Human Practices

Overview

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As a team, we pushed ourselves to engage in holistic, integrated human practices to best serve our local community and other communities impacted by water pollution. Given that the 1,4-dioxane plume is such a local issue, we focused on gathering unique local stakeholder input and spreading awareness to our community. We hope that our human practices efforts and bioremediation technology will not only help the residents of Scio Township and Ann Arbor, but reach numerous communities worldwide.

We aimed to include every layer of our community in our efforts, from seasoned experts to grade school students. We began our project by learning all that we could about the history and legal background of the 1,4-dioxane plume by speaking with experts and attending meetings with a local advocacy group, even before starting synthetic biology experimentation. This helped us define the problem we aimed to address and understand its importance. Then, we consulted several scientists in various fields to guide our experimental planning and construct our goals. Finally, we conversed with everyday people to spread awareness on 1,4-dioxane contamination and gathered their feedback on how we could adapt our design to be as effective and implementable as possible.

Our project begins and ends with the community surrounding it; at each step of our design process we consulted and re-consulted experts, policymakers, and locals. Ultimately, the strong bonds we developed with the community helped our efforts grow into a solid foundation for remediation of 1,4-dioxane.

Expert and Stakeholder Consultations

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CARD

Our relationship with the Coalition for Action on Remediation of Dioxane, or CARD, was one of the longest-standing and most regular of our expert and stakeholder consultations. We were directed to CARD by Dr. Rita Loch-Caruso from the University of Michigan, who is a member of their executive board. CARD describes itself as a “partnership of local governments and citizens that look at strategies to address the groundwater contamination from the industrial solvent 1,4-dioxane”¹. We attended monthly virtual meetings from February 2024 to October 2024 to better understand Ann Arbor and Scio Township residents’ concerns about 1,4-dioxane and to elicit feedback on our project.

While attending CARD meetings, we heard about current well monitoring data from the Michigan Department of Environment, Great Lakes, and Energy (EGLE), and participated in real-time conversations between state policymakers and citizens. EPA representatives attended meetings to discuss the proposal to add the Ann Arbor/Scio Township plume to the Superfund National Priorities List during the proposal’s mandatory 60-day public comment period². Additionally, conversations with the CARD Executive Board after meetings helped us understand the history of the plume and the current standing of remediation efforts both in terms of 1,4-dioxane sampling data and policy.

We also had the opportunity to present our project and get feedback from all CARD members, including scientists, local citizens, and policymakers. This experience helped us better shape the technical goals of our bioremediation system to match the needs of the community.

February Meeting

At the February meeting, the EGLE monitoring plan was presented and discussed. CARD members believed that monitoring regulations were not as tight as what they wished them to be. Moreover, it was discovered that sampling levels varied based on the time of the year. Members also discussed the fact that 1-4,dioxane has been found in groundwater closer to the surface than previously thought. The National Pollutant Discharge Elimination System (NPDES) permit into Honey Creek Tributary was also reviewed. The NPDES regulates point sources that discharge pollutants into the water at Honey Creek Tributary. Members also discussed how current technology is not the best for dealing with the current situation.

March Meeting

Members sent a letter to a U.S. congresswoman petitioning lowering of the Michigan limit for 1,4-dioxane.

May Meeting

At the May meeting, our members presented a slideshow sharing an introduction to ourselves and a synopsis of our project. We posed questions to the community members including:

• What can a student group like ourselves do to spread awareness about this issue? What is important to include in that message? What would we want to ask people to do to get involved?
• In what areas of the city could you see our solution implemented?
  • Do you think that a small-scale bioreactor for individual wells would be more useful? Or would a large-scale industrial process integrated into current water treatment be better?
• If we plan on interviewing residents about their experiences with 1,4-dioxane and if they think our bioreactor could be useful, what would be the best way to gather participants? Are there any specific questions or considerations we should include?

In discussing the first question, we were informed that public involvement takes much time and effort but is incredibly important for an issue like this. Much of the public is not aware of the severity or even presence of the 1,4-dioxane plume, so it is difficult to gather support for regulation and remediation efforts. As a result, we designed a survey that doubled as an educational device to gather information and promote awareness of the plume. We also designed and hung up an educational poster series around the University of Michigan campus and Ann Arbor in order to expand our reach.

The second inquiry elicited the most discussion and most impacted our engineering design. CARD members seemed to be split on a small versus large-scale bioreactor design. In favor of a small-scale household bioreactor design, it was argued to be the most feasible given our short project cycle. This could be marketed as a stop-gap measure, fitting into existing household drinking water systems and addressing residents’ immediate needs, until much broader infrastructure and policy issues are solved. On the other side of the issue, it was argued that individuals may not want to personally take on bioremediation and that a bioreactor integrated into city-wide infrastructure would be more implementable. In designing a product marketed to individuals, it has to be determined that people would have the motivation and ability to use the product. However, given that the plume was created by a company and not individuals, it is unlikely that individuals would feel a responsibility to fix the issue. Additionally, some could be uncomfortable handling a system containing live bacteria, even with sufficient safety information. In contrast, a city-wide system places the responsibility of bioremediation on the local government and allows the system itself to be operated by trained professionals. Given all of these considerations, and after surveying and interviewing community members, we decided to focus on a larger-scale bioreactor design to be implemented in city-wide water treatment infrastructure. The members of CARD also mentioned a scale-model testing facility within the Ann Arbor water treatment plant. We later toured the plant and discussed possible areas of implementation for our bioreactor.

Finally, the third discussion point directly informed our Human Practices initiatives. After this meeting, we began working on our survey “Michigan Synthetic Biology Team: How does 1,4-dioxane affect you?” with the intent of reaching Ann Arbor and Scio Township residents and homeowners. More information about our survey can be found below. CARD advised that we communicate the impact of the plume on members of our local community so that participants are made aware of the importance of addressing the issue. We incorporated this advice from CARD by including information about the history, health considerations, and policy issues surrounding 1,4-dioxane in the introduction and questions in the survey. Regarding the collection of participants, CARD offered to spread the word and suggested reaching out to local organizations for further distribution of the survey.

June Meeting

The US EPA superfund site designation updates and EGLE updates were discussed.

July Meeting

At the July meeting, we presented brief updates about our experimental process and shared our survey with the CARD community via a short slideshow. We greatly appreciate the members of CARD for allowing us to share our work with them and for helping us distribute our survey.

September Meeting

At the September meeting, we presented a slideshow of updates about our scientific progress and our future intent with the project, being to integrate our bioremediation system into existing water treatment infrastructure.

JAAGS

JAAGS was a group of master’s students in user experience research and design through the University of Michigan School of Information (UMSI). Members of the group recently graduated and won an award at the 2024 UMSI Student Project Exposition for their project “Empowering Well Owners: Trusted Resources and Community Support for Safe Michigan Water.” We first learned of the JAAGS group from attending monthly CARD meetings where they collaborated with the CARD community on their project. We were inspired by their objectives of speaking directly with community members in the forms of surveys and interviews and shaping their project around their needs. Following this, we decided to design a survey to gather information from local people who may be impacted by 1,4-dioxane, and we enlisted help from the JAAGS team to get started. We met with the group on Zoom, and they gave us many helpful tips and resources to guide our survey design and execution. Considering their expertise in UX design, we learned much about how to center the user of our proposed bioreactor to enhance the user experience. Thus, we decided to allow space for survey participants to elaborate on any of the questions and tell us their thoughts on a personal-scale bioreactor proposal, to enable us to further investigate suggestions given to us through our CARD meeting discussion. See below for more information on our survey of local residents and homeowners.

Survey and Interviews

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Local Residents Survey

We were inspired by our conversations with CARD and JAAGS to design and distribute a survey gathering information on local residents’ and homeowners’ thoughts on the 1,4-dioxane plume and our proposed bioremediation system. We designed our survey to walk participants through the issue, gather categorical data, and provide the choice to interview with us to elaborate on their responses. After reviewing guidelines on best practices and approval by multiple advisors, we distributed the survey to the local community through CARD, posters, and other local groups. See the survey questions here.

Survey Results

Thirty-one community members from a wide array of backgrounds responded to our survey. Their responses helped inform our bioreactor design and validated the need for advocacy and education for 1,4-dioxane remediation.

Previous Awareness

The results of our survey demonstrated that many residents did not know much about the 1,4-dioxane plume, but were open to supporting cleanup efforts. Surprisingly, 38.7% of respondents were not aware of the 1,4-dioxane plume, despite being local to Ann Arbor. Additionally, only 32.3% of respondents had previous knowledge about bioremediation which highlights a gap in community knowledge about environmental cleanup methods.

Policy Change

When given more information about 1,4-dioxane pollution in their community, most respondents believed that policy changes should be made to address the issue. 77.4% of respondents supported lowering Michigan’s limit for 1,4-dioxane in water, and 73.3% believe the issue needs more attention/funding. This suggests that educational efforts on the issue could build support for policy change, so advocates for remediation should strongly consider educational initiatives to aid their cause.

Adopting Bioremediation

Regarding our bioremediation system, the majority of respondents showed interest in adopting an additional water filtration system. Importantly, one respondent commented that although they would like to be personally protected, they believe that small-scale personal protection is inadequate to solve the issue on a community level. This sentiment was echoed in many of our one-on-one interviews with community members. We considered these responses when designing the final implementation of our system. To satisfy personal safety concerns on a larger scale, we adjusted our bioreactor design to fit into existing water treatment infrastructure to provide drinking water protection to the entire community.

Increased Interest

72.4% of responders indicated that our survey increased their interest or awareness of the local 1,4-dioxane plume, demonstrating that our survey successfully educated the public about the dangers of the plume and possible solutions. We have aimed to continue spreading awareness by posting informational posters and holding community events.

Overall, respondents seemed enthusiastic about our proposed system. However, many had questions about implementation and technical details, and we attempted to work through these questions in one-on-one interviews. Some questions we received included:

• Will the proposed method cause any unintended consequences?
• Will this be used in conjunction with existing treatments? How long will the process take? Is there anything residents can do to help?
• What happens to the microorganisms after treatment? Where are they being discharged?

Overall, this survey helped us learn more about what an ideal 1,4-dioxane remediation system may look like for the Ann Arbor community and significantly influenced our engineering process.

Followup Interviews

To gain more in-depth feedback from community members, we included an option on the survey to participate in a follow-up interview. These interviews functioned as an extension of the survey in which we got deeper, more personal responses from the participants. Setting up this dialogue helped us learn more about the views held by those in the affected community, and allowed us to get further feedback on our project. The interview questions can be found here.

The interviews opened our eyes to many different perspectives surrounding the 1,4-dioxane plume. New perspectives included: worry about the effects on food grown in soil above contaminated groundwater, concerns about real estate values, and attitudes of both responsibility and ambivalence towards remediating the pollution. We were able to incorporate the knowledge we gained into the design of our bioreactor system. We found that interviewees prefer a large-scale system for 1,4-dioxane remediation over a small-scale at-home system, which confirmed some of the sentiments shared with us in the May CARD meeting. This led us to refine our bioreactor design to integrate into existing infrastructure in the area. We then decided to tour the Ann Arbor Water Treatment Plant to learn more about existing large-scale water treatment infrastructure and how to potentially integrate our project into it.

Comprehensive notes from four select interviews are linked below:

• Interview 1
• Interview 2
• Interview 3
• Interview 4

Scientific Consultations

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• Who: Dr. Xuan Wang is currently a professor at Arizona State University whose research focuses on the metabolic engineering of bacteria for the production of valuable chemicals from renewable sources with the potential to replace petroleum-derived counterparts.
• Why we reached out: We reached out to Dr. Wang early in our research process to help us choose our bacterial host, as well as to get feedback on our proposed THFMO plasmid design.
• What we learned: During our meeting, we explained to Dr. Wang that we had identified 2 bacteria of interest for use as our host – Pseudomonas putida and Shewanella oneidensis. Dr. Wang recommended using P. putida as our host because it is well-studied and relatively easy to engineer. Additionally, based on his experience, S. oneidensis proved to be incredibly difficult to work with and grow in the lab. We also mentioned to Dr. Wang that we needed to develop a method to detect the activity of the THFMO enzyme in terms of testing dioxane degradation. Our initial idea was to use gas chromatography and qPCR. Dr. Wang suggested that we put our enzyme under an inducible promoter such as IPTG, transform the plasmid into E.coli, and then measure dioxane degradation to make sure the enzyme has the desired activity. The degradation assay he suggested was an endpoint assay, where enzyme activity could be measured via the quantity of dioxane consumed during the reaction over a fixed period, compared to a control condition in which uninduced bacterial cells are incubated in buffer and dioxane. Dr. Wang brought up that if we don’t observe degradation activity, then we would need to troubleshoot whether it is a transcription or translation issue, cofactor issue, or protein folding issue.
• What it changed in our design: Our meeting with Dr. Wang helped us solidify our choice of P. putida as the bacterial host of the THFMO enzyme and also helped us to devise a simple method to measure the enzyme activity of THFMO.

• Who: Dr. Navaratna is a postdoctoral researcher in the Bardwell lab at the University of Michigan who focuses on enzyme discovery and directed evolution to identify nicotine-metabolizing enzymes.
• Why we reached out: Dr. Navaratna currently works with P. putida S16 and has a great deal of knowledge in plasmid assembly and expression in this strain.
• What we learned: Dr. Navaratna recommended the use of S16 for our experiments, due to the ease with which it can be manipulated. He also gave us insight into developing an electroporation protocol as well as allowing us access to the electroporator in the Bardwell lab.
• What it changed in our design: We met with Dr. Navaratna multiple times over the course of our experiments to discuss protocols, plasmid assembly, and culturing conditions. After these meetings, he generously provided us with P. putida S16 and the plasmid pJN105-NicA2 in E. coli cells. We used the plasmid backbone for all of our THFMO experiments by mini prepping, cutting out the NicA2 gene using a restriction digest, and inserting our THFMO gene using Gibson Assembly.

• Who: Dr. Loch-Caruso works with citizens and regulatory entities on environmental health protections related to groundwater contamination with 1,4-dioxane. Dr. Loch-Caruso is very knowledgeable about 1,4-dioxane and has published papers relating to the Ann Arbor plume. She is also an executive member in CARD.
• Why we reached out: We reached out to Dr. Loch-Caruso before beginning wet-lab experimentation to better understand the background of the Ann Arbor 1,4-dioxane plume and to determine whether our method for degradation may be a feasible option.
• What we learned: Dr. Loch-Caruso helped us gain a better understanding of the history of the 1,4-dioxane plume and the current policy change initiatives.
• What it changed in our design: Dr. Loch-Caruso introduced us to the CARD group, whom we collaborated with throughout our project. She also inspired us to pursue our Sustainable Development Goals and focus on education in our human practices.

• Who: Dr. Nina Lin is a professor of Chemical Engineering at University of Michigan whose research focuses on designing and engineering synthetic microbial consortia for sustainable production of biofuels and commodity chemicals.
• Why we reached out: We met with Dr. Lin and members from her lab multiple times to discuss our bioreactor design and the specifics of how we would run the bioreactor in accordance with the biofilms.
• What we learned: During our meetings, Dr. Lin helped us fundamentally understand bioreactor function and the various components that were essential to the development of a potential prototype. We introduced our goal of using engineered P. putida in a closed system to clean contaminated water.
• What it changed in our design: Dr. Lin helped us reach the decision that a continuous stir batch reactor made the most sense for our goals. We also decided on the usage of slow stirring to prevent biofilm dispersal.

• Who: Dr. Rickard is an associate professor in epidemiology at the University of Michigan’s School of Public Health. His research largely focuses on the coaggregation and biofilm formation abilities of various strains of freshwater and dental bacteria.
• Why we reached out: We reached out to learn more about the best growth conditions for P. putida, including broth choice and temperature, as well as competence for the uptake of vector DNA.
• What we learned: We learned that P. putida grows well in a variety of media, including LB and R2A, a type of minimal nutrient media. We also learned about multispecies biofilms that could potentially be implemented to enhance a potential bioreactor design.
• What it changed in our design: Our discussion with Dr. Rickard solidified our use of LB as our main growth media and eventual usage of R2A to promote dioxane as the primary carbon source for our engineered P. putida strain.

• Who: Dr. Katherine Manz is an assistant professor at the School of Public Health at the University of Michigan. She studies environmental chemistry and develops remediation technologies. Rachel Klein is a research lab specialist associate in Dr. Manz’s lab who also attended our meetings and advised our project experimentation.
• Why we reached out: We reached out to learn more about using Gas Chromatography (GC) testing to quantify 1,4-dioxane levels.
• What we learned: Through many meetings during the course of our experimental work, Dr. Manz and Rachel helped us select a GC procedure, choose the most effective column, and optimize our procedure for the greatest dioxane peak separation.
• What it changed in our design: Dr. Manz and Rachel were instrumental in our ability to quantify 1,4-dioxane levels in our samples. Rachel aided us with using the GC-FID instrument and advised us throughout the process.

• Who: Dr. Photenhauer is a research fellow in the Ohi lab at the University of Michigan studying microbiology and immunology.
• Why we reached out: Our team reached out to Dr. Photenhauer because we wanted to ensure that our THFMO construct for the gene was designed correctly and to review our experimental plan with her.
• What we learned: Dr. Photenhauer brought up the question of whether 1,4-dioxane can passively enter gram-negative bacteria. She believed that 1,4-dioxane could likely enter gram-negative bacteria, but encouraged us to plan experiments to validate this. Moreover, she reviewed and supported the rest of our experimental plans.
• What it changed in our design: After our meeting with Dr. Photenhauer, we decided that the team should begin collecting cells at the end of every experiment so that we could perform further validation testing to ensure that 1,4-dioxane had actually entered the bacterial cells.

• Who: Dr. Subramaniam Pennathur is the Chief of the Division of Nephrology at the University of Michigan and the Norman Radin Chair of Nephrology Research. Dr. Jaeman Byun is an associate research scientist who has focused on the applications of biological mass spectrometry in disease pathogenesis with an emphasis on oxidative mechanisms and biological mass spectrometry. Dr. Saroj Chakraborty is a research fellow in the Pennathur lab investigating the effects of ketone bodies on the progression of diabetic kidney disease and their epigenetic effects on histone bodies. Dr. Manikanta Arnipalli is a research fellow in the Penathur lab who focuses on the development of complementary metabolomic techniques based on mass spectrometry for the study of diabetic kidney disease.
• Why we reached out: We contacted Dr. Pennathur, Dr. Byun, Dr. Chakraborty, and Dr. Arnipalli because they work with the mass spectrometry core at the University of Michigan’s Kellogg Eye Center. Following a failure to create a proper standard curve and analyze samples using the Manz lab’s GC-FID machine, we decided to pivot to GC-MS analysis.
• What we learned: We were able to learn how to use the GC-MS instrument and optimize existing protocols for our experiments. We confirmed through GC-MS analysis that we could achieve a much better resolution limit than GC-MS (17.622 ppb) as well as the opportunity to perform a more analytical finish on dioxane. We learned about how GC-MS differs from GC-FID, and we learned how to implement selective ion monitoring mode (SIM) to improve resolution, which is not present in FID. We also confirmed that we were able to successfully degrade dioxane.
• What it changed in our design: By pivoting from GC-FID to GC-MS, we changed our analytical approach to dioxane quantification.

Public Engagement & Education

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School Visit

Ann Arbor STEAM is a local elementary and middle school with a project-based learning approach centering an integrated curriculum with real-world applications. As a result, the school's mission statement aligns very closely with our team's, as well as the mission of iGEM as a whole. We connected with Ms. Jennie Allan, a 7th and 8th grade science teacher, when she was planning the 8th grade genetics unit. Their unit focused on the myostatin gene for hypermuscularity in cattle as found in the Belgian Blue breed. After a discussion with Ms. Allan about the appropriate difficulty level and scientific focus, we organized materials for and taught three eighth grade science classes about key concepts used in our project. Our lesson plan included bacterial transformation with plasmids and the central dogma of biology to highlight how DNA and genes can be manipulated to solve real problems. While this lesson was quite advanced for the students, they were very engaged and asked great questions.

In addition, we designed a worksheet as well as pre- and post-surveys to maintain student interest and gauge how our research was communicated to and understood by the students. We specifically designed the worksheet as recommended by Ms. Allan to help students connect the topics we presented about to other information they have learned in past class sessions. The surveys helped us see how the students' perceptions and understanding changed throughout the course of the lecture. From these surveys, we were pleased to see that students felt they gained a greater understanding of the 1,4-dioxane issue and molecular biology from our class session. The student feedback was valuable for our future educational initiatives.

• Presentation
• Pre and post survey
• Worksheet
• Student samples of pre and post survey
• Student samples of survey and worksheet

Undergraduate Education Event

Considering that our team has mainly completed educational events targeting middle school and high school students in the past, we decided to expand our educational initiatives this year to include undergraduate students at our university. We invited students in several departments and programs to an informational session in which we discussed synthetic biology in general, its applications including bioremediation, and our current project. It was extremely important to us to facilitate a two-way conversation between us and the attendees, so we included a pre- and post-survey and built-in time for questions. We aimed to both share knowledge about synthetic biology and get feedback on our project from students with many different perspectives and academic backgrounds.

• Presentation
• Pre-survey
• Post-survey

Science Olympiad

Interacting with Middle and High School Students

Science Olympiad is a nationwide competition in the United States in which students compete in events of several different scientific fields. Each year, our university hosts the University of Michigan Science Olympiad Invitational which includes a STEM expo where Michigan student organizations can talk to students about their respective fields and/or projects. We designed and constructed an informational poster about our team and our project, and conversed with students and their families about our organization. This experience was enriching because we got to share our love of synthetic biology and opportunities at the University of Michigan with the students. We were happy to meet so many talented and intelligent students who may be the faces of our team in the future.

Poster Series

One primary focus of our outreach efforts was to educate local residents about 1,4-dioxane contamination. As a part of this effort, we designed a three part poster series to inform and engage with the public about the issue and our team’s proposed solution. We designed the posters to be informative and impactful with both the visuals and the text. The content of each poster walks the viewer through the basic concepts of synthetic biology, the severity of the plume, and our proposed bioremediation system. Given that our target audience for the poster series would likely have minimal science background, we aimed to communicate the content in easy-to-understand terms while still conveying the key details, such as important statistics and area covered by the plume. Our visuals were designed to be captivating and representative of many aspects of the issue. The plume map communicates the scale of the issue, and the garden visual provides an example of how 1,4-dioxane plume can affect residents’ daily lives. We displayed our posters in high-traffic public areas including the Ann Arbor District Library, the Farmer’s Market, the Natural History Museum, and various university buildings. Importantly, the final poster in our series includes explanations of our team’s efforts to engage in research, sustainable development, and policy change. We hope that these explanations inspire poster viewers to engage in local efforts that contribute to these areas and more.

A2 Green Fair

Seeking opportunities to personally interact with sustainability-minded individuals in our local community, we hosted a table at the A²ZERO Green Fair. This annual event is hosted in downtown Ann Arbor by the local government and brings together thousands of attendees and more than 100 exhibitors focused on various aspects of sustainable development and living. Our team hosted an exhibit for three hours where we fielded questions from fair participants at our table. We educated participants about how the 1,4-dioxane plume originated, the status of current cleanup efforts, and how we propose our project fit into the existing water treatment pathway. Through the event, we realized how important visual explanations were to facilitate participant understanding. In particular, showing participants a basic schematic was incredibly helpful in explaining how bacteria would live and function in our bioreactor without ending up in the treated water. Ultimately, our team interacting with the local community at the A²ZERO Green Fair helped us promote knowledge about local applications of synthetic biology while also gathering expertise about how we could best present our findings and proposed implementation in a way which is understandable to local citizens and allows them to feel empowered to ask questions regarding potential concerns they have.

Site Visits

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Third Sister Lake

Our team first heard of Third Sister Lake when we asked the members of CARD where we could take samples of 1,4-dioxane contaminated water to test in our bioremediation system. Third Sister Lake is located in Saginaw Forest, a University of Michigan owned property and hiking ground which borders the Gelman Science’s property on the west side. In fact, the 1,4-dioxane plume was discovered there in 1984 by Dan Bicknell, a graduate student in public health at the university. A few months later, the lake came up in conversation with Dr. Lin and her research group. Given these mentions of the site, we decided to visit the property and take water samples for testing. This visit helped solidify the gravity of the 1,4-dioxane issue for our team.

Ann Arbor Water Treatment Plant

In order to learn more about how our bioreactor system can best integrate into existing infrastructure, our team visited the Ann Arbor Water Treatment Plant, the facility where drinking water is treated for the communities that surround the 1,4-dioxane plume. The Ann Arbor water treatment plant sources water for drinking from both the Huron River and groundwater from local wells. The 1,4-dioxane plume has not yet spread into these sources, but continued drawing of well water could contribute to pulling the contaminated groundwater closer to these sources. The Ann Arbor water treatment plant has already taken actions to prevent this, including the closure of a source well near the edge of the plume.

We had the opportunity to receive feedback from the Assistant Plant Manager about where a 1,4-dioxane remediation system may be integrated into existing treatment infrastructure. The Ann Arbor Plant treats water using rapid and solid mixing, settling, and filtration steps. In addition, the location utilizes multiple disinfection methods including ozone (O₃), chloramine (NH₂Cl), and UV disinfection. The best step in the process to integrate a 1,4-dioxane remediation technology like ours would be prior to ozone treatment. This would allow our technology to interact with water that is free of large particulates, and the effluent from our treatment would still undergo ozone disinfection to kill any residual bacteria.

The Ann Arbor Water Treatment Plant also has a Pilot Plant system, a smaller scale model of the water treatment plant that tests new technologies before implementing them in the main plant. Many different filtering methods, such as the use of granulated activated carbon filters, have been tested in this facility in the past. We learned that a previous project run at the Pilot Plant found that filter backwash water used to clean matter off of the filter contained trace chloramines that were harmful to the beneficial bacteria behind the filter. This applies to our bioreactor system, and is a consideration we took into account during our bioreactor design.

In addition, the Pilot Plant has begun experimenting with advanced oxidation processes for the treatment of 1,4-dioxane. Our contacts mentioned that the equipment for running these tests are expensive and difficult to attain. These are areas where our technology may be advantageous for implementation into a wider treatment process. Learning more about the local water treatment procedures around the plume area helped us shape our technology to be useful for integration into existing systems and to provide solutions to problems with efficiency and cost that the water treatment plant has seen with other remediation technologies.

Deliverables

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Bioremediation Database

The 1,4-dioxane plume is a unique issue to the Ann Arbor and Scio Township area, but environmental pollution is a huge problem all around the world. We hope that our bioremediation technology will not only impact our local community, but aid in remediating sites globally in the future. The goal of our bioremediation database was to provide future iGEM teams or research groups with avenues for adapting bioremediation technology, including our bioreactor design if they choose. We completed a thorough literature review and cataloged a variety of pollutants as well as species and enzymes that could be used for degradation. We hope that our database provides a starting point for researchers to address pollution issues in their local communities.

Ethics Review

As researchers in synthetic biology we have a responsibility to analyze and understand the ethical considerations involved in our work. Our research on the ethics of bioremediation and pollution cleanup allowed us to stay conscious of possible areas of moral concern, and helped us conduct genetic engineering in the safest and most ethical manner possible.

Our first consideration was the ethics of using living organisms in water treatment. Bioremediation in water treatment is not a new technique; bacterial flora in activated sludge is widely used during secondary wastewater treatment. Our approach similarly uses bacteria, however proposes the use of a single bacterial species in an enclosed bioreactor system. Literature review on best practices of bioremediation reveals that context and choice of bacteria are highly important. Lawrence Johnson’s system of environmental ethics suggests that the most ethical type of biological control technology is a natural biological control³. This led us to choose a bacteria native to soil and water, Pseudomonas putida, as our host species, to ensure that the host is natural to the area where the contaminated water is being sourced.

Our second consideration was the ethics involved with genetically engineering bacteria. The four pillars of bioethics are autonomy, beneficence, nonmaleficence, and justice⁴. We have taken care to align our project with these pillars. The beneficence of our genetically engineered bacteria is the potential treatment of 1,4-dioxane contaminated water. Our system aims to provide a just method for providing communities with effective water treatment technologies. In addition, ensuring the nonmaleficence of our system is crucial. Some of the concerns of genetically modified bacteria include the risk of ecological disruption and mutation. Our modified bacteria is intended to be used in a self-contained bioreactor system with UV treatment prior to release of effluent to remove all traces of bacteria before water use and to limit the risk of bacterial escape into the environment. Additionally, we intend to implement our bioreactor in an existing sequence of water treatment which is already highly regulated. In order to further reduce environmental risk as much as possible, we weighed the benefits and drawbacks of multiple methods of genetic engineering. Direct modification of the genome of our host bacteria offered the most efficient option, yet posed the risk of creating a recombinant bacteria that might mutate to result in a more dangerous strain with the potential for greater ecological disruption if accidentally released into the environment. Alternatively, the use of a plasmid carried less environmental risk. Our conversations with scientific advisors also supported the use of a plasmid. Ultimately, this led us to choose a plasmid vector over altering the bacterial genome itself as our approach for engineering our freshwater bacteria.

We also reviewed water ethics to help us better understand the moral implications involved in pollution scenarios such as Ann Arbor’s 1,4-dioxane plume. One ethical dilemma is the assignment of responsibility for pollution remediation⁵. In 1992, the Washtenaw County Circuit Court ruled that Gelman Sciences Inc. should be responsible for managing the cleanup of the plume, as they were responsible for the pollution⁶. This cleanup is enforced by the Michigan Department of Environment, Great Lakes, and Energy (EGLE). While this ruling ensures that Gelman must do the minimum required by EGLE for 1,4-dioxane remediation, our conversations with local citizens showed us that the affected communities are not confident in Gelman’s limited efforts and want to have the security of additional measures to prevent contamination of municipal water sources. Treatment systems like ours should be available to complement existing drinking water treatment infrastructure and give citizens added dependability in the safety of their water source. This led us to focus on implementation of our technology in a large-scale bioreactor integrated into existing water treatment. Overall, ethical considerations played a major role in the ideation of BioXane. Our research into the ethics of bioremediation, genetic engineering, and water security significantly helped shape our final project.

Collaboration

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UChicago

In April, our team met with the UChicago iGEM team to share about our project progress. This meeting provided us with a chance to get feedback from fellow iGEM competitors’ perspectives early in the season. Up until this point, we mainly had experience discussing our project in a social science context, as with the CARD group. This collaboration helped us better frame our proposal for a scientific audience. Additionally, in the month of September our team assisted UChicago with executing molecular dynamics simulations using the GROMACS software via the cluster computer we have access to.

Jamburrito Symposium

We had the opportunity to attend an iGEM symposium called Jamburrito run by the Stony Brook University iGEM team where we presented a bit about our project and heard from other iGEM teams. We also had the opportunity to get feedback from scientists and other attendees. This feedback helped us work on presentation clarity and provided us practice answering questions about our experiment.

KU Leuven

In August, we met virtually with a representative from KU Leuven’s team, based in Belgium. Our teams have similar interests in bioremediation of industrial water pollutants and we were able to share information and feedback on our science processes and outreach ideas.

Environment Symposium

We feel that pollution remediation is an issue that extends far beyond our local community and iGEM project. As a team, we felt it was important to contribute to discussions addressing pollution and other environmental concerns at a global scale. Our past collaborations with iGEM teams has shown us that many teams share an interest in bettering our local environments. We decided to host a virtual environmental symposium for iGEM teams to converge and share information about how their projects aim to address pressing environmental issues while utilizing synthetic biology, as well as host a discussion space where teams could ideate and plan future joint community initiatives. The teams we reached out to that signed up for the symposium were Fudan, Athens, KULeuven, BOKU-Vienna, MSP-Maastricht, Uni-Padua-IT, City of London, IEA Caltech, and Concordia. See our team’s presentation slides here.

The symposium opened up great discussion and teams shared many ideas. Attendees expressed their agreement that learning about projects with diverse approaches to topics like bioremediation introduced new perspectives and promoted creative thinking. Our team learned about how other teams approached obstacles similar to those we experienced and we brainstormed with other teams about various educational and scientific initiatives to promote application of the intersection of synthetic biology and community action. In addition, the symposium was helpful for sharing ideas for impactful human practice initiatives. We discussed ideas for future collaboration early in the next project season when project feedback may be most useful. Overall, we feel that the virtual environmental symposium succeeded in its goal of fostering collaboration amongst iGEM teams and promoting dialogue in environmental remediation research.