Team:Hong Kong HKUST/Human Practices
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.
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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.
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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.
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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.
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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.
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 Vmax 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.
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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 to our system, we again approached Prof. Bookhart to update him with our progress and to show him the data we collected. We asked for further advice on how to integrate the public opinion we obtained to help define our future direction with the project. Prof. Bookhart’s main concern still lied in the fact that CO2 was our end product. He commented that our added nitrogenase system 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 our exhibition, we came up with the biosphere-MFC conjugation and the nitrogenase system to fully sequester CO2.
For the former, we will let CO2 pass from the anode chamber that houses Shewanella into an adjoining compartment via a semipermeable membrane. This adjoining compartment will house a self-contained biosphere, where plants, shrimp and potentially fish can be kept. The plants inside can take up the CO2 that crosses over via photosynthesis, hence fixing the CO2 and therefore sequestering it as plant biomass. 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 primary 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, this MFC/biosphere conjugation could generate enough electricity to charge a power pack, through the help of a capacitor. On a small scale, it could be used in households to charge batteries or power packs for electrical devices. While on a large scale, the MFC/biosphere conjugation could be used in malls to serve as a charging station as well as a mini aquarium or terrarium to be exhibited.Business Plan
Background
The world nowadays faces 3 major environmental problems, namely the uneven distribution of energy resources, severe marine pollution, and the imbalanced carbon cycle. The uneven distribution of energy arises due to the unique and non-uniform geological distribution of fossil fuel. Hence, energy resources are not equally shared among all the people. Besides energy imbalance, plastic pollutants are also another significant problem. For instance, there are around 8 million metric tons of plastic generated to the sea per year, which greatly harm the marine life in general and upsets the ecosystem. Furthermore, the use of fossil-based products, such as plastic, imbalances the carbon cycle by introducing an excess amount of carbon that was previously hidden away into the atmosphere. This exacerbates the problem of global warming, which affects ecosystems and encourages tropical diseases such as malaria, posing a huge threat to human health.
On the business side, in this day and age, products promoting sustainability are valued as they are seen as an effort to save our Earth. It appeals to the better nature of people to be environmentally friendly. On an economic aspect, it also allows people to utilize fewer resources, hence, saving money.
Our product utilizes the principle of Microbial Fuel Cell to solve all these problems. An MFC houses a microbe capable of generating electricity in a fuel cell and connects it to an external circuit to produce electricity. The microbe in question is a genetically modified Shewanella. It utilized enzymes from E. coli to fragmentize the PE and enzymes from D. Alkenovorons to metabolize the fragments of PE. These two ideas will be compiled with the PE degradation and streamline the two processes in just one product. Besides that, our product also utilizes biosphere components to sequester any excess carbon from the breakdown of PE, to prevent an imbalance of the carbon cycle.
Technology:
Our proposed product, the Microbial Fuel Cell- Biosphere conjugate, is an alternative way to address these three pressing environmental issues and meet market demands in the future. Our system degrades Polyethylene plastic with a GM Shewanella, which was transformed with the genome of E.coli to fragmentize the PE and metabolize them with the enzyme from D. Alkenovorons; then, electricity is generated through the respiration of Shewanella Oniedensis that generates electrons to be captured by the fuel cell. The carbon in the PE is processed into a respiration side product: carbon dioxide, which is then sequestered into the Biosphere connected to the MFC. The nitrogenase in our product will capture the CO2 generated during the process of metabolism and recycle it into one of the food sources of our Shewanella with the built-in conversion system. As the fuel cell continues to function, more CO2 would be sequestered in a form of plants inside the biosphere, that makes the overall CO2 content in the environment decrease. This way, a major marine pollutant such as PE can be largely eliminated. Meanwhile, the power generated from MFC can also address energy shortage.
Product Portfolio
Our product, the microbial fuel cell, combines three processes – degradation, generation and sequestration into just a single set-up. It can be used as a charging station for electronic devices. On a small scale, it can be a household appliance as well as a mini-aquarium. On a large scale, it can be used in malls as a feature to demonstrate its innovation as well as a central energy source for the mall.
Our product is featured to be self-contained. That is the product itself is already the whole system we need and there is no need for further input to operate the system . Besides, the MFC would serve as a closed system when it is operating, making it no harm to the users and the environment, because nothing would escape from the system. On the other hand, our MFC will sustain itself as long as PE is provided, making it extremely convenient, and users are not required to replenish anything as the system is self-sustainable. Moreover, as mentioned before, our product contains a genetically modified strain of Shewanella that is adaptable to alkane metabolism. This raises another problem to the customers of whether it is safe. The original bacteria strain is long confirmed to be not related to any human diseases and the inserted genome part is also harmless to human. All the materials that construct the MFC are also verified to be innocuous to the environment and human. We could conclude that the product we designed will not negatively impact human and society. However, there are some technical weaknesses to construct an applicable MFC, which are the low output and the long degradation period. Therefore, one of our objectives is to focus on enhancing the overall efficiency of the MFC.
Opportunity
Our product could gain traction from the customers in the market since the fundamental idea of our product has never been introduced. PE is one of those man-made polymers that have no specific solution to it before. However, our team engineered a new method that could be applied to this situation, using our engineered Shewanella. Besides, our product compiles of three processes in series and they were done automatically, which is totally revolutionary to other similar products we have in the market. Other than the technical aspect, this product is also supported by the general economic condition in the Great Bay Area of China because the biotech industry is flourishing in the area and people are willing to invest on some prospective successful product. Therefore, it is our opportunity to gather funding to realize our idea. In the environmental aspect, the ultimate goal of our product is to address plastic pollution and recycle the materials in order to relieve some burden on the environment. Hence, our product is actually a result of considering how to resolve the impact of excessive PE pollution to the environment so as to sustain our future, and this concept abides to the mainstream opinions that we should protect our environment, which is appealing to the general public.
Finance:
As we are a university team, we may apply for HKUST’s Innovation and Technology Scholarship award which awards us $150,000. We may also apply for the Hong Kong government’s Innovative Technology Fund in order to finance our activities.
Besides these funds, we may also look into the Hong Kong Science and Technology Park, which provides an incubation program for technology startups. Through this incubation program, our future company can be introduced to investors with the Science and Technology Park as a middleman, thus securing future investment. The incubation program also provides office as well as lab facilities that ensure we can have access to vital resources. It also lowers our operational costs by offering these facilities for free or at a low rate.
Prospect:
Research and Development
In terms of R&D, we can focus more on how to create conditions to increase the exo-electrogenicity for Shewanella. By increasing the native electron generation ability, a higher voltage can be achieved such that our product can be more efficient in providing electricity. We may choose to look into increasing the rate of plastic degradation as it is a major limiting step in our product currently. should this limiting factor be lessened or removed, the entire process can be further streamlined, which is favorable to our goal of solving the world’s environmental problems. Also, by mass production of the hardware of our product, we hope that the production cost can be significantly lowered.
Market Expansion
Besides selling our product in Hong Kong, we can also look towards the Greater Bay Area of China. China has a huge population which alone provides a huge market for our product. Especially since the central government of China strives for environmental sustainability by 2035, our product will be very favorable in the Greater Bay Area of China.
partnership
As mentioned, we can collaborate with some of the startups in Hong Kong Science Park (HKSTP). HKSTP hosts a variety of biotech and green technology startups. We aim to collaborate with companies in both tracks such that we can augment our biological systems in the MFC and also increase its capacity so that it may serve as a better charging station or power supply. To be more specific, we could perhaps look into companies with experience in metabolomic engineering so that we may streamline our alkane metabolism module. There are also companies in HKSTP that cater exclusively to charging services. We may be able to elicit their help to improve the charging capabilities of the MFC. Furthermore, we could even contact Hong Kong aquaculture companies that utilize the concepts of the biosphere in their work, asking for advice on the biosphere components in our product.
Challenges:
Up to this moment, all the technical details of all three processes have been designed and verified, but there are still some challenges to our product, including the efficiency, the time consumed and the public acceptance. Beginning with a technical problem, the overall efficiency of our product is still far from applicable and more works to be done to solve this problem. Besides, the PE degradation still takes too much of time and it is no way to be adopted by the market, which will also be our focus in the further researches. Furthermore, besides the technical aspect, public acceptance will also be another great concern to our product. As the mainstream perception on bacteria is negative, which most non-technical people will have a deep-rooted mindset that all the bacteria are bad, it would be an even greater challenge to us to promote and persuade our customers to adopt it; for this part, we could see that it is necessary to align our product promotion with the general education on biotech and genetic modification so that people could be more acceptable to use a bacteria-contained product.
Conclusion
To conclude, our MFC-biosphere conjugate can solve many environmental problems. In an era where sustainability and environmental concerns are highlighted, we believe our product can be promoted easily. At such an early stage, the MFC-biosphere can certainly be improved in terms of efficiency. Nevertheless, it still has a huge potential to be a viable product.
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. Bibliography:
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