Team:BUCT-China/Implementation
| Engineering design |
Based on the project we are pursuing, we can apply our technique on everywhere which encounters with the Polythene waste. To make our technique to be more flexible and applicable at various situation, we setup a unit system containing a biodegradation reactor, filtration device, a biosynthesis reactor, cells breaking device and a control part.
To begin with, the polythene waste from no matter dumping ground or pollutions in oceans need to be preprocessed before inserting into the unit system to turn Polythene (PE) waste into Polyhydroxy fatty acid (PHFA) wealth. Since the efficiency of degradation and synthetic is highly related to pretreatment, there are two basic standards should be considered. First is the magnitude, using extrusion and micronization[1][2] to make PE waste to be a certain size which performs a good degrading efficiency and can’t go through the ultra-filtration device. Second is clean, the PE waste should be cleaned to ensure the toxic chemicals won’t invade the bioactive reactor. Besides, the purification of feed PE determines the purification of final PHFA product to some extent.
To begin with, the polythene waste from no matter dumping ground or pollutions in oceans need to be preprocessed before inserting into the unit system to turn Polythene (PE) waste into Polyhydroxy fatty acid (PHFA) wealth. Since the efficiency of degradation and synthetic is highly related to pretreatment, there are two basic standards should be considered. First is the magnitude, using extrusion and micronization[1][2] to make PE waste to be a certain size which performs a good degrading efficiency and can’t go through the ultra-filtration device. Second is clean, the PE waste should be cleaned to ensure the toxic chemicals won’t invade the bioactive reactor. Besides, the purification of feed PE determines the purification of final PHFA product to some extent.
In the biodegradation reactor, the spores with oxidase fusion protein displaying on the surfaces[3] are cultivated as immobilized cells[4]. The spores of bacillus subtilis may can be immobilized on PVA-cryogels beads[5] and the activity of extracellular enzyme could be stable[6]. In the case that the user can insert PE waste that has been preprocessed¬——solid powder of polythene with buffer into biodegradation reactor, we choose fluidized bed reactor (FBR) to be the reactor for immobilized cells, which is appropriate to dispose substrate that has high viscosity or insoluble powder.
After a certain duration (mainly depends on the efficiency of oxidase, quantity of input fragment of PE, cost and effect of the whole reaction), PE powder will be degraded to alkane and the buffer will be put into filtration device to separate spores with oxidase surfaces, semi-decomposed PE fragments and the alkane. Because the alkane chain is the smallest molecular among these three components, it can go through the filtration membrane easily and leave others as concentrated solution. We decide to apply tubular filter having microfiltration film with a 0.04~20μm bore diameter. Although the filtering area of unit volume is relatively small for tubular filter, it has very simple structure which gives the convenience to clean and reuse immobilized spores.
Then, the alkane will be inserted into biosynthesis reactor, the E. coli with artificial metabolic pathway will utilize alkanes to synthesize PHFA which is the product of the unit system. The whole process is inside the engineering bacteria, so we need a special fermenter known as airlift fermenter to support the fermentation process of E. coli. The airlift fermenter can blast sterile air by nozzle in 250~300m/s flow velocity, then the air flow is able to cause circulation inside reactor. And mostly important, airlift filter fermenter has a simple construction and costs less energy.
In the biosynthesis reactor, alkane in buffer turns to PHFA inside engineering E. coli. To utilize the PHFA as a product, breaking cells device is inevitable contained in the unit system. The methods to break cells are divided into mechanical and non-mechanical. We would like to use high pressure homogenizer of mechanical method. High pressure homogenizer has a high rate of breaking gram-negative bacteria and capability to operating at large scale.
| Possible end users |
It is known to all the application of Polythene is widely spread, and the most common products are plastic bottle, bottle cap, plastic bag. The diversity of utilization means PE waste can be found at every garbage ground or in rivers[7] and oceans[8]. Our unit system of turning PE into Polyhydroxy fatty acid (PHFA) gives it possibility that any organization who need to deal with PE waste can put the unit system into their whole process as one step.
Take the waste hierarchy as reference, the unit system of our project belongs to RECYCLING (turning waste into new product or substance). In this case, any organization that has recycling step or want to add recycling step can use our unit system to enhance or reach their goals. The organization can be a bureau of environmental protection in government or a recycling company like Carbios in France who has already put PET recycling on their table[10].
| Developing idea |
Like we discussed above, our unit system is the vector and pathway to use our technique. The unit system can be put into any organization’s engineering project or design as a recycling step. When it comes to our collaboration with KEYSTONE, the utilization becomes more specific that our unit system design can be added into their hardware directly. KEYSTONE provides a thought of hardware as a novel garbage can. The garbage can has five areas: control zone, motor zone, energy zone, crushing zone and degrading zone. The can is designed to collect PET bottle at natural scenic spot. The can is able to collect solar energy for sustain degrading PET bottle.
With KEYSTONE’s help, they suggest us to add filtration zone, synthesis zone and cell breaking zone to the garbage can, if we want to solve the PE waste problem at natural scenic spot or other places. Since the synthesis part’s feasibility has be proved, as the CoA ligase and acylase system of polymerization. Besides, the main product of KEYSTONE’s project is MHET which has carboxyl and hydroxyl. Based on that, our polymerization part may also take advantage of their product by add only synthesis zone and cell breaking zone to the designed garbage can.
| Safety concern |
Using the unit system won’t cause damage to nature and humans directly, but it makes the genes of novel oxidase and other proteins invading the gene pool of bacillus subtilis and
E. coli. If the spores or E. coli inside the system are leaked out, it may influence the ecosystem in unpredictable way. It may just act as artificial accelerating evolution of these bacteria by having a better PE degrading efficiency compare to natural species, or it may cause PE material unreliable under natural condition due to the existence of novel bacterium.
| Future challenges |
In accordance with all above, we don’t what to restrict our imagination by the current experiments and knowledge. However, we also are self-awareness towards what we need to prove and what challenges that we are going to face. Here, we list three main points. The most important when we want to industrialize our technique is efficiency, which is determined by the efficiency of the novel oxidase we used, the cost of equipment and PE waste, the income of Polyhydroxy fatty acid (PHFA) product. We still need more experiments to support or improve the degrading efficiency of the novel oxidase. Besides, more data should be collected and more communication with experts should be set to let us know how much and how difficult to make the unit system come true and whether it worth the cost. The last but not the least is how to use the PHFA that synthesized by E. coli with artificial metabolic pathway. These are key questions for our project and what we are going to think deeply and try to solve in the future development.
| Reference |
[1]Barboza Neto, E. S., Coelho, L. A. F., Forte, M. M. C., Amico, S. C. & Ferreira, C. A. Processing of a LLDPE/HDPE pressure vessel liner by rotomolding. Mater. Res. 17, 236–241 (2014).
[2]Awaja, F. & Pavel, D. Recycling of PET. Eur. Polym. J. 41, 1453–1477 (2005).
[3]Lin, P., Yuan, H., Du, J. et al. Progress in research and application development of surface display technology using Bacillus subtilis spores. Appl Microbiol Biotechnol 104, 2319–2331 (2020).
[4]Nur Royhaila Mohamad, Nur Haziqah Che Marzuki, Nor Aziah Buang, Fahrul Huyop & Roswanira Abdul Wahab (2015) An overview of technologies for immobilization of enzymes and surface analysis techniques for immobilized enzymes, Biotechnology & Biotechnological Equipment, 29:2, 205-220.
[5]M Szczęsna-Antczak, E Galas. (2001). Bacillus subtilis cells immobilised in PVA-cryogels. Biomolecular Engineering, 17(2), 55-63.
[6]Mirosława Szczęsna-Antczak, Tadeusz Antczak, Stanisław Bielecki. (2004). Stability of extracellular proteinase productivity by Bacillus subtilis cells immobilized in PVA-cryogel. Enzyme and Microbial Technology, 34(2), 168-176.
[7]G. Kalčíková, B. Alič, T. Skalar, M. Bundschuh, A. Žgajnar Gotvajn. (2017). Wastewater treatment plant effluents as source of cosmetic polyethylene microbeads to freshwater. Chemosphere, 188, 25-31.
[8]Eriksen M, Lebreton LCM, Carson HS, Thiel M, Moore CJ, Borerro JC, et al. (2014). Plastic Pollution in the World's Oceans: More than 5 Trillion Plastic Pieces Weighing over 250,000 Tons Afloat at Sea. PLoS ONE 9(12): e111913.
[9]Directive 2008/98/EC on waste (Waste Framework Directive) https://ec.europa.eu/environment/waste/framework/
[10]Tournier, V., Topham, C.M., Gilles, A. et al. An engineered PET depolymerase to break down and recycle plastic bottles. Nature 580, 216–219 (2020).
[2]Awaja, F. & Pavel, D. Recycling of PET. Eur. Polym. J. 41, 1453–1477 (2005).
[3]Lin, P., Yuan, H., Du, J. et al. Progress in research and application development of surface display technology using Bacillus subtilis spores. Appl Microbiol Biotechnol 104, 2319–2331 (2020).
[4]Nur Royhaila Mohamad, Nur Haziqah Che Marzuki, Nor Aziah Buang, Fahrul Huyop & Roswanira Abdul Wahab (2015) An overview of technologies for immobilization of enzymes and surface analysis techniques for immobilized enzymes, Biotechnology & Biotechnological Equipment, 29:2, 205-220.
[5]M Szczęsna-Antczak, E Galas. (2001). Bacillus subtilis cells immobilised in PVA-cryogels. Biomolecular Engineering, 17(2), 55-63.
[6]Mirosława Szczęsna-Antczak, Tadeusz Antczak, Stanisław Bielecki. (2004). Stability of extracellular proteinase productivity by Bacillus subtilis cells immobilized in PVA-cryogel. Enzyme and Microbial Technology, 34(2), 168-176.
[7]G. Kalčíková, B. Alič, T. Skalar, M. Bundschuh, A. Žgajnar Gotvajn. (2017). Wastewater treatment plant effluents as source of cosmetic polyethylene microbeads to freshwater. Chemosphere, 188, 25-31.
[8]Eriksen M, Lebreton LCM, Carson HS, Thiel M, Moore CJ, Borerro JC, et al. (2014). Plastic Pollution in the World's Oceans: More than 5 Trillion Plastic Pieces Weighing over 250,000 Tons Afloat at Sea. PLoS ONE 9(12): e111913.
[9]Directive 2008/98/EC on waste (Waste Framework Directive) https://ec.europa.eu/environment/waste/framework/
[10]Tournier, V., Topham, C.M., Gilles, A. et al. An engineered PET depolymerase to break down and recycle plastic bottles. Nature 580, 216–219 (2020).