment stage in wastewater treatment plants (WWTPs). Implementing it at this treatment point prevents the development of antibiotic resistance in bacteria, inhabiting the activated sludge. Sulfamethoxazole-free water leaves the WWTPs and does not pose an ecotoxic threat to the aquatic environment when released. The immobilization of our mutant enzyme on magnetic beads and the smart employment of a magnet makes Biotic Blue energy efficient and cost-effective. However, the greatest potential of Biotic Blue to be broadly adopted by WWTPs lies in its capability to embed a combination of immobilized enzymes, enabling it to purify wastewater from a mixture of pharmaceuticals.

Problem Description

Over the past years, global consumption of pharmaceuticals has been on the rise. This will continue to increase as lifespan increases, boosting chronic and age-related diseases [1]. The majority of these pharmaceuticals end up in the aquatic environment after their usage, either in their initial biological active form or as metabolites. Indicatively, between 30 and 90% of orally consumed pharmaceuticals are excreted in the urine as active substances and end up in the water reservoirs after they pass through the sewage system [2].

Pharmaceutical usage in veterinary activities, such as factory farms, aquacultures and pet treatment, contributes to aquatic pharmaceutical pollution. However, this usage is not considered to be the primary source route. Neither is the improper disposal of unused or expired pharmaceuticals, which are flashed down or poured in the drain, or residues leaking from pharmaceutical manufacturing [3],[4]. Excretion of active pharmaceutical substances after human usage is documented to be the primary source of pharmaceuticals in the aquatic environment [4]. From Figure 1 presented below, it becomes apparent that this route would not have been the main one if WWTPs were capable of removing pharmaceuticals adequately from sewage. In the words of Christian Baresel expert, research coordinator and project manager at IVL (Swedish Environmental Research Institute): “There are pharmaceuticals in the effluent of WWTPs. Today there is a high removal of microplastics, but not for pharmaceuticals’’ (Personal Communication C Baresel 20 Jun 2018).

Figure 1. Entry routes of pharmaceuticals to the aquatic environment. (Redesigned from Boxal AB 2004 [5])

One of the most significant implications of pharmaceutical pollution in the water is the development of antibiotic resistance, posing an apparent and substantial danger to human health. The presence of antibiotics in the environment promotes acquisition or independent evolution of resistance from bacteria that normally lack innate antibiotic capacity [6].

It is worth noting that resistance in bacteria can be developed even in exposure levels lower than the Minimal Inhibitory Concentrations (MIC) [7]. Thus, aquatic environment contaminated with antibiotics can serve as a reservoir for resistance that can be transferred to clinically vital bacteria through horizontal gene transfer mechanisms [8]. Eventually, these resistant bacteria with clinical importance disseminate and reach humans either via food or direct exposure to animal hosts [9, 10].

Another important implication of pharmaceuticals in the water is the ecotoxic effects they have on marine organisms. Pharmaceuticals are designed to intervene in biological pathways, some of which are evolutionary conserved across different species. Hence, pharmaceuticals that were designed to have an effect on some species (including humans) may also exert significant effects on the physiological function of other organisms [11]. Due to their low but consistent concentrations, concerns arise primarily for the chronic rather than the acute ecotoxic effects, which are substantially more difficult to be defined and proved [12]. In addition, mixtures of active substances are present simultaneously in the aquatic environment and generally cause higher effects than each of their comprising components alone (synergistic effect), increasing the combined toxicity [13].

In the Baltic Sea, the effects of pharmaceutical pollution in the ecosystem are remarkably high due to the low water exchange rate, low biodiversity and the physiological stress induced by brackish water conditions. Therefore, the Baltic Sea ecosystem is particularly susceptible to hazardous pharmaceutical substances [14].

One of the main substances that contributes to pharmaceutical pollution and its implications in the Baltic Sea ecosystem is the antibiotic sulfamethoxazole (SMX). SMX has been detected to be one of the most prevalent Active Pharmaceutical Ingredients (APIs) not only in WWTPs’ effluent streams but in rivers located in the Baltic Sea region too, as presented in Figure 2 [14].

Figure 2. The top 20 pharmaceuticals measured in highest concentrations in river water samples in the Baltic Sea region [14].

It has been found that SMX may alter the catabolic function of bacteria comprising marine biofilms by altering their carbon source utilization (15). Changes on the transcriptional profile of genes associated to cell envelope and outer membrane were observed, as well as changes of genes involved in replication when freshwater biofilms were exposed to SMX concentrations as low as 1.94 nmol/L (16).

SMX has even been detected in flounders (Platichus flesus) inhabiting the Baltic Sea in concentrations exceeding 50 μg/kg, meaning that SMX can even move up in the food chain (14).

Additionally, SMX contributes to the rising problem of antibiotic resistance when it is released in the aquatic environment. It has been shown that resistance to SMX alone is one of the most common single antibiotic resistances and remarkably, is associated with the main cross-resistant profiles. Consequently, it becomes apparent that SMX except for its ecotoxicological effects also drives antibiotic resistance by its presence in aquatic environments (17).

Given that the removal rates of SMX in WWTPs are inadequate to substantially prevent or limit its release in the aquatic environment of the Baltic Sea, SMX’s occurrence in the region will continue to persist. In support to this conclusion comes the fact that the biggest WWTP in Sweden, Henriksdal, located in Stockholm, removes 48% to 73% of SMX existing in the influent (18). As Sofia Andersson, a process development engineer in Henriksdal WWTP stated when we visited the facilities in Stockholm: “Pharmaceuticals, in general, are not degraded in a WWTP, they go either to the effluent water or the sludge’’ (Study visit S Andersson 10 Jul 2018). A statement aligned with the monitoring data on the removal rates of SMX.

Altogether, for these reasons, we believe that SMX occurrence in an aquatic environment is one of the key drivers of pharmaceutical pollution in the Baltic Sea. It poses an implicit and imminent threat to the regional ecosystem as WWTPs are, in their current state, unable to sufficiently remove SMX from the wastewater.

Existing Solutions

To remove pharmaceuticals from the wastewater, advanced (tertiary) treatment procedures need to be implemented in WWTPs. Several ready-to-implement technologies have been developed in the past for treating pharmaceutical residues, but each one of them bears some restrictions that prevent its broad adoption and implementation in WWTPs.

To the extent of our knowledge, there is only one WWTP in the Baltic Sea region employing advanced treatment methods to remove pharmaceuticals from wastewater, and it is located in Linköping, Sweden.

The most preeminent methods for treating pharmaceuticals in wastewater are displayed and briefly described in Table 1 and Table 2 .

Note that the emerging solution pCure in Table 2, which was developed by Pharem Biotech, is a solution designed to be implemented in the topmost upstream level, which is the level of the toilet and not in the WWTP [19].

Table 1. Existing solutions for treating wastewater from pharmaceuticals [19].

Table 2. Emerging solutions for treating wastewater from pharmaceuticals [20].

Note also that one common system including ultrafiltration (or micro-filtration) in conjunction with a biological wastewater treatment (activated sludge) process is called Membrane Bioreactor (MBR), and it is not displayed separately as it is represented in Table 1 by the ultrafiltration method.

Combinations of the existing solutions can be implemented in WWTPs, either integrated into the primary treatment stages or as an extra stage of treatment. With this tactic WWTPs try to leverage the main advantages from different solutions, achieving sufficient removal rates of pharmaceuticals. As Sofia Andersson from Henriksdal WWTP disclosed: “With Ozonation, there is a risk of receiving compounds you do not want, but Linköping WWTP has moving Biofilm Membrane Bioreactor (MB-MBR) carriers implemented after the Ozonation” (Study visit S Andersson 10 Jul 2018). However, as it is logical to assume, this approach leads to an increased treatment cost as well.

Overall, the two most easily accessible and realistic solutions for the removal of pharmaceuticals are Ozonation and Activated Carbon (GAC & PAC). On the one hand, the by-products produced after the redox reaction catalyzed by ozone (O3) have unknown ecotoxic effects. Coupled with slack control of removal rates due to the variability of wastewater content, skepticism is raised regarding Ozonation’s suitability. Commenting on Ozonation, Christian Baresel voiced: “Ozonation of untreated wastewater leads to the ozone targeting other carbon sources than the ones you want (i.e. pharmaceuticals), which leads to high ozone levels needed, which is not economical. Ozonation large scale system like the size of Linköping (serving approximately 100,000 people) is 0.1 SEK/m3 which is relatively affordable. However, you need polishing after Ozonation, and that pushes the cost quite higher’’ (Personal Communication C Baresel 20 Jun 2018).

On the other hand, despite sufficient efficiency of Activated Carbon on removing pharmaceuticals from wastewater, financial concerns associated with its usage keeps its implementation limited. Regarding Activated Carbon Christian claims that: “Activated Carbon is a better solution than Ozonation because combined with biological filters (MBR) tenfold increase can be achieved in the lifetime of activated carbon granules. However, you need to have really clean water in order to use it. Otherwise clogging becomes an issue” (Personal Communication C Baresel 20 Jun 2018). Even in this case, the increase of activated carbon granules’ lifetime cannot offset MBR’s installation and operational costs.

In our quest to better understand the profile of the existing solutions we visited Kungsängsverket WWTP, located in Uppsala. There, we learned that investigator engineers, Elin Kusoffsky and Jesper Olsson, concluded, after conducting a thorough assessment, that either Ozonation, Activated Carbon or a combination of both could be the chosen solution for treating wastewater from pharmaceuticals in their WWTP. Specifically, the designated investigators estimated for Uppsala WWTP that 15gr of O3 per m3 of wastewater and approximately 20 minutes retention time will be needed for sufficient removal of pharmaceuticals, resulting in acceptable costs for Kungsängsverket WWTP standards. Although it was clearly admitted that the energy consumption will be increased from 0.05 to 0.1 kWh/m3 (between 10 and 30 % more of the existing value without advanced treatment) the investigators still considered Ozonation as a satisfactory solution. Additionally, Jesper Olsson’s statement “With PAC, the pharmaceuticals are not eliminated, they end up in the sludge’’ spotlighted another flaw of Ozonation (Study visit J Olsson 15 Aug 2018). When the conversation ended up on Activated Carbon the investigators voiced that: “The biggest concern is the production of PAC if we are going to implement both technologies, which is a very energy consuming process. With GAC, a technique similar to the one the drinking water plants use to take away organic pesticides, the cost is much higher’’ (Study visit J Olsson, E Kusoffsky 15 Aug 2018).

Taking into account all the relevant data and information we managed to collect about the existing solutions, we considered useful to create a landscape map in order to synthesize an overview of the field.

Figure 3. Landscape of the existing solutions for treating wastewater from pharmaceuticals in WWTPs.

From the first sight of the landscape map in Figure 3, it is apparent that there is a great need for a new solution that will meet the capabilities of Activated Carbon for removing pharmaceuticals from wastewater. In addition, the solution would need to be energy efficient without creating unknown by-products with unknown ecotoxic effects and it would not need to be coupled with another solution in order to sustain low costs.

In support to this conclusion comes the statement from Stefan Berg, SYVAB’s process engineer at Himmelfjärds WWTP during our skype meeting: “Ozonation can be good, but not enough if you have tough restrictions. Although it is very common to have Ozonation followed by PAC or GAC, I would prefer one technique and not a combination’’ (Skype call S Berg 29 Aug 2018).

Consequently, it is reasonable to assume that WWTPs are indeed examining their available options for removing pharmaceuticals from wastewater and are susceptible to implement a solution that covers their unmet needs.

Our Solution - Biotic Blue

The bead...

In order to limit the occurrence of pharmaceuticals in the aquatic environment we developed Biotic Blue, an enzymatic solution exploiting synthetic biology - based techniques. Diving into the molecular level, Biotic Blue comprises of a mutant oxidoreductase, the laccase enzyme from the polypore mushroom Trametes versicolor, immobilized on magnetic beads. In more detail, our mutant laccase, approximately 55 kDa in size and improved for degrading SMX, differs from the wild-type laccase by three amino acids. The bulky, volatile Phe162, Phe332 and Phe337 amino acids were replaced by isoleucine residues. These modifications were suggested by the results of rational design analysis. The core of the beads, which is approximately 200 nm in size, consists of magnetite (Fe3O4) giving ferromagnetic properties to the final product. The recovery from the water and re-use of Biotic Blue is based on these magnetic properties, as it is illustrated in Figure 4.

Figure 4. The immobilization process of mutant enzymes on the magnetic bead.

The magnetite core is coated by a layer of polystyrene sulfonate (PSS) which in turn is covered by Chitosan (Chi). PSS is an ionomer and mediates the adherence of Chi to the magnetic core since Chi cannot be adsorbed or form covalent bonds directly with the magnetite. Chi, a polysaccharide, offers a suitable surface to the bead for covalent bond formation. Although part of the Chi surface may be covered by adsorbed or covalently bound enzymes, the largest surface area is covered by Glutaraldehyde (GA), an agent used for the cross-linking of the enzymes. Cross-linking increases the capacity of each bead in regard to the number of enzymes it can bear, vastly improving its efficiency. Macroscopically, Biotic Blue has the form of a black powder and can be effortlessly discerned from clean water. The powder form enables uncomplicated integration of Biotic Blue in every wastewater treatment process system and makes its operation fairly handy. As Christian Baresel voiced: “Every plant is unique, thus, it is important to have a tailor-made solution for each one. The treatment technology is based on dosing, motion, etc., and therefore the tailor-made solution makes the most impact. For example, some hospitals have infection departments, and some do not, which requires different treatment of the waste” (Personal Communication C Baresel 20 Jun 2018).

... in the wastewater treatment process

The implementation site of Biotic Blue is the WWTP. Although the wastewater treatment process is not the same in every WWTP, in general, three stages can be distinguished: the mechanical, the biological and the chemical as it is illustrated in Figure 5. After considering all the feedback we received from experts in the field of wastewater treatment management, we decided that the point of implementation of Biotic Blue will be right before the biological stage.

Figure 5. Integration of Biotic Blue in the wastewater treatment process. (The top part of the illustration i.e. the main treatment process, is a redesign from Natuvardverket: Wastewater in Sweden 2016 [20].

Since radical changes in WWTP infrastructure are impractical and highly costly, implementation of Biotic Blue was designed to be compatible with the existing equipment and practices. More specifically, its implementation as an additional step before the flow of the sewage to the biological treatment can be materialized by deploying in essence, an extra water tank, a magnet, a collection chamber and a pump. An integrated aeration system would need to be added to the water tank, which will bear the wastewater with the magnetic beads as well, in order to adequately secure mixing conditions. Therefore, with air bubbles spurt from the bottom of the tank, the possibilities for the enzyme-substance reactions to take place increase, and the precipitation of the magnetic beads is avoided. Besides, continuous supply of oxygen to the laccase is essential for its activity, and the aeration system provides it in a constant manner. The magnet that would be used needs to have an elliptical shape and its strength needs to be sufficient to attract and separate the magnetic beads from the water. It is important to highlight that the pipes bearing the wastewater along with the beads after the tank, would be Polyvinyl Chloride (PVC) pipes. Unlike steel and iron, materials commonly used for water pipes, PVC (plastic polymer) has low magnetic permeability and, therefore, does not have any magnetic shielding properties. Due to the strong ferromagnetic properties of the beads and the smart structure of the magnet and pipe setting, the beads separation process would be smooth and continuous, as it is illustrated in Figure 5. The collection chamber in which the magnetic beads will end up after their separation from the water needs to have an inlet for the washing buffer used for the recovering of the enzymes before their re-use. Finally, the chamber will be connected with a pump transferring the recovered beads to the initial water tank. It must be highlighted that the proposed closed system is modular and it can function in fully automatic mode.

Below, in Figure 6 our 3D printed prototype is displayed. Note that the prototype does not bear all the features of the implementation of Biotic Blue as it was developed due to time requirements before its design reached the current state.

Figure 6. Prototype of Biotic Blue

Comparative analysis

In the following Table 3 the advantages and disadvantages of Biotic Blue are presented, indicating the points of superiority compared to existing solutions and the features that need further development. Table 3 can be used as a valuable guide for future improvement driving through different stages the evolution of Biotic Blue.

Table 3. Advantages and disadvantages of Biotic Blue implementation in WWTPs.

Although Biotic Blue may not be the ultimate solution for treating pharmaceuticals in WWTP, undoubtedly, it bears significant competitive advantages against its two major competitive methods, Ozonation and Activated carbon. Our solution has a higher cost-effectiveness profile, lower operational energy consumption, does not pose any threat to its handlers and the environmental risk of degradation residues is less than Ozonation by-products.

In comparison to Activated Carbon, Biotic Blue has significant cost benefits since the magnetic beads can be retrieved in a more efficient way and they can be regenerated with lower capital demands as well. Furthermore, our solution offers lower installation costs and space requirements, making it particularly attractive for WWTP with limited available space.

However, at this point it is important to highlight that the implied capability of Biotic Blue to immobilize any enzyme on the magnetic beads, allows it to potentially treat wastewater contaminated by any mixture of pharmaceuticals. This unique tailoring capability according to the synthesis of the contaminants in the inflow is what makes Biotic Blue resource-efficient, outperforming the other solutions.


We have successfully immobilized the fungal laccase on magnetic beads. The following images show that the immobilized enzymes were functional (Figure 7) and that the magnetic beads can be captured and separated from water solution using a magnet (Figure 8).

Figure 7. On the left: Control.
On the right: Immobilized laccases showing activity (color change) when ABTS (a laccase model-substrate) was added.

Figure 8. Recovery of immobilized magnetic beads on a magnetic rack.

Impact on Society

"Our feet may stand on Stockholm, surrounded by the water of the Baltic Sea but our eyes rest on our whole planet."

With this in mind we considered the United Nations 2030 Sustainable Development Goals as the ideal framework to show the impact of Biotic Blue on society.

The Sustainable Development Goals (SDGs), also known as Global Goals, are a set of universal goals, targets and indicators that meet the most urgent challenges facing our world. SDGs came into effect in January 2016 and since then, with 15 years horizon, set the framework for the United Nations Development Programme. SDGs follow and expand on the Millennium Development Goals (MDGs) in which all 191 United Nations member states declared commitment from 2000 to 2015 (21).

The success of the goals depends unsurprisingly on their translation into the introduction of new legislations, improvement of existing regulations and the implementation of action plans in both governmental and sub-governmental level. Therefore, as it seems paradoxical at first, “localization’’ is the key to attain these ambitious global goals. Localization means to put everything within subnational frameworks, from setting targets to deciding the mode of action and selecting indicators for monitoring progress. And from this local perspective, we would like to show Biotic Blue potential to contribute to fulfilling these goals, which in our eyes mirrors its impact on our planet and society in the most accurate way (22).

Biotic Blue, to different extent contributes to attain 10 out of the 17 goals, demonstrating its capability to influence society on multiple levels (Figure 9).

Figure 9. The Sustainable Development Goals 2030 Biotic Blue contributes to be attained. (The illustration is a redesign of UN SDGs 2030 [23])

By minimizing the levels of hazardous pharmaceuticals in effluent wastewater and consequently in the aquatic environment, Biotic Blue increases safe re-use of wastewater and reduces pharmaceutical pollution. It also protects and restores water-related ecosystems including rivers and lakes.

Helps human settlements, adopting and implementing integrated policies towards inclusion. In addition, Biotic Blue enhances the capacity for more sustainable urbanization.

Helps the development of advanced regional infrastructure to support sustainable economic growth by upgrading the technological capabilities of WWTPs to treat wastewater.

Helps the achievement of sustainable management of consumed pharmaceuticals. Biotic Blue minimizes the adverse impacts on human health and the environment derived by the presence of pharmaceuticals in the wastewater.

Biotic blue helps in preventing and reducing marine pharmaceutical pollution protecting sensitive coastal and marine ecosystems. It stops the exposure of marine organisms to chemical entities not occurring naturally in the aquatic environment.

Helps to slow down the development of antibiotic resistance, which comprises a major and imminent threat to human health, by minimizing uncontrolled exposure of environmental bacteria to antibiotics.

Helps to preserve marine ecosystems that play a significant role in our effort to restore our planet's climate and mitigate the environmental impact of the human activity-pharmaceutical consumption.

Biotic Blue is an innovative solution with high added value that contributes to decoupling pharmaceutical consumption, an indicator of economic growth, from environmental deterioration.

By having lower energy needs than the competitive solutions, Biotic Blue contributes to more efficient energy usage.

Biotic Blue is an environmentally sound technology derived from a developed country and can be transferred and diffused to developing countries in order to counter the emerging problem of aquatic pharmaceutical pollution.

Figure 10. Impact of Biotic Blue on SDGs


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