As many Finnish social, economical and cultural values are deeply connected to the Baltic Sea, our
thinking quickly turned towards protecting water resources. In recent years there have been growing
concerns about micropollutants, due to the fact they are very persistent and biologically
active. They include industrial chemicals, pesticides and pharmaceuticals. The latter are
thought to be particularly harmful since they are designed to affect organisms at low doses [1].
Sources of pharmaceuticals include effluent water from wastewater treatment plants, human and animal
excreta, industrial, medical or veterinary waste, as well as landfill leachate (Fig. 1).
Figure 1. Selected sources of pharmaceuticals in natural waters [2;
modified].
Even though there is no evidence that such low amounts of pharmaceuticals in drinking water may be a
threat to human health [2], these compounds may have a negative impact on the environment [1]. For
that reason, the EU ‘watch list’ of pharmaceuticals for EU-wide monitoring in aquatic environments
was created. It includes substances that are thought to be high-risk and high priority to monitor
and to establish reliable methods of their degradation [3]. These substances are present in rather
low concentrations, which makes their monitoring challenging. In addition to that, after reaching
out to experts working in the wastewater treatment industry, we have realised that the detection is
not the only issue: removal of pharmaceuticals is very resource-consuming. It requires the use of
ozone and activated carbon, which are associated with a high carbon footprint and
energy-consumption. We came to the realization that the biosensor may have a better
implementation. It could be used to optimize the removal process of pharmaceuticals and thus,
limit the energy and chemicals needed. One of the substances present on the EU ‘watch list’
since 2015 are macrolide antibiotics [4]. In addition to issues mentioned above, their presence in
the environment carries another risk: development of antibiotic resistance by microorganisms that
interact with it. In fact, antimicrobial resistance was listed by WHO [5], as one of top 10 current
health concerns.
Why Macrolides?
Macrolide antibiotics include, among others, erythromycin and semi-natural azithromycin and
clarithromycin, which are compounds characterized by a macrocyclic lactone ring (Fig. 2). They are
said to have antibacterial, antifungal and anticancer qualities [6].
Figure 2. Chemical structure of selected macrolide antibiotics.
Macrolides are considered broad-spectrum antibiotics; they are capable of inhibiting gram-positive
and selected gram-negative bacteria. They are usually prescribed for various human infections, such
as pneumonia [7]. Due to their common use in hospitals and veterinary, especially in animals such as
poultry and cattle [7], there is a growing resistance towards them [8]. Macrolides are often
detected in effluent wastewaters and drinking water. The sources of this contamination mostly come
from their use in healthcare and agriculture. The biggest part comes from faeces and using manure as
fertilizer, however a portion of macrolides in water can be contributed to their improper disposal.
Another significant source [2, 9] of macrolides includes hospitals and facilities producing
pharmaceuticals. Since a lot of drug production takes place in developing countries the
concentrations of macrolides may be higher there. However, it can’t be fully known since macrolide
concentration in wastewater is not monitored [9].
Macrolides have been recognized as hazardous substances not only in the EU [4, 10], but also in the
US, where they are included in Contaminant Candidate list 3 [11] and in China, which has given them
a high priority based on their presence in water [12]. They are the second most widely used group of
antimicrobials in EU past beta-lactams and penicillins [13].
SUSTAINABLE DEVELOPMENTAL GOALS
An important part of Integrated Human Practices is ensuring our projects tackle actual local or
worldwide problems. Inspired by iGEM & The Sustainable Development Goals seminar, we decided
to evaluate if and how our project addresses the goals for 2030.
SINISENS would help to monitor macrolide antibiotics pollution in aid the improvement of their
removal process. This aspect aligns with ③ Good Health And Well-Being, which aims to reduce
the number of illnesses and deaths arising from pollution of soil, air and water. In addition to
that, prevention of antimicrobial resistance is crucial to reduce the number of deaths from
infectious diseases. A macrolide biosensor could also help with goals ⑥ Clean Water And
Sanitation, ⑭ Life Below Water and ⑮ Life On Land, by improving the quality of
water. Optimizing the removal process would ultimately help reduce the amount of pharmaceuticals
released from wastewater treatment plants, which will end up in sea, fresh water and land. It is
also worth noting that the removal process of micropollutants is quite energy-consuming. SINISENS
would help to optimize this treatment by allowing easy and cost-efficient measurements of macrolide
concentrations, which could help conserve energy, thus addressing the goal ⑫ Responsible
consumption and production.
BIOSENSOR ETHICS - BIOCONTAINMENT
To further discuss
potential negative social and environmental impact of our biosensor, we hosted an ethics workshops
for Nordic iGEMs, all of which had a biosensor development as the end goal. The main discussed
topics were the life cycle of the biosensor in the terms of sustainable production and proper
disposal, risks of GMO, guidelines for the potential end users and interactions of the projects with
the society.
Life Cycle of the Biosensor
Even though our biosensor aims to optimize highly energy consuming pharma removal processes and in
that sense improve the environmental friendliness of the wastewater treatment, we have to ensure
that the production of the biosensor itself is sustainable. The strip material should be
carefully selected, nitrate cellulose as a one option, as it can be re-used for manufacturing
of new strips. In addition, the one-time use biosensor strips could be sent back to the producer,
where they could be properly sterilized. The other important factor to consider is
the management of the wastewater sample that might have been contaminated by the modified organism.
Here,
minimizing the sample size is the key in terms for example of the energy required for the
waste management.
Risks of GMO
Implementing the biosensor into the wastewater stream might cause possible leakage to the
environment. For that reason, developing a separate system where the sample water is
collected, for example as a 24-hour representative sample, is crucial. When considering the
risks of GMOs, education plays
an important role since lack of understanding might lead to negative reactions, prevent further
research and use of GMO products.
Guidelines for the End User
In terms of safety, the prevention of physical contact is important to keep in mind already in the
sensor design. The system could also be automated in the part of the process that poses most
of the risk, for example periodically getting the strips in and removing them. In the sensing
element itself, it would be possible to build a kill switch-system so that the microorganism
survives only in the controlled environment. In addition to mentioned points, training of the
maintenance staff is important especially during malfunctions in order to be aware of the
risks.
Interaction with the Society
Environmental applications seem to be more acceptable than human or food-related applications.
Moreover, GMOs used in the pharmaceutical or chemical industry usually are not met with strong
resistance from the public. Usually, the most questioned aspect in the discussion of GMO is the
safety. Open discussion should highlight the benefits of GMO and why, in some cases, they may be
necessary for sustainable development. Moreover, the discourse should stay clear on
possibilities, reality now, and risks that there might be. Overstating what science is able
to do might lead to not meeting set goals and losing credibility.
To conclude, throughout our project we kept in mind the values we set in the beginning of the
project.
Environmental: During the ideation it became clear that all our team members are
concerned about current environmental issues. We settled on protecting water resources.
Social: In recent years, the concern of pharmaceuticals in water resources has risen as a
global problem. Natural waters are used for recreational purposes and are economically valuable,
which is why we need to step up with their protection.
Moral: We have wanted to be responsible and consider our project's consequences from many
aspects, such as ethics. We have had the courage to think "outside the box", but on the other
hand, we also wanted to do a project that has a real-world need. Here we have been assisted by
several experts.
Scientific: Our project is not limited to one discipline; we wanted to combine many. We
have ensured good scientific practice and honesty. Protecting the ecological status of Baltic
Sea requires collaboration and open science between the Baltic Rim countries.
STORYLINE
In order to ensure that our project is relevant and has a potential use in the real world, we contacted
various experts from academia, industry, environmental organizations and legislation.
“The wastewater treatment plants play a key role in keeping our waters
clean. As part of our national identity, surrounding natural waters have a strong social value, not
only by supporting the livelihoods of many people, but also by creating a place of memories and
adventures. Therefore, we settled on protecting water resources.”
— Aalto-Helsinki 2020 team
“Pharmaceuticals in wastewater currently pose a challenge due to their
potential risks already in low concentrations, unclear regulations regarding their monitoring and
removal requirements, and lack of cost-effective detection methods.”
— Anna Mikola, Aalto University
“Utilization of biosensors in environmental monitoring is a “topical
topic”. Macrolide antibiotics are most likely an important group of pharmaceuticals, since they were
selected to the EU ‘watch list’.”
— Matti Leppänen, Finnish Environment Institute
“The additional purification step, ozonation, is highly energy consuming
and the production of activated carbon has high environmental footprint. Implementation of a
biosensor before and after the purification step could be used for the optimization of the process.”
— Paula Lindell, Viikinmäki wastewater treatment plant, HSY
“If regulations come that require the removal of pharmaceuticals, a device
will be needed to measure whether this removal process succeeds. In that sense, your innovation
would be useful.”
— Ari Kangas*, Ministry of Environment, Finland
Below you can read the detailed storyline of SINISENS, which was refined by our interaction with
stakeholders
and the community. In the storyline, we use colours to describe the different phases of the project
and
to highlight our conclusions and the actions we have done after
specific steps. In the future
section, we
summarize the compromises and possibilities for future development of our innovation.
GETTING STARTED
In order to get to know each other better and draw a very rough outline of what we envision our
project to be, each of us picked the Standard Track Awards which we are the most interested in. We
chose the tracks based on our study fields and personal interests. The
common categories included: diagnostics, environment, food & nutrition and therapeutics.
PREVIOUS iGEM PROJECTS
We had a couple meetings where each person prepared presentations about previous iGEM projects and
explained why they caught their attention. Later we analyzed which
aspects of them could be implemented in our project.
BRAINSTORMING
We divided ourselves into three groups and tried to come up with as many ideas as possible, no matter
how outlandish they felt. Then we rotated to see what the other teams came up with. At the end we discussed all of the ideas we liked and wanted to research
further.
PROJECT POTENTIAL MATRIX
From all discussed ideas, we selected six potential topics that should be explored further. Each team
member had an opportunity to vote on the topics they believed to be most promising. After presenting
the research we had done, we voted on the topics so that each team member could rank the potential
project ideas from one to six, one being the most and six the least favorite. The criteria we chose
included practicality, usefulness, do-ability, and enjoyment. We
decided to pick three out of these project directions to read more in-depth about.
NARROWING THE TOPIC
After exploring chosen topics, we ruled out designing a quick
diagnostics
device for tick borne diseases, as they are already available on the market and did not
seem to require a significant improvement.
When it comes to the two remaining ideas, we found out that there is a strong connection between the
microplastics and wastewater related topics, since a primary source of microplastics in soil comes
from wastewater. Therefore, we decided to focus more deeply on
wastewater treatment. The WWTPs (wastewater treatment plants) play a key role in keeping
our waters clean. These facilities prevent most of the pollution from entering the sea. However,
certain types of contamination, known as micropollutants, are currently hard to detect and
completely remove from wastewater.
OUR VALUES
It was important to us that this project reflects our values. During the ideation it became clear
that all our team members are concerned about current environmental issues. As Finland is known as a
land of thousands of lakes and is situated by the vulnerable Baltic Sea, we settled on protecting
water resources. The Baltic Sea compiles different environmental, social, moral and
scientific
values. Protecting its ecological status requires collaboration and open science between the Baltic
Rim countries. In addition, as part of our national identity, surrounding natural waters have a
strong social value, not only by supporting the livelihoods of many people, but also by creating a
place of memories and adventures. It was shown best on the Baltic Sea Day, launched by the John
Nurminen Foundation, where our team, along with hundreds other people, shared our “Sea of Memories”.
EUROBAROMETER: ATTITUDES OF EUROPEANS TOWARDS WATER - RELATED ISSUES
According to Europeans, the main three concerns relating to water issues in their countries are
chemical pollution (84% of participants), climate change (55%) and changes to the water ecosystem
(49%). Chemical pollution has been increasingly considered as a threat since 2009. It became
clear that the general public has identified it as an important problem.
After deciding on a general topic on wastewater treatment, all our
team started to research various environmental problems involving waters in Finland, the Baltic
Sea region and the European Union in general.
CONCLUSION: WASTEWATER TREATMENT
Next: Find the route in the sea of possibilities.
REMOVAL, RECYCLING OR DETECTION?
We came up with three possible directions to consider: removal of compounds, their recycling or
detection. We needed to decide which one was the most feasible. Based on the research we conducted,
our focus was mostly divided between microplastics, PFAS (per- and polyfluoroalkyl substances) and
pharmaceuticals, but we were also playing with the idea of a system for pathogen detection. To get some insight from the field, we decided to contact and discuss
with experts.
Degradation of PET, one of the most common microplastics in soil, could be promising with enzymes
called cutinases, for example from Thermobifida or Ideonella sakaiensis. However, degradation of
microplastics in soil can be time consuming and three months of lab work may not be enough to
get
meaningful results. An additional challenge can be the analysis of microplastics from soil
samples,
which is lacking standardized methods.
Microplastics seemed to be a hot-topic, but determining the amount of
them in the water was a major issue, since they are particles of very different sizes. We were
also considering whether the project concerned microplastics degradation was manageable, given
limited time frame.
BIGGEST PROBLEMS IN MUNICIPAL WWTPs - MICROPOLLUTANTS
There are concerns about micropollutants and how they end up in the environment via WWTPs.
Microplastics may accumulate in the soil if treated sludge is land applied and many
pharmaceuticals
and PFAs are hard to remove from the effluent water. Pharmaceuticals in wastewater currently
pose a
challenge due to their potential risks already in low concentrations, unclear regulations
regarding
their monitoring and removal requirements and lack of cost-effective detection methods. Their
metabolites need to be taken into account because they transform in the wastewater treatment
process. From the environmental point of view, it is challenging to find effective and
sustainable
methods for micropollutant removal.
When it comes to uptake or degradation of micropollutants, it does not
seem plausible for our project. The development of a quick, cost-effective and reliable
detection method for pharmaceuticals could be meaningful to counter the presented
challenges.
PFAS are one of the challenges in the industry of wastewater treatment. When PFAS
concentrations are on relatively high levels, the current detection methods are quite
efficient.
In a previous iGEM project, a
biological detection method for some
PFAS has already been under development.
PROSPECTS OF SYNTHETIC BIOLOGY IN WASTEWATER RELATED APPLICATIONS
In iGEM competition, it is important to seek novel synthetic biology ideas. The unusual global
situation allows the project to focus more on modelling. The detection of micropollutants is an
interesting topic and synthetic biology provides excellent possibilities to detect compounds
present
in low concentrations in wastewater. For pharmaceuticals, the modelling would have wide
potential.
Both topics of microplastics and pharmaceuticals in wastewater could be
promising. However, it seems that a biosensor for pharmaceuticals would allow us to model more
aspects of the project.
1,8 THOUSAND TONS OF PHARMACEUTICALS END UP IN THE ENVIRONMENT YEARLY
UNESCO & HELCOM (2017) [14] and SYKE (Finnish Environmental Institute)
[15] reports have pointed us in the
directions of pharmaceuticals. Increasing amounts of drug residues end up in the
wastewater treatment plants as the usage of pharmaceuticals keeps growing. According to the HELCOM
report, municipal WWTPs release 1,8 thousand tons of pharmaceuticals to the environment yearly.
Furthermore, a significant part of all pharmaceuticals are only partially removed during the
wastewater treatment process. The SYKE report states that the pharmaceutical industry, hospitals and
WWTPs should monitor their pharmaceutical emission. This requires the determination of limit values
for these compounds.
There are a lot of elements that make it hard to get reliable, representative samples, when
measuring the concentration of pharmaceuticals in wastewater. One of them is the release of
pharmaceuticals: the amount of pharmaceuticals related to the usage is quite stable, but the
manufacturing-related release depends greatly on the time the given compound is being produced.
The
composition of wastewater might also contribute to variability between samples, especially when
the
samples are taken at different steps of the process, since it can affect specificity of liquid
chromatography analysis.
Many factors need to be taken into account during pharmaceutical product development. In order
to
receive marketing authorization for a product, it always requires the evaluation of the
environmental risks. However, in different market areas there are different authorities and
marketing authorization processes.
The removal of micropollutants, such as pharmaceutical residues, fragrances, detergents and
pesticides etc. is an issue that is increasingly drawing attention, especially in the municipal
wastewater sector. During the wastewater treatment process, some of the micropollutants are
removed,
reduced or transformed into other compounds. Especially the transformation makes the study topic
very challenging, as it expands the already vast group of target analytes even more.
Currently, only a few countries, like Switzerland, have opted for regulation and technology to
remove such pollutants from water. Newly implemented purifications steps for micropollutant
removal
are most often either activated carbon or ozone treatment. Most of the contaminants, such as
antibiotics and other pharmaceuticals are present in the wastewater in very small concentrations
(around 0.01–10 mg/L). Therefore the analysis requires liquid chromatography combined with mass
spectrometer (LC-MS).
We decided to contact Swiss WWTPs to get more insight on the methods
they are using for pharmaceutical removal (see our final
design section). In addition to that, current detection methods used
for pharmaceuticals, such as LC-MS, are expensive and time-consuming.
CONCLUSION: BIOSENSOR FOR PHARMACEUTICALS
Next: In search for the right compound.
DISCOVERING THE 'WATCH LIST' OF PHARMACEUTICALS FOR EU WIDE MONITORING
UNESCO & HELCOM (2017) [14] report is a potential tool for guiding the selection of the right
compound. Substances present at high concentrations in wastewater effluent, such as diclofenac
or
amoxicillin, could be possible targets for a biosensor. However, using a biological sensor can
come
with certain constraints, such as susceptibility to antimicrobials. Additionally, the presence
of
mixtures, including metabolites, as well as variable conditions make achieving good sensitivity
and
specificity challenging.
Based on these reports, we selected 20 pharmaceuticals present in
highest concentrations in municipal WWTP effluents. The HELCOM report also mentioned the
‘watch
list’ of pharmaceuticals for EU-wide monitoring, which caught our attention.
OUR RESEARCH
We researched the basics of biosensor design and average concentrations of pollutants that we could
detect. According to the HELCOM report, the most frequently measured pharmaceutical compounds in the
Baltic sea marine environment are analgesics, anti-inflammatory, cardiovascular and central nervous
system agents [14].
The European Commission has created a ‘watch list’ of pharmaceuticals for EU-wide monitoring in
aquatic environments [3]. It includes substances that are thought to be high-risk and high priority
to monitor, and to establish reliable methods of their degradation. We
included these pharmaceuticals in our research.
We ranked these pharmaceuticals based on the efficiency of their
removal at WWTPs, the environmental impact and currently available methods to quantify them. To
better understand compounds that are problematic to WWTPs, we contacted stakeholders in academia
and industry.
For wastewater treatment plants, many pharmaceutical compounds, such as diclofenac, certain
antibiotics and X-ray contrast media are difficult to remove. Biggest load of pharmaceuticals to
WWTPs comes from the households and pharmaceutical industry. Another major source of the
emissions
are hospitals.
Based on our previous research, we wanted to know if antibiotics,
painkillers, BPA (Bisphenol A) or carbamazepine would be meaningful targets for our
biosensor.
Carbamazepine is relevant because it is not biodegradable and it is not removed at all in the
existing treatment processes. From the environmental perspective paracetamol and ibuprofen are
quite
easily bio-degraded and not persistent in wastewaters and environment. BPA is persistent, but it
seems that the environmental concentrations are decreasing. This indicates that BPA might be
efficiently controlled and replaced in different materials. Antibiotics are very relevant not
only
from the persistence point of view, but also due to the concern about antibiotic
resistance.
During
these COVID-19 times this is of course even more acute than earlier, as we have seen a
significant
increase in antibiotic use.
Currently, one of the most discussed issues in WWTPs is the detection and removal of
pharmaceuticals. At the moment, the analysis methods are insufficient, removal is challenging
and
energy-consuming. It would be essential to understand which of these compounds are
harmful in the
receiving waters and make people aware of pharmaceuticals' proper disposal in order to limit
their
presence in wastewater in the first place.
As for the legislation, there are no limit values for the effluent water from WWTP. However, the
EU
legislation regarding water issues is under fitness check and some of the directives will be
revised. Moreover, WWTPs are required to operate in terms of environmental permits.
A biosensor for detecting pharmaceuticals sounds like an exciting idea for monitoring the
concentration levels. The biosensor could be used as a tool for operating the process,
especially if
implementing new purification steps for pharmaceutical removal. It would be relevant to
detect the
compounds that are most difficult to remove. The EU ‘watch list’ could work as a good starting
point
when searching for the right pharmaceutical. These compounds often lack sufficient research and
their behaviour in natural waters is unknown, which is why it would be reasonable to pick one of
these compounds for monitoring.
The implementation of new micropollutant purification steps pose a
significant challenge: the treatment methods are highly energy consuming or resource
heavy. The meeting confirmed a potential need for a biosensor and guided us to
start its development for optimization of the removal process of pharmaceuticals in WWTPs. In
addition, we decided it would be essential to research how aware people are on how to dispose of
pharmaceuticals properly. After all, prevention is better than cure: limiting the amount
of
pharmaceuticals in water is more resource-efficient than their removal. Also, it would be
interesting to find out how disposal methods differ between countries.
There is a watch list for microcontaminants, which are currently not monitored. The reasons for
that
include technical difficulties, as there are many substances in the background, as well as no
direct
legislation concerning their allowed concentrations. Considering the above, a biosensor is a
very
interesting idea, this kind of device would have a potential to be used at WWTPs.
As an important topic, there has already been a discussion concerning pharmaceuticals in
wastewaters
for several years. However, the WWTPs are operating according to the environmental permit. The
change in legislation has to come first if new process steps for the removal of pharmaceuticals
from
wastewater are to be introduced. Also, implementation will probably take place first in the more
modern and the bigger WWTPs in Finland.
For us, it was important to hear about the situation regarding
pharmaceuticals in wastewater in smaller WWTPs in Finland. Also, we noticed that there is a big
difference in the environmental permits between WWTPs and which compounds they are required to
remove. We wanted to research more deeply what are the reasons behind these differences.
A biosensor could be interesting, especially if it is a quick and cost effective
technique. Generally,
it is expensive to analyse micropollutants, thus any new solutions are welcome.
A biosensor could be useful for WWTPs as a detector of the presence or absence of specific
compounds.
Right now, there are no regulations regarding monitoring pharmaceuticals in the Spanish WWTPs.
It could
be beneficial for a biosensor to have a fast output, since some of the compounds in the
wastewater are
not very stable and tend to change rapidly. Macrolide antibiotics could be interesting compounds
from
the EU ‘watch list’.
Based on the feedback from wastewater treatment industry and academia,
a
biosensor for detecting pharmaceuticals could be a relevant project since the analysis methods
are currently very challenging due to a lack of legislation and low detection limits. We also
found out that antibiotics are problematic for WWTPs and could be a potential group to
target.
When it comes to pharmaceuticals in wastewater, there is a need for further monitoring studies.
It
would be important to understand these compounds’ behavior in WWTPs. Thus, developing a
biosensor
for pharmaceuticals could be relevant, but very challenging. In the water ecosystems,
pharmaceuticals and their transformation products might cause hormonal and toxic effects or
changes
to the behavior of organisms. There is discussion about the “pharmaceutical cocktails” and that
their collaborative effects are unknown. Residues of antibiotics are one of the important
classes of
harmful compounds and the development of antibiotic resistance is a great global
concern.
Utilization of biosensors in environmental monitoring is a “topical topic”. One of the biggest
challenges of pharmaceuticals in wastewater is the mixture toxicity. For example, if it can be
proved that the group of chemicals have a common, measurable influence on a given species,
especially in a chronic time frame, it could be implemented in legislation using a threshold
approach. Macrolide antibiotics are most likely an important group of pharmaceuticals, since
they
were selected to the EU ‘watch list’.
DECIDING OF THE PHARMACEUTICAL GROUP - MACROLIDE ANTIBIOTICS
Antibiotics, macrolides being a major class of them, have become a
global environmental and public health concern due to their possible contribution to the
development of antimicrobial resistance. They might also have the above mentioned common effect
on a selected species. WWTPs are considered as the main source of spreading macrolides and other
antibiotics into the environment. The majority of macrolide pollution begins via excretion in
feces and urine of humans and animals. Moreover, pharmaceutical production facilities and
hospitals are point pollution sources for macrolides [7]. In addition to the accelerating
antibiotic resistance, macrolide antibiotics among the other micropollutants can persist in
nature, accumulate in organisms, and remain biologically active [16]. In the further development
of the project, we decided to concentrate on the three most common macrolides: erythromycin,
clarithromycin and azithromycin.
DISPOSAL OF PHARMACEUTICALS - DISCOVERING THE NEED FOR A CAMPAIGN
Indian iGEM team IISER Pune
In the meeting we have learnt that India's regulations regarding pharmaceutical disposal are much
different than the ones in Finland. This got us interested: what does the legislation look like
around the world?
We decided to dive in. The aim was to find out how significant these
differences are, since improper disposal may cause drug residues to end up in the environment.
Here you can find our map containing a summary of disposal
guidelines around the
world. Not only do these guidelines differ greatly between countries, but also people
are often
not aware of them.
We concluded that it might be beneficial to spread awareness of these
guidelines, along with
information on effects of improper disposal of pharmaceuticals. Thus, we created a campaign.
Here you can find our campaign material, flyers, and a
video.
CONCLUSION: MACROLIDE BIOSENSOR
Next: The heart of the matter - Biosensor Design.
MEASURING POLLUTION IN WASTEWATER - SEMINAR WITH INDUSTRIAL WATER LTD.
(TEOLLISUUDEN VESI OY)
We attended a seminar which focused on current trends in water analytics, contaminant removal and
water treatment chemicals. It was useful to hear about the regulations concerning wastewater and
about the different techniques used in measuring pollution in wastewater. One of the main things we
learned from these presentations was that it is difficult to predict the effects of most substances
in the environment. This makes it hard to impose new regulations concerning pollutants.
One of the most interesting presentations was from Law & Water Ltd
regarding the regulation of micropollutants in the environment. We decided to contact this
company later on in our project to find out more about the regulations concerning
micropollutants in wastewater at the EU level and in Finland. See the “a real-world need”
section of
the project timeline here.
OUR RESEARCH IN BIOSENSOR DESIGN
What are the types of biosensors?
Biosensors can utilize, for example, whole cells, antibodies, or enzymes as sensing elements. Even
though enzymes are most widely used for recognition, protein purification is time and cost
consuming, which doesn’t make them ideal for an affordable biosensor. Another challenge is to create
a sufficient signal for a particular target. Often it requires multienzymes or cofactors [17].
We determined construction of whole-cell biosensor is more feasible for
us, since they are cheaper to make and can respond to a wider range of stimuli.
What issues can we anticipate?
A common mistake during biosensor design is the fact that the chosen organism does not survive in
the environment it is supposed to be used in. Another problem might be plasmid loss due to lack of
pressure to keep it.
Since we are using our biosensor to detect compounds in wastewater, we
ensured that our organism can survive in this environment. Wastewater contains a vast amount of
different and also toxic compounds that can affect the viability of the organism used. See our
viability tests here.
Which features are the most relevant?
Based on our research the most relevant aspects of biosensors seem to include sensitivity,
stability, specificity and time of response [17].
We contacted the WWTPs to verify that these features are most relevant
for them and is there something else we should take into account.
What have previous iGEM teams done?
We prepared an overview of all biosensor-related iGEM projects to see which compound was detected,
what was the incentive for the project, was the biosensor whole cell based and which organisms were
the most commonly used. Also, we looked at what kind of sensing systems were used and which of them
we could possibly utilize. We were also very interested to see what we would be able to model, which
softwares were used and which common mistakes we should avoid.
We went through almost 70 previous projects and highlighted the ones we
found most inspiring. Most projects utilized E. coli which is a well characterized
organism and there are a lot of synthetic biology tools available for its modification. We found
that transducers were almost without exception optical: colorimetric or fluorescent.
For our mathematical model we took inspiration from the 2016 Australia
team's "internal cellular
modelling". We also got inspiration to use Rosetta for protein modelling from a couple iGEM
projects, one of which was from the 2018 Washington
iGEM team.
We found it challenging to find information on some of the wiki sites, and we decided to keep in
mind that good documentation is essential for someone to build upon our project.
At this point of time, we set three sub goals to the project:
KEY POINTS TO KEEP IN MIND WHEN DESIGNING A BIOSENSOR
Biosensors can be used to detect small molecules. Electrochemical biosensors are rather robust
and
sensitive. Further, they can be used to detect both electrochemically active substances directly
as
well as products of an enzymatic reaction in case the target molecule is not itself redox
active.
One of the biggest issues with electrochemical biosensors is the selectivity owing to the
complex
matrix where the measurements are carried out. Interfering elements often show redox peaks that
overlap with those of the target molecule(s): you need a filtering mechanism or increase
specificity
by affecting the peak potentials by surface modifications of the electrode, for example.
Modelling biosensors is demanding, and most important is to formulate specific questions at
different levels (e.g. electrode material, electrochemical interface, electrolyte, mass transfer
etc.). Each level of these phenomena give different type of information that needs to be coupled
together. By systematically proceeding step by step through the entire phenomenon, one can
obtain a
lot of useful information.
VIRTUAL WWTP TOUR IN HSY - STARTING POINT OF OUR MOBILE GAME "FIX THE
FLOW"
Paula Lindell from HSY held us an online WWTP tour and explained to us in detail about the
wastewater treatment in Finland. Viikinmäki, as the largest WWTP in the Nordic countries, receives
270.000 m3 of wastewater every day. After the treatment, the effluent water is released to the
Baltic Sea. In the current process, phosphorus, nitrogen and organic matter are the main components
to remove so that these don't end up in the sea. As a side product from the treatment process,
Viikinmäki produces treated sludge and biogas. Sludge is used in production of fertilize products
and biogas for energy production. Viikinmäki WWTP produces almost all the energy it is consuming
[18].
For a detailed description, see our implementation page.
Mechanical treatment
In the mechanical treatment, the non-degradable solids, such as sand, grease,
and
other mixed waste are separated from the wastewater. Mixed waste is often referred to as
screening. This step also includes primary sedimentation, in which finer solid matter is
removed.
Chemical treatment
In the chemical treatment, the phosphorus is removed by precipitation. It
originates from washing detergents, for example. Ferrous sulfate is used as a detergent which
causes matter containing phosphorus to sink in the bottom of the treatment pools. There it can
be collected to be treated further in the sludge treatment process.
Biological treatment
In the biological treatment step, the bacteria in wastewater are used to
decompose organic matter that is rich in nitrogen. At this step, small bubbles of air are
introduced into wastewater to allow bacteria to grow and multiply. This forms activated sludge,
and bacteria can start utilizing and breaking down the organic matter. As organic matter
decomposes, nitrogen gas is released into the air.
After this, the water passes to the secondary sedimentation step. In this
step, the sludge and clean water are separated. The sludge sinks to the bottom of the pool, from
where it can be collected and recycled, for example, back to the beginning of the biological
treatment process. Clean water remains on the top of the pool. It is clean enough to be
conducted to the sea at this point.
In the Viikinmäki treatment plant, the biological removal process is continued
with biological filters. This step further enhances the removal of nitrogen.
Many people are not aware how the wastewater treatment process actually
looks like, which ends up with many things, such as pharmaceuticals or diapers, being disposed
improperly. We thought it might be helpful to transform this rather unattractive topic into
something more exciting and approachable. Following the iGEM goal of promoting synthetic biology
among the general public, we also wanted to show how microorganisms can be used in industry, as
well as highlight the fact that they can be improved using biotechnology. We developed
“Fix the
Flow”, which is a strategic and informative tower defence game to educate about
wastewater
treatment process and synthetic biology. See “Fix the Flow”here.
Future removal methods for micropollutants in Viikinmäki WWTP would most likely include activated
carbon and ozonation. However, the additional purification step ozonation is highly energy
consuming
and the production of activated carbon has high environmental footprint. Implementation of a
biosensor before and after the purification step could be used for the optimization of the
process.
The sensor and future micropollutant removal steps would be located at the end of the process
before
releasing treated wastewater to the environment. An additional advantage to locate the biosensor
at
the end of the treatment process is the uniform structure of effluent wastewater, which makes
the
measurement more reliable.
Ideal sensor would be located in the process and water itself. It should be easy to clean and
maintain. Most importantly, it should be reliable and cost-effective.
The detection limits of pharmaceuticals are minimal and the concentrations vary significantly
from
time to time. It would be beneficial to be able to compare the content of influent and effluent
water. It would be most convenient if the sensor was "online," easy to maintain and
cost-effective.
As a conclusion, our innovation would be beneficial to guide the
optimization of future micropollutant steps in WWTPs that are highly energy consuming. The
sensor should be cost-effective, reliable and easy to maintain. The ideal sensor would locate in
the process and wastewater itself with a continuous measurement feature. However, due to the
risk of releasing GMO to the process, we chose to concentrate on developing a one-time use
biosensor in the separated system from the process.
SWISS WWTPs - FIRST IN MICROPOLLUTANT REMOVAL
As mentioned earlier, we found out that only a few countries, such as Switzerland, have opted for
regulation and technology to remove such pollutants from water. We
wanted to take a deeper look at current treatment methods for micropollutant removal in Swiss
WWTPs, with emphasis on how they are monitoring the process. Additionally, we asked if there are
any decentralized treatment methods in point pollution sources, such as in hospitals.
According to the Swiss law, those WWTPs which have to remove micropollutants, must remove
micropollutants by 80%, measured on the basis of selected indicator substances. The water
treatment
methods, ozonation or treatment with activated carbon, being applied in Switzerland are
applicable
to any country. Before implementing the new purification steps it is recommended to have a well
working biological treatment with nitrification beforehand.
Removal performance is monitored by the authorities via 48-hour composite samples and the
indicator
substances are analysed with LC-MS/MS. UV measurement (254 nm) is currently the best method for
online supervision and online control. Energy consumption is one reason for online control of
micropollutant removal to ensure the optimal dosing for powdered activated carbon or ozone.
Dilution
and loads may vary daily, seasonally as well as due to long term change. Thus, reliable and
cost-effective methods to assess and control the required dose are of relevance.
Highly toxic wastewater is being treated at the source, in the industrial facilities or also in
hospitals (e.g. chemotherapy wastewaters). However, loads of the average wastewater of hospitals
is
not sufficient to justify a separate treatment: for cost efficiency most wastewater from
hospitals
is treated at the municipal WWTPs.
MEASURING MACROLIDE ANTIBIOTICS IN LABORATORY ENTERPRISES
The challenges of wastewater analysis come from the fact that wastewater can contain a wide
variety
of solid and liquid components as a matrix. The assay limits are very small for pharmaceuticals,
so
even small interfering factors in the matrix pose challenges. This requires samples to be
subjected
to various purification steps before the actual analysis.
We do not yet measure macrolide antibiotics. The compounds that are analyzed are based on
current
legislation. There will definitely be a demand for this kind of product since it is currently
more
and more important to get results as quickly as possible.
BASICS OF ELECTROCHEMICAL BIOSENSORS AND A MORE IN-DEPTH VIEW ON MTR PATHWAY
While researching biosensors, we noticed that in addition to optical biosensors, electrochemical
biosensors are very popular. Electrochemical biosensors can measure turbid samples, which could be
an advantage for us when working with wastewater [19]. According to our research, electrochemical
biosensors tend to show higher sensitivity than optical ones, which is very important since
macrolide concentrations are very low in wastewater. Based on our
findings, we proceeded to search
for a pathway that could provide E. coli with the ability to give an electrical output.
Also,
we contacted some experts in the field to get more perspectives.
When comparing electrochemical and optical biosensors, sensors relying on electrical output may
lack
specificity. In electrochemical biosensors, the creation of a signal is proportional to chemical
reactions that take place on the electrode. Thus, anything that resides in the sample, including
impurities, might cause current. There is a risk of unknown compounds getting oxidized on the
electrodes and a risk of electrode fouling with wastewater. Many compounds might be able to
attach
to the electrode better than cells. Therefore, for analytical purposes, electrodes are often
modified with molecules that are sensitive to one analyte only.
After searching for different options, we came across the Mtr pathway
of Shewanella oneidensis. As E. coli is a model organism and our universities did
not have experts working with S. oneidensis, we decided to research E. coli as a
heterologous host for the Mtr pathway. To read more about the Mtr-pathway and our research with
this topic, visit our contribution page.
The immobilization technique should be considered carefully, as conductivity might decrease
because
of certain materials' properties. For E. coli to utilize the Mtr pathway, there should be
an
anaerobic or anoxic environment. In wastewater treatment plants, the specific conditions might
limit the applicability of the electrochemical biosensor. For instance, the presence of
dissolved oxygen gas in wastewater might make the measurement challenging.
The Mtr pathway should work properly in E. coli before it would be sensible to use it for
a
biosensor application. The electrochemical biosensor seems challenging to be executed in three
months. Also, the low concentration of macrolides in the sample might be a more significant
issue
than the sensitivity of the output signal.
The main identified obstacles with the Mtr-pathway were the
immobilization of the cells to the electrode and the biosensor working in an anoxic environment.
We decided that due to the limited time of three months in the laboratory and our lack of
electrochemical background, we will focus on developing a macrolide-sensing circuit with optical
output rather than improving the Mtr-pathway in E. coli.
IN SEARCH FOR A SENSING ELEMENT FOR MACROLIDES - DISCOVERY OF MphR REGULATORY
SYSTEM
With an intensive literature research we found MphR, a transcription factor that can bind to
macrolide antibiotics [20]. MphR acts as a repressor protein binding to pMphR promoter, see more on
our parts page. Using this transcription
factor, we designed our genetic circuit for the optical
biosensor to detect and quantify macrolide antibiotics in wastewater. See our detailed
design here.
INTELLECTUAL PROPERTY RIGHTS
During our research, we found that the only known transcription factor that detects and binds
macrolide antibiotics is MphR. We found that a research group has applied for a patent regarding
this natural gene. The patent application claims the use of any mutated form of MphR and its use in
biosensor. We contacted an innovation expert and the iGEM
HQ to identify if we still can use this transcription factor in our project.
A patent is used to protect a solution if it is new and inventive compared to the previous ones,
and can be used industrially. Patents apply to commercial use; patented solutions can be applied
freely in research. Patent applications do not yet preclude commercial use before granted.
Natural phenomena cannot be protected, per se. Still, technical solutions that exploit them can
be, if the solution is novel and innovative, and therefore not evident from the available
information. For example, several patent applications and patents related to blood glucose
measuring strips use the same enzyme and reaction (e.g., glucose oxidase). The protection scope
can be connected to other sensor features that achieve, for example, smaller size, better
accuracy, shorter measurement time, or better manufacturing.
Patent publications must carefully determine claims. They define the scope of the patent. If the
case is made in a manner that does not comply with the other party's claims, it does not violate
the patent. Claims are also included in the applications and can be evaluated. They often become
narrower during the processing of an application before it becomes a granted patent. Patents are
also geographically limited. The PCT (Patent Cooperation Treaty) application phase is an
international intermediate phase from which it is possible to proceed to different regions.
The document [21] would appear to be an international PCT patent application, not yet a patent. By
using a different gene manipulation than that described in that application, it may be possible to
protect the innovation. In any case, the matter in that publication is public, that is, it can no
longer be protected like any other public information.
The application does not (yet) have patents granted. However, one US application is pending, and
that may be accepted. It is worth following this in what form it will be granted and what its exact
scope is.
As iGEM HQ and the expert confirmed, we were allowed to use MphR in
our project since the patent application is still pending. However, we need to follow the
process and consider our actions if the patent would be issued.
SINISENS - A STEP TOWARDS CLEANER WATERS
SINISENS, our solution, is an optical biosensor to detect and
quantify
macrolide antibiotics in wastewater. The end-user we designed our biosensor for are
WWTPs to
optimize the micropollutant removal process. Below is a schematic picture of our device.
A sample of wastewater is to be placed in the biosensor: if macrolide antibiotics are present,
green fluorescence would be created.
We decided to have fluorescence as an output for the biosensor. Since
macrolides are present in extremely low concentrations in wastewater, we set our third sub
goal to focus on increasing the sensitivity of the biosensor. This altered our initial
experimental plan.
CONCLUSION: OPTICAL BIOSENSOR
Next: Improving the key points, sensitivity and specificity, of the sensor.
MODELLING AND SOLVING THE CHALLENGE WITH MACROLIDE CONCENTRATION
The most challenging aspect of macrolide antibiotics quantification is their low concentrations in
wastewater. Using modeling, we confirmed that in order to achieve
satisfactory detection limits and reliable output signal, concentration of macrolides may be
necessary.
We also considered a different way of improving biosensor sensitivity, which included selecting MphR
mutants with alterations at the binding site that would increase the binding affinity of MphR for
macrolides and, thus, potentially lower the detection limit. We used
the Rosetta software to predict these mutations. We sought guidance to confirm that our approach
is feasible and how to decide between different protein modifications due to limited lab
time.
Since the macrolide antibiotics are typically present in very small concentrations in wastewater, it will be important to ensure that the MphR regulatory system can be used to detect them. Using sophisticated Rosetta modelling and site-directed mutagenesis of MphR should allow improving the binding affinity towards the three macrolides if necessary. It would also be useful to test whether E.coli cells can accumulate the macrolides as this could potentially increase sensitivity of detection.
When comparing the results in Rosetta, the overall score should not be calculated as an average
of all the scores: it is important to evaluate which of the characteristics actually differ
significantly. One of the most important variables to consider is the dG_separated, which
measures the binding energy of the protein. However, it is impossible to predict which ones will
work best in practise and therefore as many mutations as possible should be tried out in the
lab.
We decided to focus more on the recommended parameter to predict the
best mutations for the MphR transcription factor in order to increase the binding affinity to
macrolides (see our modelling page). After obtaining the modified sequences from
Rosetta, we chose the six best ones and ordered them to be tested in the laboratory.
MICROFLUIDICS IN INCREASING SENSITIVITY
We considered microfluidics as one possible solution for concentrating macrolides.
Microfluidics has three classes of benefits: portability, improved performance, and the fact that
the structure can be the same size as target molecules. The portability aspect would most likely
benefit your project.
A sample can be concentrated by evaporating the solvent or by using absorption-based techniques.
If microfluidics were involved, maybe the most feasible technique to solve the macrolide
concentration issue would be the gradient technique.
From the gradient techniques, we decided to test isoelectric focusing
(IEF). With IEF, the analyte can be concentrated up to a thousand times [22]. This method would
allow us to increase the macrolide concentration in wastewater to more easily detectable levels.
As a proof of concept, we tested this technique.
CONTROL BACTERIAL PROLIFERATION
We found out that controlling the number of cells in our biosensor could be crucial for the
measurement to be successful. The cell proliferating could affect the measurement as the amount of
fluorescence correlates with the number of cells present. Also, considering our final product, we
would want to integrate our cells on a hardware platform, and we are considering immobilization as
the technique. We have considered different matrices and methods for immobilization.
Immobilizing the cells would be advantageous if one would like to integrate the cells on some
hardware platforms. If the sensor cells would be prepared in advance on a microtiter plate, for
example, and keep it refrigerated until use, encapsulation is probably a very good idea.
By immobilizing cells on a suitable polymer, the cell growth could be minimized, and then the
proliferation should not interfere with the measurement.
After confirming with experts that immobilization on a suitable polymer
could be advantageous for our SINISENS biosensor, we decided to test it. To find
potential immobilization methods for our biosensor, we tried immobilizing our sensor cells on
different matrices. See our results here and
more of the immobilization experiments here.
KEY FEATURES FOR THE SENSITIVITY OF THE BIOSENSOR
To our final design, we would immobilize our biosensor cells on a hardware platform using entrapment
technique to inhibit cell proliferation from interfering with the measurement. By site-directed
mutagenesis to MphR transcription factor and suitable microfluidics technique we can increase the
sensor’s sensitivity to desired level.
CONCLUSION: FINAL DESIGN FOR THE BIOSENSOR
Next: Taking a deeper look in the legislation field.
LEGISLATION PERSPECTIVE IN THE PROJECT
We contacted the following experts in the field of environmental law
with three different areas in mind: EU level decision making, pharmaceutical industry and
wastewater treatment.
In Spain, the agency that regulates the life cycle of pharmaceuticals is the Spanish Agency of
Pharmaceuticals and Sanitary Products (Agencia Española de Medicamentos y Productos Sanitarios
(AEMPS)). This agency depends on the Sanity Ministry and it is coordinated with the European
Medicines Agency (EMA), to respect the rules and regulations established by the European Union
regarding pharmaceuticals. AEMPS evaluates every drug and gives the authorization to
commercialize it based on quality, security and efficiency criteria.
Regarding the environmental impact of the medicine production, lots of institutions are focusing
on carrying out “green chemistry”. With this, they want to (1) reduce the chemical risk
associated to the use and manufacturing of chemical products, (2) reduce the impact of
wastewaters and the dispersion of pollutants to the atmosphere, (3) reduce the intensive use of
water and energy, (4) reduce the environmental impact of the chemical products once used and (5)
minimize the flux of matter from the not renewable natural resources to the productive
processes.
Wastewater Treatment:
Pharmaceutical compounds are conceived mainly to perform physiological processes and to resist
their inactivation before they do that. This is what also makes them difficult to biodegrade and
gives their potential toxic effects in aquatic and terrestrial ecosystems. Also, these can
bioaccumulate and manifest in conditions that could drive some aquatic populations to
extinction. Medicines (including antibiotics) arrive to WWTPs as emerging contaminants, and
normally they are only partially degraded because WWTPs don’t have specific removal systems for
them. Currently, there are studies going on about the introduction of a combination of
treatments called tertiary treatments, which would be able to remove emergent compounds and
would improve the quality of the effluent water.
Could there be Green Stickers for pharmaceutical products to enable consumers to take
environmental considerations into account?
In order for consumers to take into account the environmental impact of a medical product, the
development of a green labeling system for drugs is viable. It could be based on this green
chemistry of pharmaceutical producers.
At the EU level, there are only common environmental laws for micropollutants regarding water
ecosystems. WWTPs are also able to show responsible actions, even though there would not be new
laws to guide the operation.
Pharmaceutical Industry:
The operation of pharmaceutical manufacturers is regulated by environmental permits. One tool to
control the environmental impact in terms of released wastewater could be industrial wastewater
agreement. The industrial wastewater agreement is settled between an industrial enterprise and a
corresponding treatment plant. In the agreement, limit values for wastewater and a monitoring
program are drawn up. However, these agreements are not required by the law; moreover,
pharmaceuticals are rarely part of them. If there would be limit values for micropollutants for
wastewater in the future, WWTPs probably have pressure to determine the sources of pollutants.
In order for official limit values to take place in legislation, monitoring studies about
harmful effects of pharmaceuticals are needed.
Wastewater Treatment:
Pharmaceuticals in wastewater have not received much attention in environmental permitting to
date and more information about the amounts of them should be gathered systematically. However,
currently even ordering the correct analysis for micropollutants is challenging and not the top
priority for WWTPs.
It was highlighted that more monitoring studies and information about
pharmaceutical’s environmental effects are needed in order to determine the official limit
values for them in wastewater. To get more insight for EU level decision making, Vieno suggested
us to contact Ari Kangas from the Finnish Ministry of Environment.
*Note: The following is the opinion of an official individual and does not represent the Ministry
of the Environment's general position.
Wastewater Treatment:
Switzerland is the first country to force the requirement to remove micropollutants from
wastewater. Currently, the removal methods are activated carbon and ozonation, which are
non-specific removal methods. They use particular indicator compounds to monitor the progress
of the removal process. These indicator substances have been selected based on their
chemical, biological, and physical properties as they can represent certain chemical groups. By
tracking that these substances are removed in the process, it is relatively certain that other
harmful substances have been removed.
EU Legislation:
The European Union has published a communication on its strategic approach to reducing the
environmental impact of pharmaceuticals [23]. It is to be expected that a similar trend to
Switzerland’s is coming elsewhere as well.
The list of priority substances includes substances for which there is proof that they harm the
environment. More research information is needed for substances on the watch list before
assessing whether some of the compounds should be transferred to the priority list. Adequate
research data is the keyword in this matter. It should also be noted that in the wastewater
treatment plant’s environmental permit the emission limit values are set according to the
environmental quality standards.
The reform of the Urban Waste Water Treatment Directive, on which wastewater treatment is based,
is currently underway. It sets minimum requirements for what should be removed from wastewater.
In Finland, the environmental permits require much stricter wastewater treatment than required
by the current directive.
Ten years could possibly be the timeframe for setting new requirements for wastewater
treatment in the EU framework. There are a few projects ongoing in Finland to study
removal methods of pharmaceuticals from municipal wastewater and hospital wastewater.
Macrolide antibiotics are on the EU watch list of harmful substances. If regulations come that
require the removal of pharmaceuticals, a device will be needed to measure whether this
removal process succeeds. In that sense, your innovation would be useful.
How can science affect the creation of a new environmental policy?
Eventually, legislation is politics. However, no political decisions can be made without
research information. In decision making, those who have evidence based data are most likely the
ones to make the change.
As a conclusion from these meetings, there is a lot of discussion on
the topic of pharmaceuticals in water resources today, showing the possible direction of
decision making. Decision-makers seem to be aware of the problem of pharmaceuticals ending up in
the aquatic environment. Currently, many drugs, such as macrolide antibiotics, are being
considered for transfer from the EU ‘watch list’ to the list of priority substances.
The tertiary treatment processes for micropollutant removal, including ozone and activated
carbon treatment, are being studied and piloted in some Finnish wastewater treatment plants. If
Finland follows the techniques used in Swiss WWTPs, the efficiency of the micropollutant removal
process could be monitored by using indicator substances. Clarithromycin, belonging to the
group of macrolide antibiotics, is used as one of these indicator substances [24]. Therefore,
SINISENS has potential use in wastewater treatment plants to monitor the removal performance
of micropollutants [24].
CONCLUSION: A REAL-WORLD NEED
Next: As excited as we are about our work, it is still a long way to go!
FUTURE
The storyline summarizes the steps we have taken during the development of SINISENS. The following
section discusses the potential advancements that can be done to improve our innovation in future as
well as all the necessary compromises.
It should be considered that wastewater may contain inhibitors that may interfere with the GFP
signal. In addition to that, loss of plasmid may be an issue. Final product may have to have the
sequences integrated into the bacterial chromosome. The proliferation of bacteria can be
controlled by using poor media or lowering the temperature of the culture.
TEST IN NON-STERILIZED WASTEWATER AND VARYING CONDITIONS
Due to safety reasons, we used sterilized wastewater for our experiments when developing and testing
our biosensor. However, the biosensor should be tested in fresh and non-sterilized wastewater to
optimize its performance further. Also, it should be noted that in the wastewater treatment process,
both pH and temperature may vary slightly. Therefore, it would be beneficial to study more closely
how these variations affect the biosensor's performance.
OTHER OUTPUT SIGNALS
Electrochemical biosensors may be more sensitive, which is an important feature given the low
concentrations of macrolides in wastewater, but the development is more resource-consuming.
Moreover, we are aware that it would be easier to quantify the output with an electrochemical
biosensor, which is an essential feature for the end-users. Therefore, we could consider
improving our biosensor by changing the output signal.
BIOSENSOR FUTURE - CONTINUOUS MEASUREMENT
As discussed in the storyline, the ideal sensor would locate in the process and wastewater itself
with a continuous measurement feature to meet the WWTP’s need for operating the removal
process of pharmaceuticals. Due to the risk of releasing GMO into the process, we chose to
concentrate on developing a one-time use biosensor in a separated system outside the process. The
sensor could be located next to the water flow, where the sample is collected and pumped into the
device. In addition, the process of replacing the sensor could be automated. See more about the
implementation here.
In order to assess feasibility of our project and make sure we are utilizing all of the synthetic
biology tools that are available, we contacted various experts working in academia, that specialize
in microplastics, pharmaceutical research, biosensor design and many others.
Jukka Hassinen
Aalto University, Finland
Jukka Hassinen, D.Sc. (Tech) in Applied Physics, has expertise in the
preparation and advanced characterization of nanomaterials, and their applications in
water purification and photonics.
Benjamin Wilson
Aalto University, Finland
Dr. Ben Wilson is a Staff Scientist at Aalto University who has an
extensive international research experience in materials and the circular economy of
metals. He is currently involved in research related to the sustainability of
batteries/WEEE, wastewater purification and use of biorefinery wastes for value added
applications.
Anna Mikola
Aalto University, Finland
Anna Mikola is responsible for wastewater related research in the
Group of Water and environmental engineering in Aalto University School of engineering
in Finland. Her main research topics at the moment are biological nitrogen removal and
gaseous emissions, nutrient recovery processes and removal of micropollutants and
microplastics from wastewater and sludge.
Merja Penttilä
Aalto University, Finland
Merja Penttilä is a research professor in biotechnology at VTT
Technical Research Centre of Finland and an adjunct professor in synthetic biology at
Aalto University. Her interest is in using cell factories to produce chemicals, fuels,
materials and proteins. She has been promoting and developing synthetic biology
technologies and concepts in Finland and led many large academic and industrial projects
on these topics. Recently, she established with Wihuri foundation funding the Centre for
Young Synbio Scientists (CYSS), which also includes a BioGarage space.
Dr Henri Xhaard, Docent, PhD in computational modelling (Åbo Akademi
University). More info can be found here.
SUDDEN is a research project that aims at reducing the environmental
hazards related to the life cycle of pharmaceuticals.
Tomi Laurila
Aalto University, Finland
Tomi Laurila is an Associate Professor in Microsystem technology
in the Department of Electrical Engineering and Automation at Aalto University.
Lasse Murtomäki
Aalto University, Finland
Professor of Physical Chemistry and Electrochemistry, Aalto
University, Dept. Chemistry & Materials Sci. Lasse Murtomäki received his doctoral
degree at Helsinki University of Technology in 1992; the topic of the thesis was
electrochemistry in immiscible water-oil systems. Prof. Murtomäki’s expertise covers, in
addition to electrochemistry, transport processes and pharmaceutical applications of
physical chemistry.
Michael Lienemann, Dr. rer. nat., is an Academy of Finland Research
Fellow, affiliated with the VTT Enzyme and Material Biotechnology research team and has
previously engaged in international collaborations at the Lawrence Berkeley National
Laboratory, Montana State University and Stanford University. During his early academic
career, he has investigated the structure-function relationships of carbohydrate-binding
and amphiphilic proteins and currently investigates the structural basis of CO2
reduction by cathode-bound enzymes.
VTT Technical Research Centre of Finland Ltd. is one of the leading research and
technology companies in the Nordic countries that performs demonstrations, piloting and
proof-of-concepts.
Silvan Scheller
Aalto University, Finland
Silvan Scheller is currently working as an Assistant Professor in the
Department of Bioproducts and Biosystems at Aalto University. The biochemistry group,
led by Prof. Scheller, uses metabolic engineering to construct new pathways for the
conversion of CO2 to fuels. They utilize biocatalytic transformations that occur in
nature, but that are currently not possible in classical chemistry. In order to utilize
such new metabolisms and enzymes, we first need to understand them in detail. For this
reason, they carry out fundamental research in microbial physiology and mechanistic
enzymology.
Norman Adlung
Aalto University, Finland
Norman Adlung is currently working as a postdoctoral researcher in the
biochemistry group led by Prof. Scheller at Aalto University. He has a background in
molecular biology and uses the methanogenic model organism Methanosarcina acetivorans to
study regulatory processes and anaerobic oxidation of methane. Previously, Norman
achieved his PhD in the field of molecular plant-pathogen interactions.
Ville Paavilainen
University of Helsinki, Finland
Ville Paavilainen is a tenure track group leader at the Institute of
Biotechnology at University of Helsinki-HiLIFE. The Paavilainen lab uses chemical and
structural biology to study protein subcellular targeting and biogenesis. In this
project, he has provided advice and feedback on various aspects of the project,
including functional test of reporter cell lines.
Maria Sammalkorpi
Aalto University, Finland
Dr. Maria Sammalkorpi leads the Soft Materials Modelling group in
Aalto University School of Chemical Engineering, Finland. Her cross-disciplinary
research program bridges biotechnology and bio- and polymer materials. She uses
molecular level and mesoscale computational approaches to study e.g. colloidal
assemblies, films and molecular coatings, and biomacromolecular solutions for materials
applications. More information available at: https://www.aalto.fi/en/department-of-chemistry-and-materials-science/soft-materials-modelling.
Shimshon Belkin
University of Jerusalem, Israel
Dr. Shimshon Belkin is a Professor of Environmental Microbiology and
the Ministry of Labor & Social Welfare Chair in Industrial Hygiene at the Hebrew
University of Jerusalem, Israel. A microbiologist with over 25 years of experience in
the development of genetically engineered whole-cell biosensors, he is an international
authority on the synthetic biology of microbial-based biosensors.
Ville Jokinen
Aalto University, Finland
Dr. Ville Jokinen is a University Lecturer at Aalto University School
of Chemical Technology. His fields of interest are microfabrication, microfluidics and
wetting, especially when these are applied to biomedical applications.
Tomasz Płociniczak
University of Silesia in Katowice, Poland
Tomasz Płociniczak is a PhD in the department of Microbiology at
University of Silesia in Katowice, Poland.
Industry
We wanted to make sure our product could be utilized in the industry. For that reason, we have
contacted WWTP from different countries, pharmaceutical and other chemical manufacturers. We
inquired about the most important issues in the industry and asked what they think of our biosensor
idea.
Otto Järvinen has studied environmental engineering focusing on water
and waste management in Tampere University. He has interests in environmental
engineering and sustainability. He specializes in industrial wastewater management and
life cycle of pharmaceuticals.
Orion is a globally operating Finnish pharmaceutical company - a builder of well-being.
Orion develops, manufactures and markets human and veterinary pharmaceuticals and active
pharmaceutical ingredients. The company is continuously developing new drugs and
treatment methods. The core therapy areas of Orion's pharmaceutical R&D are central
nervous system (CNS) disorders, oncology and respiratory diseases for which Orion
develops inhaled Easyhaler® pulmonary drugs.
Katriina Rajala works as a research scientist at Kemira and she
focuses on water and wastewater treatment, especially on emerging contaminants. She has
studied water and environmental engineering in Aalto University and chemistry in
University of Helsinki.
At Kemira, we use our chemistry to improve your everyday. We add optimal quality,
functionality and strength to paper and board products, we ensure safety and hygiene of
water and food packaging, and we maximize yield from energy resources. Our areas of
expertise are pulp & paper, water intensive industries & municipal water treatment, and
oil & gas. Through our global network of dedicated experts, production facilities and
R&D centers, we serve customers around the world and improve their product quality,
resource efficiency and sustainability.
Paula Lindell (MSc, Chemistry) works as Group Manager in the
wastewater division for the Helsinki Region Environmental Services Authority HSY and has
over 10 years of experience working in the water sector. She has a diversified
background in the water sector on engineering, operation and research and development on
national, Baltic sea Regional an European level.
Helsinki Region Environmental Services Authority HSY is the most prominent environmental
public body in Finland. It produces waste management and water services for over 1 M
residents throughout the Helsinki Metropolitan Area. In addition, it is responsible for
air quality information, and it provides geographic information about the regional
development and promotes climate work. Greater Helsinki’s wastewaters are treated at two
of Finland’s largest wastewater treatment plants: Viikinmäki in Helsinki (PE 1,3 M) and
Suomenoja in Espoo (PE 0,4 M). Plants are operated by the Helsinki Region Environmental
Services Authority HSY. Through collaboration HSY works to meet the vision, to transform
the Helsinki Metropolitan area into the world’s most sustainable urban region. The
prerequisite for reaching this goal is to be at the cutting edge of environmental
responsibility and resource efficiency, high reliability in performances, smoothly
running services and a stable economy.
Engineer with degrees in both Chemical and Environmental Engineering.
Currently works as a Specialist Engineer in New Technologies Centre of MPWiK where she
took part in several research projects co-financed by the National Centre for Research
and Development. Her free time is spent on various crafts and conducting a PhD in
Mathematical Modelling of Wastewater Treatment at Wrocław University of Technology.
MPWiK S.A. in Wrocław is a Polish municipal water and wastewater company. The New
Technologies Centre (Centrum Nowych Technologii – CNT) is an R&D department of MPWiK S.A
in Wrocław. A basic goal of CNT is to work towards technological development of the
company in order to improve efficiency of water and wastewater management for the sake
of environment.
Sara Rantamäki works as a Production Engineer in WWTPs of Kurikan
Vesihuolto Oy. She is M.Sc. (Tech.) in Environmental Engineering and has a major in
Water and Waste Management Engineering.
Kurikan Vesihuolto Oy produces water services in Kurikka, Jalasjärvi and Jurva in
Finland. Their customers are households, industry and cooperatives in the area. In
addition, the company sells water to Poronkankaan Vesi Oy. Supply water is produced by
their own groundwater intake stations. The wastewater is treated with biological and
chemical based treatment methods in two operating plants. Kurikan Vesihuolto Oy works
with quality service, economical and sustainable values in mind.
Jarkko Laanti (M.Sc. (Tech.) in Industrial Engineering and Management)
is an experienced Quality And Environmental Manager with a demonstrated history of
working in the environmental services industry. He has 15 years experience in Quality
Management, Risk Management, Operations Development and Continuous Improvement with 9
years in the sector of water services.
Turun seudun puhdistamo Oy is a wastewater treatment service provider that is owned by
14 municipalities and offers high-quality wastewater treatment services to its owners.
The company is responsible for the operation and treatment results of the Kakolanmäki
wastewater treatment plant, with the aim of providing an optimal purification
performance. The plant processes the wastewater of almost 300,000 residents in the Turku
region, in addition to the industrial wastewater of the region.
Christoph Ort is a Group Leader in the Department Urban Water
Management in Eawag. He’s interests lie in source typing and tracking of water-borne
pollutants such as personal care products, pharmaceuticals and illicit drugs in both the
built and natural environment. He combines full scale measurements, modeling
(deterministic and stochastic) and laboratory experiments. Furthermore, he develops new
measurement devices and work flows to gain unprecedented insight into pollutant dynamics
and the spatio-temporal variability of environmental variables.
Eawag is the Swiss Federal Institute of Aquatic Science and Technology and is part of
the ETH Domain, which includes the two universities of ETH Zurich and ETH Lausanne
(EPFL), the four independent research institutions of Empa, PSI, WSL and Eawag. Firmly
anchored in its home country of Switzerland, but with a global network, Eawag is
concerned with concepts and technologies for dealing sustainably with water bodies and
with water as a resource.
Dr. Adriano Joss works in the Department of Process Engineering in
Eawag. He expertises in wastewater, micropollutant and ozonation.
Eawag is the Swiss Federal Institute of Aquatic Science and Technology and is part of
the ETH Domain, which includes the two universities of ETH Zurich and ETH Lausanne
(EPFL), the four independent research institutions of Empa, PSI, WSL and Eawag. Firmly
anchored in its home country of Switzerland, but with a global network, Eawag is
concerned with concepts and technologies for dealing sustainably with water bodies and
with water as a resource.
Christa is a Senior Scientist and Group Leader in the Department
Environmental Engineering in Eawag. Her research interests lie in input and fate of
organic contaminants, e.g. pharmaceuticals, from urban settlements in the aquatic
environment, wastewater treatment processes and strategies for the removal of organic
micropollutants and development of analytical techniques for the determination of
organic pollutants and transformation products in sewage and ambient waters.
Eawag is the Swiss Federal Institute of Aquatic Science and Technology and is part of
the ETH Domain, which includes the two universities of ETH Zurich and ETH Lausanne
(EPFL), the four independent research institutions of Empa, PSI, WSL and Eawag. Firmly
anchored in its home country of Switzerland, but with a global network, Eawag is
concerned with concepts and technologies for dealing sustainably with water bodies and
with water as a resource.
Jaana Saukko graduated from the University of Helsinki with a master's
degree in food sciences focusing on food chemistry. Bachelor's degree studies Saukko
performed at Seinäjoki University of Applied Sciences in the biotechnology and food
engineering program. At present, Saukko works at MetropoliLab Oy as one of the persons
in charge of water analyzes in the organic chemistry team and as the leader of method
development projects for various analyzes of organic chemistry.
MetropoliLab is an impartial, independent laboratory T058 accredited by Finas, the
Finnish Accreditation Service according to the standard SFS-EN ISO 17025 located in
Viikki Campus area. Our analytical strengths are: foods, their microbiological and
chemical quality, contaminants, composition and additives; chemical and microbiological
analyses of different environmental samples (water, soil etc.); the quality of indoor
and outdoor air; microbes causing moisture damage; and contaminated soils and sediments.
Our laboratory services cover the most important analyses, in situ measurements and
sample taking and/or collection tasks for food, water and environmental testing and
inspections.
Environmental Organizations
The value we had at heart when we were developing our biosensor was protecting the environment. We
contacted water protection-related organisations to ask about the most relevant issues that
should be focused on.
Jari Männynsalo has finalized his studies in limnology and currently
works as an environmental expert in The Water Protection Association of the River
Vantaa and Helsinki Region. He specializes in wastewater related topics.
The River Vantaa is the closest river system to a million inhabitants in the most
densely populated area of Finland. The 100-kilometre long river bed flows into the Gulf
of Finland in Helsinki.
The Water Protection Association of the River Vantaa and Helsinki Region monitors
the water quality, biota and the operation of wastewater treatment plants in the
catchment area of the River Vantaa. Monitoring is conducted objectively at a river basin
scale for different activities requiring environmental permits. The Water Protection
Association is also actively implementing various projects to improve the status of the
river system. The non-profit association was founded in 1963. The Association is a
member of the Federation of Finnish Water Protection Associations whose members comprise
12 regional water protection associations
Matti Leppänen (Senior Research Scientist, PhD) works as an
ecotoxicologist in the Ecotoxicology and Risk Assessment –group in SYKE where the major
tasks relate to biotesting of environmental samples and chemicals, expert guidance for
the environmental administration (local and central) and research in aquatic
environment.
SYKE (Finnish Environment Institute) is both a research institute and a center for
environmental expertise, providing knowledge and solutions enabling sustainable
development. SYKE forms part of Finland's national environmental administration and
works under the auspices of the Ministry of Environment. With the staff of ca. 570
employees, SYKE covers all aspects of our living environment from urban areas to
pristine locations and from environmental policy to laboratory analysis.
Legislation
To make sure our biosensor is compliant with current regulations that WWTPs have to obey by, we
contacted environmental lawyers and the Finnish Ministry of the Environment. We also wanted to
confirm our work does not infringe on any intellectual property rights, so we consulted with a
patent expert.
Josep Maria Prat Garcia
Lawyer specializing in Environmental Law
Josep Maria Prat Garcia, Lawyer specializing in Environmental Law, Graduated from the University of Barcelona (1995), Postgraduate in Environmental Management and Administrative Law; Research proficiency for the doctorate in Law from the University of Barcelona; WEEE Auditor (Waste Electrical & Electronic Equipment) and Environmental Management Systems Auditor; Collaborating professor in different university centers in Environmental Legislation, Environmental Law and Administrative Law (Fundació Universitat Politecnica de Catalunya, IL3 - University of Barcelona, Private Foundation University Institute of Applied Technology IMF, Fundació Layret - Camilo José Cela University); Author of the book "The Ecological Crime" CEDEX Editorial (2.000)
Niina Vieno has a Doctor of Technology degree in Environmental
Engineering. She has worked within the environmental sector for 20 years, mostly related
to water treatment and hazardous substances. Her passion is to improve waste water
treatment of industry and municipalities, to cut down environmental burden caused by
hazardous substances and to inform and educate people how to reduce their chemical
footprint.
Law and Water offers legal and water technology related services for companies and
communities. Company offers the combination of juridical and engineering know-how and
looks into subjects of water quality, industrial waste waters, public procurements and
environmental permits.
Ari Kangas
Ministry of Environment, Finland
After finalizing his studies in environmental protection technology,
microbiology and chemical engineering at the Helsinki University of Technology Mr.
Kangas has been working around wastewater issues the whole of his professional career.
He has worked as a research engineer, operational chemist, and operational chief at the
WWTP and then as a consultant and private entrepreneur before changing over to
permitting authority, supervising authority and finally to the Ministry of the
Environment. In his present position, Mr. Kangas is coordinating national projects
covering wastewater treatment, nutrient recovery and sludge utilization.
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