SulFind 2021

Human practices

"Human Practices is the study of how your work affects the world, and how the world affects your work." — Peter Carr, Director of Judging

Fish has been an important source of food throughout history, and in the last 50 years the average person has almost doubled their consumption of fish and other aquaculture products [1]. Keeping the fact that it is estimated that the world population will reach 9.9 billion people in 2050, the fishing sector will be important in the years to come [2]. However, there are several high-profile issues land based aquaculture must overcome to support sustainable growth:

Fish welfare

The use of RAS gives control over many parameters that play a part in fish welfare, such as pH, temperature and dissolved oxygen, and RAS thus has potential to maintain high fish health. However, H₂S toxicity is an obvious hindrance to this, which decreases fish welfare. Even at H₂S concentrations that are lower than lethal, damage may be done to the fish, and it is likely that the fish will become stressed.

Sustainability

In Norway RAS is often used in combination with sea-based aquaculture, so that most fish spend some of their younger life in RAS facilities [3]. This means that the problem of H₂S toxicity extends to almost all Norwegian aquaculture, and thus have a large impact on the sustainability of Norwegian aquaculture.

Should H₂S toxicity lead to fish death in RAS facilities, it goes without saying that the resources put into rearing the fish go to waste.

As a result, solving this issue would decrease the waste of resources, making the industry more sustainable - and RAS a more viable fish farming method both in Norway and globally.

Integrated Human Practises

“The delay to our project caused by the covid-19 pandemic, gave us the opportunity to fully integrate human practises into our idea and project design.”

SulFind was originally intended to be the NTNU iGEM team for 2020. However, with the coronavirus pandemic striking Norway in March and our university closing its laboratories over the summer, we decided to postpone our iGEM participation one year. Still being in the brainstorming phase, the delay gave us the opportunity to explore our project idea whilst spending time engaging with stakeholders, incorporating the information we gleaned into our project.

By engaging with human practises throughout our project, we were able to gain valuable insight into the problem of H₂S toxicity in RAS facilities, incorporate our findings into our project and experimental design, as well as receive feedback from industry stakeholders.

Brainstorming

When the NTNU iGEM team first was assembled in the winter of 2019, we leaped right into discussions about the kind of impact we wanted to make with our iGEM project. It soon became apparent that we were all passionate about the environment, and that we wanted to use our iGEM project to tackle local issues that affected the environment.

Living in Norway, the world's second largest exporter of fish and seafood [4], we quickly knew that we wanted to contribute to solving a problem in the fish farming industry.

We went through a lot of brainstorming sessions with discussions of how our iGEM project could contribute to a more sustainable fish farming industry. During these sessions we had different activities to prompt ideas, talked to our supervisors, and reached out to different researchers at our university.

Learning about the problem

Olav Vadstein
March 2020
Olav Vadstein is a professor in microbial ecology at NTNU. He is involved in work concerning the microbiota in RAS facilities. He is teaching a subject in microbiology at NTNU. Several of our team members followed his lectures at the time, where he briefly mentioned RAS facilities. Wanting to know more about RAS, we reached out to him via email.
Key insights: In Norway RAS is often used in combination with sea-based aquaculture. Most of the fish spend part of their young life in RAS facilities before being transferred to fish cages in the sea. This means that the majority of the Norwegian aquaculture industry somehow is dependent upon RAS. Water filtration is an integral part of the functioning of RAS, as compounds formed in the facility, for example from bacterial processes and fish waste, accumulate in a way that would otherwise not be a problem in open systems. Careful monitoring of the water composition is therefore vital to ensure good water quality in RAS facilities. Hydrogen sulfide toxicity is a current hot topic in Norwegian RAS facilities, due to several cases of acute fish death where elevated levels of H₂S in the water was the suspected cause. Current H₂S-detection methods are often insufficiently sensitive to detect the low concentrations that are toxic to fish.
Impact on our project: After corresponding with Vadstein, we wanted to learn more about RAS and the problem of H₂S toxicity. The insight given to us by Vadstein directly led to brainstorming around H₂S toxicity in RAS facilities.

Carlo C. Lazado
August 2020
Carlo C. Lazado at Nofima (The Norwegian Institute of Food, Fisheries and Aquaculture Research) is a senior researcher at the project H2Salar, where the effects of H2S on Atlantic salmon are being studied. We came across H2Salar during our initial research on hydrogen sulfide toxicity in RAS, and contacted Lazado to learn more. Some of our team members visited him at Nofimas offices in Ås (Norwegian municipality).
Key insights: During the meeting, the general workings of RAS facilities and the problem of H₂S toxicity was described to us. We were made aware that fish death due to H₂S poisoning in RAS facilities might go unreported, because some insurance companies in Norway do not cover financial losses caused by H₂S. So far, it is a challenge to detect H₂S at low concentrations. Existing sensors with somewhat sufficient sensitivity to the low levels of H₂S that may harm fish are expensive. Consequently, only a few RAS facilities in Norway are equipped with such sensors and it is still uncertain if the sensors are sufficient. Through the project H2Salar, Lazado’s research group investigates the biological and physiological consequences of H₂S for Atlantic salmon. At the time, experiments to determine the threshold level of H₂S that is harmful to Atlantic salmon were being planned. These future results were of great interest to our work, and we were delighted that Lazado was willing to share the threshold value with us once the experiments concluded.
Impact on our project: The visit at Nofima confirmed that H₂S toxicity is a pressing issue in the RAS sector, and that the industry is in need of a sufficiently sensitive and affordable detection system. This information directly influenced our decision to focus our iGEM project on this issue. We also discovered that the issue seemed to revolve around a lack of H₂S detection, and we thereby decided to look further into how one might make a biosensor for H₂S detection. For further research, Carlos provided us with the contact information to several researchers at NTNU with more information about the topic. The visit at Nofima informed us about the negative impact H₂S toxicity has on fish welfare. This further sparked our interest in the issue.

Scale Aquaculture AS
September 2020
Astrid Buran Holan is Head of Development – Closed Aquaculture Technology at Scale Aquaculture AS , a company that manufactures RAS facilities in Norway and abroad, and delivers technology for closed aquaculture in general. Carlo C. Lazado encouraged us to contact her to learn more about RAS, and we visited her at ScaleAQ’s offices in Trondheim.
Key insights: Holand held a presentation detailing the specifics of how RAS facilities function. An important preventive measure against H₂S poisoning is maintaining a low particle concentration in the tank and in the system as a whole, which is accomplished by removing uneaten feed and faeces immediately as they are formed in the tanks. ScaleAQ have designed particle traps in the middle of their tanks, which keep debris from accumulating in the tank. However, H₂S production might still occur in the bioreactor and surrounding pipes, where there might be stagnant water. Implementation of a H₂S sensor at this location would therefore be beneficial. We were informed that measuring pH, temperature, dissolved oxygen and salinity in the tanks is mandatory for Norwegian RAS facilities, but measuring H₂S is not. Many RAS facilities forgo H₂S detection, which is likely because of the cost of H₂S sensors. ScaleAQ does not actively measure H₂S in the tanks they deliver, but focuses on other preventative measures for H₂S build-up. Still H₂S detection and other preventative measures are not mutually exclusive. Should a sensor be affordable in comparison to existing systems, it would be a welcome safeguard against H₂S build-up, as it would allow RAS facilities to increase the inflow of fresh water to lower the H₂S concentration.
Impact on our project: The meeting gave us a lot of technical insight into how RAS facilities function, as well as the current role of RAS in the Norwegian aquaculture industry. We were also given the first confirmation from within the RAS sector that today's H₂S detection systems do not cover the needs of the industry. We therefore decided to continue pursuing biological H2S detection methods in litterature to see if a biosensor could cover the needs of the industry.The insight we gained about how RAS water filtration works directly influenced our proposed implementation, which we later presented to Holand and her colleagues at ScaleAQ.

Creating a solution

dCod 1.0
September 2020
Anders Goksøyr is a professor at Department of Biological Sciences (BIO) at University of Bergen, and is the project leader of dCod 1.0, an interdisciplinary project that seeks to create a deeper understanding of the cods' adaptations and reactions to stressors in the environment. We came across dCod 1.0 during our extensive research phase on biosensors, toxicants and fish welfare.
Key insights: dCod 1.0 uses a range of techniques in their research to gain an understanding of toxicological responses in cod, and then use this knowledge in environmental monitoring. Knowing of our interest in hydrogen sulfide toxicity, Goksøyr suggested looking into heme-proteins, which bind strongly to sulfide. He recommended several articles that documented the interactions between heme proteins and hydrogen sulfide. During our discussion of using heme-binding proteins in a biosensor, Goksøyr proposed that the proteins might be immobilised on a so-called "lab-on-a-chip", and mentioned that there were projects at NTNU concerning this.
Impact on our project: Speaking with Goksøyr gave us a greater understanding of how toxicants, such as H₂S, affect fish, and prompted us to think more about fish welfare in correlation with our iGEM project. The sulfide-binding characteristic of heme proteins ended up being integral in our biosensor design. Therefore, this meeting directly influenced our project engineering. We also looked further into lab-on-a-chip after speaking with Goksøyr, reaching out to people at our university involved in microfluidics such as Husnain Ahmed, who ended up being one of our supervisors, and helped us incorporate microfluidics into our proposed implementation.

ScaleAQ 2.0
February 2021
Five months after our first meeting with Astrid Buran Holand at ScaleAQ, she and her colleagues Yngve Ulgenes, and Torstein Kristensen met with us virtually to give feedback on our tentative project design.
Key insight: The most important specifications a biosensor needs to be viable as a replacement for existing H₂S sensors in RAS were pointed out. These were high sensitivity, continuous or semi-continuous monitoring (as H₂S-levels can increase suddenly), affordability, and possibility to handle for non-technical personnel. We discussed what it would mean to place a biosensor in a RAS facility in terms of GMO regulation and biosafety. Our proposed design of testing run-off water from the tank was well received, and the team at ScaleAQ stressed the importance of not returning the water to the tank after passing through the sensor. Placing the biosensor externally to the fishing tanks would also minimize the risk of biofilm formation that could impact the sensor. Existing H₂S sensors function by measuring H₂S in gaseous form or use membrane technology. Should a microfluidic chip be used in a RAS facility, the water would need to be filtered or treated in some way, so that larger particles would not clog the chip. Ulgenes suggested that this filtration might itself be incorporated into the chip.
Impact on our project: We used the information gleaned from this meeting in the further development of our project. Specifically we moved ahead with microfluidics as part of the project because we deemed a lab-on-a-chip to be a good fit for the criteria set out by Holand and her colleagues (specifically the possibility of continuous testing). This meeting directly influenced our proposed implementation of our biosensor, and how we plan to remove water from the tank for testing. The meeting also led to a discussion regarding the use of GMO in our project, and led us to focus on the idea of using runoff water from the tanks rather than placing the sensor inside the actual tank.

Kari Attramadal
March 2021
Kari Attramadal is a researcher at NTNU, and is involved with several studies concerning RAS and the microbiota that influence RAS facilities. One of our team members took her RAS course taught at NTNU in preparation for our project, and contacted her with some questions.
Key insights: Biofilm formation in RAS facilities may pose a challenge to sensors placed in the tank. A microfilter for filtering tank water before reaching the sensor was suggested. This would require workers to frequently change the filter, but might still be worth it. There is a lot of skepticism surrounding the use of GMO in Norway that is rooted in opinion, rather than in actual risk assessment. A non-return-valve was proposed so that water that passes through the sensor may be promptly discarded. H₂S build-up can be caused by poor tank maintenance, something a sensor would not fix. Since H₂S is currently in the spotlight, other problems stemming from poor tank maintenance might be falsely attributed to H₂S.
Impact on our project: Furthermore, the meeting caused us to reflect about the impact of our project. As Attramadal made us aware, a H₂S sensor does not negate the need to keep a clean fish tank.

Stakeholder survey
Early April 2021
A stakeholder survey containing questions concerning H2S in RAS facilities was sent out via email to several Norwegian RAS manufacturers. We had already been in contact with the RAS industry, but wanted some quantity of data. We received 5 responses in total.
Key insights: The feedback from industry stakeholders was somewhat varied. One company claimed there was no H₂S problem, whilst others expressed a need for H₂S monitoring. We were made aware that the biggest danger associated with H₂S is not slow build-up over time, but rather sudden spiking levels. H₂S production is most likely to happen in the filtering systems of RAS, this is where a sensor might be most useful. Some facilities used expensive or time-consuming methods for H₂S monitoring. It was reported that the most important factors to be fulfilled for them to be interested in switching to a biosensor was affordability, sensitivity, and for the sensor to be easy to use.
Impact on our project: The survey had no direct impact on our project, but rather gave us reassurance and more confidence that we were on the right track with the focus areas of our sensor being sensitivity and accessibility.

Husnain Ahmed
Summer 2021
Husnain is a PhD Candidate at the Institute of Physics at NTNU. His work concerns microfluidics and “lab-on-a-chip” (LOC), which we previously had learned about from our meeting with dCod 1.0. As a result we contacted Husnain, and we stayed in touch over the summer. We had one crucial meeting in July that changed the entire design of our chip.
Key insights: Our initial LOC design was based on having two different chambers on the chip. The first chamber was supposed to be the mixing chamber. For our SqR approach, H2S would be mixed with GSSH in an aqueous state, and sent to the next chamber by channels. In the heme approach, this channel would only be used as a guiding channel. The next chamber we would have immobilized E. Coli or heme protein. When the aqueous solution passes by the immobilized biological entities, the H2S can pass through the immobilization substance and bind either to heme or repressor proteins. After the fluorescence takes place, it would be measured by a fluorometer. When we discussed this idea with Husnain, he told us that it would be better to exclude the immobilization chamber, and instead use a flow-through solution. By then, we had set aside the SqR-approach due to insufficient results. Our new design consisted of one inlet for the heme solution, and two for the spacer oil. When the heme solution interacted with the oil, droplets would be created. Then, by picoinjection, we would insert the H2S solution into the heme droplet. Since the reaction occurs instantly, there was no need for any incubation channels. After the picoinjection the droplets’ fluorescence would be measured with a fluorometer.
Impact on our project: This meeting turned out to be integral to our sensor, since we changed the entire design and layout.

Roger Fredriksen
Summer 2021
Roger Fredriksen is the manager at Fredrikstad Seafoods AS and the Production manager Scandinavia at Nordic Aquafarms AS, a company that, amongst other things, manages and operates the RAS facilities belonging to Fredrikstad Seafood. We were encouraged to reach out to him via a personal contact, to learn more about RAS and get the opinion of other stakeholders in the RAS industry.
Key insights: Fredrikstad Seafood had not experienced significant problems concerning H₂S poisoning in their RAS facilities. However, they were aware of the problem in the fish-farming industry, and have had some near-accidents with H₂S in their facilities. At Fredrikstad Seafood the tanks are regularly cleaned to prevent H₂S production from occurring. Current H₂S sensors do not have sufficient sensitivity for Fredrikstad Seafood to be interested in purchasing them. In order for a H₂S sensor to be useful for the industry it needs to reliably detect H₂S before a toxic level is reached, be able to monitor large parts of the tanks, and be user friendly for employees that are not engineers. The use of RAS gives control over parameters such as temperature, dissolved oxygen, and pH, which can be adjusted to ensure the best living conditions for the fish. When asked about concerns regarding GMO Fredriksen told us that Fredrikstad Seafood were not concerned with this and that we should be able to argue for the use of GMO in our project.
Impact on our project: The meeting confirmed information we previously had obtained from Nofima, ScaleAQ and the survey in that the industry wants and needs a solution to the H2S problem, and further that sensitivity and easy usage are determining factors. As several stakeholders in the fish farming industry now had mentioned sensitivity as a key feature for a sensor, this meeting led to a discussion about focusing all our energy on the heme-based approach as it had proven to give the most sensitive results in our lab at the time. This discussion, amongst the lack of good results from the SqR approach, was directly determining the direction of our laboratory work. Further, the meeting was motivational for our team members in that GMO was not necessarily as big of an issue for everyone in the industry as previously indicated by our March survey.

Nofima, concentration
Summer 2021
Carlo C. Lazado at Nofima (The Norwegian Institute of Food, Fisheries and Aquaculture Research) is a senior researcher at the project H2Salar, where the effects of H2S on Atlantic salmon are being studied. We reached out to him via email to ask for the concentration of H2S that is harmful to salmon.
Key insights: Right as we were planning our experiments to test our system response to hydrogen sulfide, we received the results of Carlo’s experiments at H2Salar. The concentrations that his research team had found were toxic to Atlantic salmon was 0-25 g/L.
Impact on our project: We adjusted the concentrations of H2S we wished to test our system on accordingly, so we would see if we could detect the concentrations that H2Salar found to be toxic to Atlantic salmon. As a result, the collaboration with Nofima directly impacted our laboratory work, as the concentration represented the goal for our biosensor-sensitivity we wanted to work towards.

Stine Egeland
August 2021
Stine Egeland is employed as Underwriter Aquaculture at the Norwegian insurance company, Gjensidige. We learned about her from Astrid Buran Holand at ScaleAQ and through research as she had spoken about the problem of H2S in RAS several times in the media.
Key insights: About 50% of insurance payouts from Gjensidige (numbers from august 2021) to smolt facilities are related to the fact that the facilities are land based. This can be caused by technical failure, human error, natural disasters and water chemical damages such as H₂S poisoning. RAS is a relatively new technology. When massive acute fish death is reported to insurance companies, H2S can be designated as the reason if there are no other obvious reasons. Hydrogen sulfide poisoning can occur in two ways: Acute hydrogen sulfide poisoning as a result of chunks of biofilm that loosens and releases toxic amounts of H2S. A sensor will not be able to prevent such poisoning as it happens too rapidly. Slow build up of hydrogen sulfide by leakage from biofilm over time. This can lead to fish death over time and reduced fish welfare. The most important means to prevent H2S production is good design of facilities, cleaning of tanks and knowledge about water chemistry amoonguss the staff. The problem of H2S is complex and will not be solved alone by a sensor. A sensor might be able to detect the slow build up of H2S and as a result improve fish welfare. In combination with other preventative methods, a sensor might help improve the RAS technology.
Impact on our project: The meeting had no direct impact on our project implementation, but made us reflect about certain aspects of the H2S problem. Even if our sensor worked perfectly, it would not be enough by itself. Awareness and other preventative methods are equally important. The RAS technology needs new and innovative solutions, and a biosensor detecting H2S is one step of the way contributing to a more sustainable industry.

AquaNor
August 2021
AquaNor is the world’s largest aquaculture technology exhibition. It is the most important meeting place for companies within the aquaculture sector.
Key insights: The team participated at AquaNor which is the world’s largest aquaculture technology exhibition. We had our own stand together with NTNU Oceans, but also used our time to visit the other stands. During the day, the team spoke with several different aquaculture companies. We learned a lot regarding aquaculture technology and the needs of the industry. The Norwegian insurance company, Gjensidige where Stine Egeland is employed, told us that our sensor would be very helpful for them, as they pay out a lot of insurance money to RAS facilities due to H2S related issues.
Impact on our project: By talking to several different aquaculture companies, we learned that our sensor was very much desired and why.

Conclusion - “close the loop”

To conclude, integrating our human practises work into our project was viable for determining several aspects of our project design, engineering and implementation. By communicating with experts, stakeholders and the industry throughout the project we were able to adjust and redesign our project based on feedback loops. In addition, it allowed us to communicate with the fish farming community and create a solution that fulfills the needs of the industry.

By continuously integrating human practises into our work we were also able to close the loop between our initial project ideas and goals, and realistic implementation in real life. Initially we wanted to remove H2S from the RAS facilities, but during the course of our human practises work we learned that detecting H2S is the first step in solving this issue, and as a result decided to focus our project on that.

We want to thank all stakeholders, experts and professors for their time and insightful knowledge that contributed to shape SulFind. Their unique perspective opened our eyes to the complexity of the H2S-issue that we could not have gleaned ourselves.

References:

Ritchie H, Roser M. Fish and Overfishing [Internet]. Our World in Data; 2021 [cited 2021 15/10]. Available from: https://ourworldindata.org/fish-and-overfishing.
World Population to Reach 9.9 Billion by 2050 [Internet]. SDG Knowledge Hub; 2020 [cited 2021 15/10]. Available from: https://sdg.iisd.org/news/world-population-to-reach-9-9-billion-by-2050/ .
Et hav av muligheter – regjeringens havbruksstrategi [Internet]. Nærings- og fiskeridepartementet; 2021 [updated 06/2021; cited 2021 17/10]. Available from: https://www.regjeringen.no/no/dokumenter/havbruksstrategien-et-hav-av-muligheter/id2864482/?ch=1.
Johansen U, Bull-Berg H, Vik LH, Stokka AM, Richardsen R, Winther U. The Norwegian seafood industry – Importance for the national economy. Marine Policy. 2019;110(103561).

Team:NTNU-Trondheim/Human Practices