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

INTEGRATED HUMAN PRACTICE

• 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 the substrate and original system design

  
  

  Of all plastics, Polyethylene (PE), is relatively harder to degrade as it is essentially a long hydrocarbon chain. There are virtually no chemical groups for enzymes to recognize and therefore no specific enzymes to digest it. It has also proven very resistant to chemical attacks. As such, polyethylene is a difficult pollutant to tackle. Major products made from PE include plastic bags and they currently take up the largest percentage of marine debris. This has caused major damage to aquatic life. In fact, in Hong Kong, the government has imposed an environmental levy scheme on plastic bags in order to minimize the impact of PE plastic bags on our environment. But still, plastic bags remain in widespread use, with those disposed of winding up in landfills, waiting for millenia to be decomposed. This shows how abundant PE plastic is throughout the world and especially in HK. It has caused so much pollution yet there lie hardly any viable solutions to combat it. The iGEM team of HKUST 2018 aims to tackle the challenge with our genetic constructs.

  Our team however realized that by simply degrading the polyethylene, it would still lie around in the environment as fragmented alkanes. Being in the environmental track, we therefore searched for some other environmental issues that our end product can possibly solve. We realize that energy security is another pressing issue countries face.

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

• 3. System consolidation – Interviewing Prof. Davis Bookhart

  
  

  As a team in the environmental track, we decided to consult Professor Davis Bookhart, the head of the HKUST Environment and Sustainability Department who is currently working on different projects to reduce or eliminate 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 comments on how our project would impact the environment and how we could ensure the sustainability of the 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.

• 4. Exchanging ideas – Meetup with teams in the environmental track

  
  

  During our meetups with other iGEM teams for collaboration, we have been exchanging ideas with the team HKJS_S and we realised that their project mechanism can be implemented into our system to tackle the concern of CO₂ sustainability raised up by Prof. Bookhart.

  One of their parts encodes a nitrogenase that could have the ability to transform CO₂ into methane. Both substrates were actually 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.

  Originally, we meant to use E coli bacteria to secrete laccase to fragment the PE and use Shewanella, stored in the MFC, to house the alkane channel and alkane metabolism pathway. But to integrate the comments from Professor Bookhart and our discussion with the HKJS_S team, we decided to incorporate our entire system 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. The new schematic is as follows.

  

  As usual, the laccase degrades PE into alkanes (please see our PE Degradation page for details). The AlkL gene (coding for Alkane outer membrane channel protein) allows larger alkanes to enter the transformed Shewanella and the Alkane Metabolism pathway allows the cell to process it (please see our Alkane Metabolism page for details). In addition, we added a nitrogenase gene to convert CO₂ into methane. The idea is that since methane can also be processed by the Alkane Metabolism pathway, CO₂ produced by Shewanella during respiration can be recycled into the MFC system. However, at this early stage, we will focus on the laccase and alkane channel first before moving on to the nitrogenase aand alkane metabolism.

  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 time 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 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.

• 5. Integrating our potential users – Market research from Info Day booth

  
  

  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 shows 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:

  - Enhance product safety and 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.
  - The size of the MFC should be scaled up in design to accommodate the slow PE plastic degradation rate.
  

  
  

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

  
  

  After the system modification using the nitrogenase system as mentioned earlier, we went back to Prof. Bookhart to update him on our progress and to show him the data we collected. We asked for further advice on the direction we could take, considering the comments from the public. Prof. Bookhart’s major concern was still that CO₂ would be our end product. The nitrogenase system added in this case would only delay the problem of CO₂ release by recycling it, instead of trapping it permanently.

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

  For the former, we would let CO₂ pass from the anode chamber that houses Shewanella into an adjoining compartment via a semipermeable membrane. This adjoining compartment would house a self-contained biosphere, where plants, shrimp and potentially fish could be kept. The plants inside could uptake and fix the CO₂ that crosses over via photosynthesis, converting the CO₂ into plant biomass, and therefore sequestering the CO₂. This will help decompose PE without releasing additional CO₂ to solve the sustainability issue raised by Professor Bookhart. The nitrogenase system, since it does not trap the CO₂ permanently, it was not suggested to be the primary way to solve the sustainability issue. Our team decided to keep this system however, to enhance our electricity generation when the plastic degradation process is limited.

  Alongside carbon sequestration, there is an added benefit of using the plants near the MFC to absorb any excess Cu ions that helps laccase enzymatic activity. This will aid the biosphere and also prevent Cu contamination to the environment.

  

  
  Hence, our modified product design will now be an MFC/biosphere conjugation. It is hoped that the mini-ecosystem can be a visual prop to educate people on the idea of sustainability and also motivate them to be more environmentally friendly as the flourishing biosphere may serve as a tangible reward for them. In addition to educating the public and raising awareness towards the environment, the MFC/biosphere conjugation can still 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. While 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 or terrarium to be exhibited.

  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.

  Carbon degradation:

  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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