Team:UCL/Poster

UCL Poster

PETZAP: Integrating enzymatic PET degradation into Microbial Desalination Cell technology

Team UCL 2020

Student Team members
Olaide Ibiyemi, Juliette Champaud, Stefan Hristov, Pedro Lovatt Garcia, Daniel Castellano Garrido, Anna Su, Li Xu and Oliver Hernandez Fernandez

Abstract
The world’s oceans are suffocating from an annual addition of 8 million tons of plastic which threaten marine ecosystems and exacerbate water scarcity affecting over 2 billion lives. Our project aims to tackle these two global challenges by integrating enzymatic polyethylene terephthalate (PET) degradation into a Microbial Desalination Cell (MDC). The system involves a 2-step process co-culturing engineered E. coli to express a PETase-MHETase fusion degrading PET and P. putida to achieve further degradation and produce lactate, which then supports the biofilm growth of exoelectrogen, S. oneidensis, generating bioelectricity for desalination. Desalination efficiency was maximised by optimising lactate secretion, co-culture design, and MDC configuration based on the results from flux balance analysis (FBA) and agent-based model, simulating bacterial plastic degradation and bioelectricity production, respectively. Insights for further technical optimisation and feasible implementation at large scales were obtained through iterative engagement with experts and stakeholders.

Problem

By 2050, it is estimated that plastic will outweigh all fish in the ocean (1). Plastic pollution of the Earth’s waters exacerbates freshwater scarcity with over 2 billion people living under high water stress (2). Recycling and desalination are current methods used to address these problems. However, only 14% of plastic packaging is recycled (1) and current methods of desalination such as reverse osmosis are highly energy-intensive and not environmentally friendly. As plastic is currently an integral part of modern life, we need to change how we see and handle end of life plastic, in a more environmental way.

Motivation

8 million tons of plastic waste are disposed in the ocean annually (3). With plastic being a common and non-degradable commodity, its linear life cycle is not sustainable for its use. Therefore, we were inspired by the possibility of synthetic biology to create a circular plastic economy by repurposing plastic waste as a resource for other uses.

Idea

We propose a technology that can combine enzymatic plastic degradation with seawater desalination in an MDC using engineered microbes to degrade plastic and generate bioelectricity to drive seawater desalination. Our project employed in silico modelling to investigate the potential of using synthetic biology to integrate enzymatic PET degradation with the MDC technology. In a three-chambered device, electricity is generated by the exoelectrogen, S. oneidensis, which relies upon lactate as its sole food source. Lactate is produced by the co-culture of PET-degrading E. coli and P. putida and diffuses to the anode chamber. The transfer of electrons to the anode creates a potential difference that drives the movement of the ions in the seawater out of the middle chamber through ion exchange membranes.

Figure 1. Schematic of a three-chambered Microbial Desalination cell

Research Questions

The research questions our project aimed to answer were:

1. Can PET be used as a food source for bacteria to generate electricity?
2. Can bacteria produce enough electricity for desalination?

Figure 2. Overview of the mechanism of the co-culture of E. coli, P. putida and S. oneidensis designed to investigate the research questions above. E. coli feeds on PET by generating Ethylene glycol (EG) and TPA. Ethylene glycol is used to feed E. coli, while TPA feeds P. putida in order to maximise lactate secretion. The lactate generated can then feed S. oneidensis, which grows as a biofilm on the anode surface and generates a current output.

Construct Design

We identified strains to express the PET degradation pathway to produce sufficient lactate to sustain the growth and electricity generation by S. Oneidensis. E. coli was engineered to degrade PET into terephthalic acid (TPA) and ethylene glycol (EG) while P. Putida fully metabolizes TPA to produce lactate for S. Oneidensis.

Figure 3. Pathway schematic representation of a model of enzymatic plastic degradation coupled with microbial electricity generation. The engineered E. coli secretes PET degrading enzymes, PETase and MHETase, to degrade polyethylene terephthalate (PET) into mono-terephthalic acid (MHET) and ethylene glycol (EG) (4)(5). EG enters E. coli via transporters to be further metabolized in the native secondary pathway expressed an operon in E. coli forming glycerate (GL). While GL enters glycolysis and gets converted into pyruvate (Pyr) to support E. coli's biomass growth, TPA enters P. putida via TPA transporter encoded by genes from Comamonas sp. strain E6 (6). Our engineered P. putida expresses transgenic genes to degrade TPA into 1,2-dihydroxy-3,5-cyclohexadiene-1,4-dicarboxylate (DCD), next into 3,4-dihydroxybenzoate (PCA) (7). PCA enters P. putida's endogenous β-ketoadipate pathway forming succinyl-CoA (SucCoA) and acetyl-CoA (AcCoA) (8). AcCoA enters the tricarboxylic acid cycle (TCA) of P. putida, generating lactate for exoelectrogen S. oneidensis. Native pathway in S. oneidensis metabolizes lactate to support biomass growth and electron production (9)(10).

PET degradation pathway construct design:
We designed constructs that would allow the selected bacteria to to fully degrade PET in the MDC. Therefore, we had the following objectives:

1. Design constructs to degrade PET in E. coli by expressing the PET-degrading enzymes, PETase and MHETase
2. Express the TPA transporter and degradation pathway in P. putida

PETase and MHETase were expressed in E. coli as a fusion protein to increase the efficiency of PET degradation. We made a structural model and consulted experts prior to designing the constructs to decide on the gene order of the enzymes in the fusion protein. The model showed that the MHETase-PETase construct had less steric hinderance which we hypothesized to perform better. The McGeehan group from the University of Portsmouth also verified our hypothesis as they found that the PETase-MHETase fusion could not be functionally expressed in E. coli due to protein aggregation (11). Therefore, we designed constructs with the individual enzymes and the two configurations of the fusion protein to test if the gene order affected extracellular secretion, functional expression or enzyme activity.

Figure 4. The 4 constructs of PETase and MHETase in Biobrick standard.

In P. putida, we designed a construct to express the TPA transporter and catabolic operon from C. testosteroni to degrade TPA into PCA which is metabolized into lactate in pathways native to P. putida.

Figure 5. Construct of TPA transporter and catabolic operon designed to degrade TPA into PCA in P. putida

Optimising Co-culture Design

Exoelectrogenic behaviour is exhibited during anaerobic respiration, where oxygen cannot be used as the terminal electron acceptor. We performed a Flux Balance Analysis (FBA) over our proposed two-species metabolic pathway to investigate the growth and lactate production of the co-culture in aerobic and anaerobic conditions.
The flux balance analysis (FBA) was performed to:

1. Model the growth of E. coli and P. putida under different oxygen conditions
2. Optimise TPA production by E. coli and lactate secretion by P. putida

Figure. 6 E. coli biomass growth simulation on varying PET and oxygen uptake rates.

Figure 7. 1. P. putida biomass growth simulation on varying TPA and oxygen uptake rates. 2. P. putida lactate secretion simulation on varying TPA uptake rate and biomass growth rate.

The results show that E. coli and P. putida can only grow and produce sufficient lactate to support the growth of S. oneidensis in aerobic conditions. In aerobic conditions, the maximum lactate secretion rate reached by P. Putida is 8.2132 mmol/gDCW/h at a growth rate of 0.2 h-1. E. Coli is unable to degrade PET under anaerobic conditions and therefore no TPA would be produced for P. Putida.

Current Output and Desalination

We built a cellular automata (CA) to investigate the 2nd research question which modelled the current density generated by the formation of an S. oneidensis biofilm on the anode surface and linked to a desalination rate model.

Aims of CA model:

• Investigate the growth of S. oneidensis biofilm on the anode for electricity generation
• Predict desalination efficiency achieved over electron transfer models

Figure 8. 1. Agent type over one a week, with time in a hours, showing active cells in blue, quiescent cells in orange, and dead cells in green. Each agent behaves as 125 cells. 2. Total current density generated by biofilm over one week

We observed a peak in the current density after about 48 hours at 1.2 A/m2. A steady state current density of approximately 0.0397 A/m2 was achieved generating an average desalination rate of 0.623 L/m2/h which is within the range of rates observed by Ramírez-Moreno et al. (12). For a lab-scale MDC with a 250 ml anode chamber and an anode surface area of 25cm2, 1.56 ml of water can be desalinated per hour. The system will have to be scaled up appropriately to desalinate enough water for irrigation.

Human Practices

In collaboration with Team Ashesi, we distributed a survey to gather opinions on plastic pollution and attitudes towards genetic engineering solutions in Ghana.
1 indicates extremely negative while 5 indicates extremely positive
Figure 9. Survey results on the use of GMOs for plastic treatment and freshwater scarcity in Ghana. A total of 99 responses were collected.

The survey showed that ~60% of the participants expressed a positive attitude to the use of GMOs to tackle plastic pollution and more than 50% are willing to purchase crops irrigated by our desalinated water. About 86% of the participants also answered that they would like further information about the safety of GMOs to feel more comfortable about consuming crops treated by the MDC technology. Therefore, we created a brochure with Ashesi to educate the end-users about synthetic biology and how they would benefit from our project.

Market Research

Our project, once implemented in the real world, would be novel pieces of infrastructure constructed at the seaside of the selected markets. We have selected Southern California as our test market while Ghana and Nigeria were selected as the final markets as they are all coastal regions and have been suffering from both plastic pollution and fresh water scarcity.

Criteria for Market Selection Criteria for market selection:

• Struggles with oceanic plastic pollution
• Coastal region with a need of fresh water for irrigation
• Strong and active NGO activities to ease up communications as we are aiming at building connections initially with NGOs for advertising of and more general suggestions on our project design, and then penetrate and present the idea to academic and industrial experts for further considerations

Selected Regions: Target Market:

• Ghana: We collaborated with the Ashesi iGEM team to gather public opinion on plastic pollution and attitudes towards synthetic biology solutions in Ghana.
• Nigeria: We consulted RecyclePoints, an NGO recycling organisation in Nigeria, to gather more information on plastic pollution in Nigeria.

Test Market:

• Southern California: We communicated with the University of California San Diego, (UCSD) and Algalita, an NGO focused on plastic pollution in Southern California

Implementation

The proposed implementation of our MDC technology would be within an industrial desalination plant to produce fresh water for irrigation. After interacting with stakeholders and experts in the desalination industry, we developed a flowsheet of the steps to be included in the MDC desalination process to ensure a safe and efficient implementation of the technology.

Figure 10. Flowsheet showing proposed implementation strategy of the desalination process

As the FBA results showed that the co-culture and desalination have to be performed separately, the process has been split into 2 steps with an aerobic chamber for the co-culture and an anaerobic MDC chamber for electricity generation. After the co-culture, centrifugation and filtration are used to remove the cells from the outlet stream containing lactate which is then transferred to the anaerobic MDC.

From the CA, we found that the MDC will have to be scaled up to increase the surface area of the anode in order to desalinate enough water for irrigation. Through our research and communications with experts, we've proposed scaling up by stacking multiple MDCs or using a tubular MDC configuration. Though, this will have to be investigated further by next year's UCL iGEM team.

Education and Science Communication

As our human practices demonstrated, public perception of new biotechnology is improved with improved education and outreach. Water our MDC produces will be used initially for irrigation, and we felt strongly as a team the importance of improving science communication. Therefore, we achieved the following:

• Engaged with high school students – the next generation of scientists and engineers - through organised webinars
• Targeted outreach activities to underrepresented students in education
• Published a fictional magazine story about a synthetic biology superhero who saves a local community from a crisis using the power of engineered bacteria
• Used written communication to convey synthetic biology to the public and the wider iGEM community.

Figure 11. Top left: Webinar presentation at UCL academy guest lecture. Top right: MisSTEM, the main character of the ficitional magazine story we published. Bottom: Our audience’s view on the effectiveness of our project after project was explained. Data from three webinars; pie charts made in PowerPoint. Young generation overall open to the implementation of this project and positive about its usefulness.

Achievements

Gold medal criteria
Integrated Human Practices

• Integrated expert feedback into implementation strategy including implementation flowsheets, seawater and plastic pre-treatments, brine treatment, cell replacement, disinfection, post-treatment and distribution systems
• Integrated expert feedback into construct and MDC configuration design
• On-site visiting of real-world water treatment facilities
• Surveys and interviews targeting end-users
• Integrated survey and interview results into project reshape, taking end-users' safety and cost concerns, lack of understanding of GMOs into account, and trying to address by using non-pathogenic microorganisms to mitigate safety issues and recycling brine for bringing down the overall costs

Modelling

• Developed flux balance analysis and cellular automata models to inform the design and operation of the MDC
• FBA provided insights into the co-culture conditions for sufficient lactate production
• CA predicted the current output and desalination rate produced by S. oneidensis

Science Communication

• Targeted outreach activities to underrepresented students in light of current racial discrimination events
• Fostered an open two-way dialogue on synthetic biology through the use of interactive polls
• Made materials accessible to a wider audience by translating to different languages and sharing recordings on Youtube

Partnership

• Partnered with iGEM Exeter in an attempt to characterise our PETase-MHETase fusion protein construct and perform Flux Balance Analysis modelling on their bacterium’s metabolism to gain critical insight into their synthetic biology approaches
• Partnered with iGEM Ashesi to target potential stakeholders to improve the human practices & implementation approaches of our projects

Silver medal criteria
Engineering Success

• Iteratively followed the engineering cycle to research, design and build PET-degrading constructs to be expressed in E. coli and P. putida
• Conducted a thorough literature research and consulted with experts for guidance on construct designs
• We sent our gene design and the plasmid backbone to GenScript and they built 4 designs for us according to our instructions
• Performed structural modelling to inform the design on the fusion protein construct design
• Designed protocols and experiments to test constructs in the lab

Collaboration

• Collaborated with iGEM MSP-Maastricht to write a peer-reviewed paper for the iGEM 2020 Proceedings Journal
• Collaborated with iGEM Exeter to support them with uploading content to their wiki

Human Practices

• Conducted a market research to identify regions that would benefit the most from our project and regions which had the required infrastructure to support our MDC operations
• Engaged with stakeholders and public in selected market locations to assess the feasibility of our project in these regions
• Reflected on feedback from survey and experts to inform project implementation

Implementation

• Designed an MDC desalination process to generate water for irrigation based on feedback from experts
• Considered safety aspects of using and containing engineered microbes – made co-culture auxotrophs to limit survivability outside the lab
• Considered additional safety aspects of pre-treatment, waste generation, and brine treatment

References and Acknowledgements

Acknowledgements
Sponsors

Student Team members

Olaide Ibiyemi, Juliette Champaud, Stefan Hristov, Pedro Lovatt Garcia, Daniel Castellano Garrido, Anna Su, Li Xu and Oliver Hernandez Fernandez

Supervisors and Instructors
Dr Stefanie Frank, Dr Kenth Gustafsson, Dr Chris Barnes, Dr Darren Nesbeth, Dr Rana Khalife, Alexander Van de Steen (Postgraduate Research student), Rory Gordon (Masters Research Student) and Neythen Treloar (Postgraduate Research Student).

Thanks to everyone that shared their expertise and supported us throughout the project.

References

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