Team:Hong Kong HKUST/Human Practices

• 
• Project
  Overview PE Degradation Alkane Metabolism Alkane Results MFC Design MFC Result Experimental protocol Notebook Parts
• Modelling
  Laccase Alkane Metabolism Alkane and MFC (FBA)
• Human Practices
  Education and Engagement Collaborations Integrated Human Practices
• InterLab
• Medal Requirements
• Safety
• Team
  Team Members Attributions

• 1. Motivation

  
  

  Environmental pollution by plastics has long been an issue of concern across the globe. According to National Geographic, an astounding 8.3 billion metric tons of plastic have been produced and accumulated since its introduction in the 1950s. Triggered by the disturbing facts of plastic accumulation, the world has come up with many solutions to tackle this problem, for example by degrading PET. But there has been no defined solution to tackle Polyethylene, one of the most common plastics, as of yet. The HKUST iGEM team hopes to solve this issue by degrading PE and turning it into useful resources for everyone.

• 2. Choosing Polyethylene as our Plastic Substrate

  
  

  Through doing more secondary research from the World Wildlife Fund and ScienceDirect, we identified Polyethyelene (PE) as one of the most prevalent sources of plastic pollution and yet seems to be one of the toughest kinds to eliminate. Plastic bags made out of PE take up the largest percentage of marine debris (90% of all floating marine debris), which caused major damage to aquatic life. On the other hand, people have found it extremely hard to degrade PE, as it is essentially made up of a long hydrocarbon chain with no chemical groups for enzymes to recognize and digest. It has also proven to be very resistant to chemical attacks.

  In 2015, the Hong Kong government imposed a levy scheme on PE plastic bags in order to minimize its environmental impacts. Nonetheless, some plastic bag use remains, along with past wastes which wound up in landfills, waiting for millennia to be decomposed. As such, our team is committed to tackle this problem and help fulfil Hong Kong government's vision of a cleaner Hong Kong using our genetic constructs to degrade PE.

• 3. Taking it a Step Further - Converting Waste to Electricity

  
  

  Our team realized, however, that simply degrading polyethylene would produce fragmented alkanes, which could potentially be harmful to the environment as well.

  We wanted to turn this problem into an opportunity by using our end product to potentially solve yet another environmental issue. We realized that energy security is another pressing issue which countries face.

  To solve these two problems simultaneously, we aim to convert the degraded plastics into electricity by feeding them as an energy source for an electricity-producing strain of bacteria, Shewanella oneidensis MR-1. Plastic degradation and electricity generation will be merged together in a Microbial Fuel Cell as described in our MFC design.

• 4. How Talking to Prof. Bookhart, an Environment and Sustainability Expert, Changed our Project Design

  
  

  To better mold our project into addressing genuine environmental concerns, we consulted Professor Davis Bookhart, the head of the HKUST Environment and Sustainability Department, who is currently working on different projects to reduce the University’s environmental impacts while addressing and managing risks that arise from climate change and resource scarcities. We thought that he would be able to give us more insights on real/current environmental challenges which we could help solve and how we could ensure the sustainability of our production system.

  We first presented our system to Professor Bookhart in July. A few concerns he had was that, from the perspective of sustainability, our project was actually converting PE, a carbon-containing pollutant, into CO₂ through the respiration of Shewanella, which in itself ıs a greenhouse gas. This actually exacerbates the problem of global warming despite removing a terrestrial and marine pollutant. Instead, Prof. Bookhart suggested that we find a way to reuse the carbon in any way or to feed back into the system for more electricity generation. We can also sequester the CO₂ in a way that the CO₂ is converted to a solid or a liquid to be stored.

  Prof. Bookhart had also asked about the threshold of electricity we expected to generate and how that electricity could be used. To really transform our project into an impactful product, we should implement an application for the electricity generated. We should as well focus on designing a device that could encourage the public to put their plastic waste to good use by generating electricity for daily use.

• 5. More Insights – Meetup with iGEM teams in the environmental track

  
  

  During our meetups with other iGEM teams for collaboration, we exchanged ideas with team HKJS_S and realized that their project mechanism could be implemented into our system to tackle the concern of CO₂ by-product waste raised by Prof. Bookhart.

  One of their parts encodes a nitrogenase, which has the ability to transform CO₂ into methane. Both substrates were intimately linked with our project: the CO₂ was the sustainability problem we had to tackle, while methane was a substrate that could be processed by our Alkane Metabolism pathway. Seeing this connection, we asked for their permission to borrow their construct design to improve our system overall. /p>

  Originally, we meant to use E. coli bacteria to secrete laccase for PE plastic degradation and Shewanella, stored in the MFC, to house the alkane channel and alkane metabolism pathway. However, to integrate insights from both Professor Bookhart and the HKJS_S team, we decided to change our final system design into one single Bacterial Artificial Chromosome, so that our individual systems could be housed in one single cell to increase the automation of our project. This modification will be implemented upon successful attempts on PE degradation and Alkane metabolism. The new schematic is as follows:

  

  As usual, laccase degrades PE into alkanes (refer to our PE degredation page for details). The AlkL gene (coding for Alkane outer membrane channel protein) allows larger alkanes to enter the transformed Shewanella and the ASS cluster allows the cell to process it (please see our Alkane metabolism page for details). In addition, we added a nitrogenase gene to convert the CO₂ products into methane. The idea is that since methane can also be processed by the ASS cluster, CO₂ produced by Shewanella during respiration can be recycled into the MFC system.

  According to our modelling, carbon dioxide can be converted into methane after undergoing reduction process, in which the molecule uses the energy from the sun / catalyst to break up the CO₂ molecule into carbon and oxygen atoms, then combine with hydrogen to form methane and water, as explained on the chemical equation below.

  

  Using irreversible Henri-Michaelis-Menten Kinetics, we tried to consolidate an enzyme-catalyzed reaction with a single reaction and and reaction rate equation with V_max of 0.8 ± 0.07 nmol/min and a Km for CO₂ of 23.3 ± 3.7 mM ^[1].

  

  
  From this graph, it can be seen that it takes over 3 hours to fully convert 10 nmol of CO₂ into methane. It appears to verify that common features of homogeneous catalysts for CO₂ reduction to CH₄ are low reaction rates (e.g., turnover frequencies) and limited number of turnovers (e.g., turnover number) before inactivation of the catalyst ^[2].

  Since laccase takes about 80 days to degrade about 40% of the sample PE, we will also consider integrating a delay module into the alkane metabolism module, inspired by the iGEM team of HKUST 2017. The idea is that the alkane channel and alkane processing proteins will only kick into effect after 80 days of PE breakdown, so as to save cellular resources. The time delay module will be enabled by the phlF inducer, which induces the pHLFp promoter in front of the alkane metabolism genes. Since the genes have a lower RBS strength than that used by the phlF inducer gene, this will create a time delay when producing the alkane metabolism proteins.

• 6. Primary market research: Listening to our potential users

  
  

  One of the aims of our public engagement and education exhibition was to find out the major concerns of the general public in choosing sources of renewable energy. This will help us to choose the focus and design our MFC to better suit the public’s concerns and aid in a more user-friendly design. The detailed analysis of our collected data can be found here

  The results show that “impacts on human health” and “constraints in the conditions/ requirements for energy production” are the public’s major concerns. There are also concerns with the release of environmental pollutants such as sodium hydroxide and hydrofluoric acid during the production of renewable energy generators. However, survey results shows that the public had regraded this issue as less important as the long term generation of renewable energies are comparatively cleaner than energies from fossil fuels where there are no chemical pollutants generated during energy production. Upon reviewing our MFC system design, the release of CO₂ from our MFC will be produced as a by-product during electricity generation, this greenhouse gas therefore will still be a pressing issue that the team needs to tackle. Involving the public engagement results into our MFC design, we have set the following design standards/requirements for our MFC:

  - Limit the impacts on human health by limiting users’ interaction with the inner mechanism of our Laccase degradation and MFC system. Chemicals and cell culturing medium should not be harmful to both the environment and humans. This can be done by making the MFC a closed system during operation, where the users need not manually operate the system at any intermediate stage.
  - Focus on reusing or fixing the CO₂ that will be released from the respiration of our cell culture to minimize environmental pollution.
  - The size of the MFC should be scaled up to accommodate the slow PE plastic degradation rate.
  

  
  

  7. Finalising our ideas – Final wrap-up with Prof. Davis Bookhart

  
  

  After making the mentioned modifications in our system, we again approached Prof. Bookhart to update him with our progress and to show him the data we have collected. We asked for further advice in integrating the public opinions we have obtained to our future direction. Prof. Bookhart’s main concern still lied on CO₂ being our end product. He commented that our added nitrogenase system will only delay the problem of CO₂ release by recycling it, instead of trapping the CO₂ permanently.

  Integrating all the concerns and suggestions given by Prof. Bookhart and our market research during our exhibition, we came up with the biosphere-MFC conjugation and the nitrogenase system to fully sequester CO₂.

  For the former, we would let CO₂ pass from the anode chamber that houses Shewanella into an adjoining compartment via a semipermeable membrane. The adjoining compartment would house a self-contained biosphere, where plants, shrimp and potentially fish could be stored. The plants inside, with the help of a lamp, could uptake the CO₂ that crosses over via photosynthesis (the MFC can power a small lamp as a light source), hence fixing the CO₂ into a plant, and therefore sequestering the CO₂. This will help decompose PE without releasing additional CO₂ to solve the issue raised by Professor Bookhart. Since the nitrogenase system does not trap the CO₂ permanently, it was not suggested to be the main way to solve the sustainability issue. Nonetheless, our team decided to keep this system,to enhance our electricity generation when the plastic degradation process is limited.

  Alongside carbon sequestration, there is an added benefit of putting plants near the MFC: to absorb any excess Cu ions from our PE degradation module (which role is to enhance laccase enzymatic activity). This will nourish the biosphere and also prevent Cu contamination to the environment.

  

  
  All in all, our final product design is modified into a MFC/biosphere conjugation. We hope that the mini-ecosystem can serve as a visual attractant to educate people on the idea of sustainability and also to motivate them to be more environmentally friendly. In addition to educating the public and raising awareness towards the environment, thisMFC/biosphere conjugation can generate enough electricity to charge a power pack, through the help of a capacitor. On a small scale, it can be used in households to charge batteries or power packs for electrical devices. On a large scale, the MFC/biosphere conjugation can be used in malls to serve as a charging station as well as a mini aquarium to be shown.

  References:
  Ojha, N., Pradhan, N., Singh, S., Barla, A., Shrivastava, A., Khatua, P., Rai, V. and Bose, S. (2017). Evaluation of HDPE and LDPE degradation by fungus, implemented by statistical optimization.
  
  Seefeldt, L., Rasche, M. and Ensign, S. (1995). Carbonyl sulfide and carbon dioxide as new substrates, and carbon disulfide as a new inhibitor, of nitrogenase. Biochemistry, [online] 34(16), pp.5382-5389. Available at: https://pubs.acs.org/doi/pdf/10.1021/bi00016a009.
  
  Yang, Z., Moure, V., Dean, D. and Seefeldt, L. (2012). Carbon dioxide reduction to methane and coupling with acetylene to form propylene catalyzed by remodelled nitrogenase. Proceedings of the National Academy of Sciences, [online] 109(48), pp.19644-19648. Available at: https://pdfs.semanticscholar.org/19f5/fc872e91b3a7259a73528eeb3b6df301ab33.pd.

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