One. Environmental Issues
Plastic and climate:
Discarded plastic products not only pollute the environment, as a petroleum product, its manufacturing process like refined oil also has an impact on the environment and climate. According to the U.S. Environmental Protection Agency, the output of plastic products now accounts for about 8% of global oil production. The process of oil drilling and processing of plastic products will emit harmful gases into the atmosphere, including carbon monoxide, hydrogen sulfide, ozone, benzene and methane (a greenhouse gas that causes a more serious greenhouse effect than carbon dioxide). The production of one ounce of polyethylene terephthalate Glycol esters (also known as PET, the most commonly used plastic for making water bottles) emit five ounces of carbon dioxide.
In summary, plastics are closely related to the issue of global warming, one of the most pressing issues facing the earth today. 
Various microorganisms can degrade bioplastics in different ecosystems (landfill, compost, sea water, river water, etc.). However, under different environmental conditions, the biological diversity of plastic-degrading microorganisms is also different. Under the conditions of soil and compost environment, a high bioplastic degradability is shown, mainly due to the high diversity of microorganisms under this condition. A large amount of plastic will enter water bodies and marine systems and cause unavoidable impacts on freshwater and marine ecosystems.
Two. The current treatment methods for waste plastics and their effects
Four modes of handling plastic wastes:
Presently, global treatment methods for discarded plastic include landfill, incineration, regeneration and granulation, and pyrolysis.
It is a traditional method widely used to dispose of waste plastic waste. This simple and direct physical treatment method has relatively high potential hazards and increases the pressure on the use of land resources. The refractory plastic severely hinders the penetration of groundwater, and the additives in the plastic cause secondary pollution of the land. Plastic waste, whether in rivers, oceans or on land, can persist in the environment for centuries. It is almost impossible for plastics to decompose completely in nature. Most plastic products will never disappear completely but become smaller and smaller.
It is also a widely used plastic waste disposal method. According to the BBC, the "incinerators" said that plastics are produced from oil and natural gas, mainly hydrocarbons. When they are incinerated, they will generate a lot of heat and then use the generated heat to generate electricity. Incineration of plastic waste in this way is currently replacing polluting fuels such as coal or oil in some places. However, burning plastic will produce toxic and harmful gases. If the incinerator is not efficient, these waste gases will enter the environment. Take Germany as an example. At present, there are 200,000 tons of PVC waste in the country every year, 30% of which are burned in incinerators. The German environment department has stipulated that all incinerators must meet the limit of less than 0.1 ng (nanogram) per cubic meter of exhaust gas. Although the air pollution standards for incinerators in Germany have been recognized as high standards in the world, it still doesn’t state that the incineration method will not release harmful substances due to mechanical failure.
Liu Jianguo, a professor at the School of Environment of Tsinghua University, said in an interview with a reporter from the Global Times that it is necessary to use a more complete control system to make plastic waste more fully burned, and a complete flue gas purification system must be set up to treat the flue gas in place so that pollution can be effectively controlled.
3. Regeneration granulation
It is a method of physical recycling of plastic waste. Most recyclable plastics are broken down into particles by mechanical processing, and then remanufactured into new plastic products, such as packaging materials, seats or clothing. However, the recycling granulation method also has limitations. The process is not suitable for plastic films, pouches and other laminated plastics, which are usually sent to landfills or incinerated.
4. Pyrolysis of waste plastics
This chemical decomposition method refers to the process of using the thermal instability of the organic matter in the solid waste and placing it in a pyrolysis reactor for thermal decomposition. This technology can convert waste plastics into high value-added energy products such as fuel oil, natural gas, and solid fuel.
Three. The recycle process for discarded PET
1. Energy recovery
Incineration of waste PET converts hydrocarbons into carbon dioxide and water, and releases heat energy. It can replace fossil fuels, and the slag after incineration can be disposed of in landfills. However, toxic gases will be produced during the incineration process, which will have an impact on the environment and people's health.
2. Physical recycling
Physical recycling refers to the process of granulating waste PET products through sorting, flotation, washing, drying, melt extrusion and other processes, and then directly using, blending, strengthening, blending and other physical methods to make new products, so as to realize the recycling of waste PET. However, it is not suitable for plastic films, pouches and other laminated plastics. The physical and mechanical properties, thermal properties, etc. of recycled PET have declined, and can be used in some occasions that do not require high material properties.
3. Chemical recovery
Chemical recovery refers to the depolymerization of waste PET raw materials into terephthalic acid (TPA), dimethyl terephthalate (DMT), and terephthalic acid through chemical methods such as saccharolysis, alcoholysis, hydrolysis, ammonolysis, and supercritical depolymerization. Chemical raw materials such as ethylene glycol formate (BHET), ethylene glycol (EG), TPA's diamines (TPD), etc., to realize the recycling of waste PET. The chemical recycling method provides a relatively complete PET recycling performance, so that the recycled products have high value-added reuse performance. However, the toxic gases released during the chemical recovery process still have a certain impact on the environment. Moreover, the chemical process requires high pressure and high temperature and consumes a lot of chemicals.
4. Bio recycling
Biological recycling refers to the complete depolymerization of waste PET raw materials into monomers through selective enzymatic hydrolysis, and then re-make PET. Enzyme is a natural catalyst with high selectivity, but the efficiency is low.
Four. Microbial degradation precedents
In 2016, Ideonella sakaiensis, a bacterium that can "eat" PET, was isolated from a PET recycling facility in the suburbs of Osaka, Japan. This strain of bacteria secretes an enzyme that can hydrolyze PET into small molecules, called "IsPETase". The decomposed small molecules MHET and TPA can be absorbed and utilized by bacteria.
Figure 3: PET biodegradation mechanism (source: interviewee)
IsPETase is the only real PET degrading enzyme produced through natural evolution process so far. However, IsPETase is not a brand-new enzyme, it belongs to an ancient enzyme species-"cutinase" (its original role is to break down plant cutin by microorganisms).
The ancient cutinase has very low activity in decomposing PET, but IsPETase can hydrolyze PET very well. And how can bacteria convert cutinase into PET degrading enzyme in a short time? In the nearly 70 years since PET came out, the mystery has never been revealed.
Figure 4: Key differences between IsPETase and other classic cutinase active areas (Source: Interviewee)
Ruiting Guo and Chunqi Chen's team have been engaged in the research of protein structure and function analysis for a long time. In 2017, they reported the first internationally on the crystal structure of IsPETase and the complex structure of enzyme and substrate analogues. This is the only structure with a PET substrate so far. Related papers were published in Nature Communications.
This scientific research team has continued in-depth research in this field for many years. By comparing the protein structure of IsPETase and cutinase, it has been found that the substrate binding area of cutinase is relatively narrow, which is more suitable for acting on slender cutin, which is not conducive to acting on PET with a larger structure.
According to reports, the research team integrated various polymer biodegradation mechanisms, especially discussing the structural characteristics of enzymes and substrate binding modes, looking for the correlation and the evolution direction of microorganisms in response to the emergence of man-made substances, for the utilization of biomass resources It provides future prospects for the biodegradation of plastics. The related paper was published in Nature Reviews Chemistry in March 2020 and became the cover article of the current issue.
Figure 5: March 2020, the cover article of Nature Reviews Chemistry (Source: Interviewee)
Ruiting Guo said that the composition of the substrate binding region of IsPETase is the same as that of cutinase, but a tryptophan 185 (W185) in the substrate binding region of IsPETase can swing freely, and in all cutinases, the direction of the side chain of tryptophan It is fixed.
The researchers further analyzed the area adjacent to tryptophan and found that in all cutinases, tryptophan was supported by two amino acids with larger side chains (histidine and phenylalanine for short). They are like stents that hold tryptophan firmly so that it cannot be rotated.
In IsPETase, under W185 are serine and isoleucine (small binary for short). Their side chain groups are small and cannot fix W185, so W185 can swing freely, and the substrate binding area of IsPETase can also "scale freely".
Figure 6: Graph丨Ruiting Guo’s team tested 8 cutinases and found that the introduction of 6 cutinases into the Ser/Ile small binary mutation can greatly increase the enzyme activity for PET degradation (source: interviewee)
Interestingly, replacing the small binary body of IsPETase with a large binary body will greatly reduce the activity of PET degradation. On the contrary, replacing the large binary body in the cutinase with a small binary body will degrade the activity of PET. Significantly improved. From this, it can be seen that the conversion of large and small binary bodies is most likely the most critical condition for the production of a PET degrading enzyme.
"Inspecting the codons, we can find that it only needs to mutate three bases to turn a large binary into a small binary, and the accumulation of three mutation sites may happen in a short time. This only requires thorough Through the use of the strategy of introducing small duality." Ruiting Guo said.
Five. Benefits of earthworms
For better application of the program, the problem of plastic waste and microplastic pollution of arable land cannot be solved by simply using engineering bacteria, because this does not solve the limitations of traditional microbial treatment. However, we thought of earthworms, a common soil ecological engineer.
1. The perfect mobile platform
Earthworms are protozoa of the soil and are very mobile in the soil. However, if it is an engineered bacterium, it cannot effectively deal with soil plastic pollution, because the scope of action of the engineered bacteria is limited. Therefore, by making earthworms carry engineered bacteria, the engineered bacteria can move together with the earthworms, thereby playing a comprehensive role in soil management in the region. In summary, through earthworms, we provide a good carrier for the spread of engineering bacteria throughout the soil.
2. Excellent host for engineered bacteria
1) Earthworm intestines. The intestinal mucus of earthworms is rich in soluble organic carbon, has a high humidity, and a stable pH value. It is generally considered to be the most suitable habitat for microorganisms. Soil microorganisms enter the intestines of earthworms with the soil. The abundant usable organic carbon, suitable temperature and pH value in the intestine of earthworms can provide good living conditions for microorganisms, promote their growth, and eliminate some dominant bacteria in the intestine, such as Bacillus subtilis and Bacillus subtilis.
2)Earthworm Feces. The digestive tracts of earthworms are small incubators for the growth and reproduction of many bacteria and actinomycetes. The rich nutrients and large surface area in earthworm feces provide good environmental conditions for the stable survival and mass reproduction of microorganisms. In addition, earthworms ingest a large amount of organic matter in the soil during their lifetime. After preliminary decomposition, under the action of phenol oxidase secreted by intestinal cells and microbial decomposition products, they will further condense to form humus. Therefore, earthworm feces also contain a lot of humus. The inherent colloidal properties of humus can improve soil structure, facilitate soil aeration and water retention, and improve soil buffering capacity. At the same time, humic acid itself is a strong adsorbent, which can adsorb soil plastic particles.
Six. Project design introduction
Plastic waste pollution has seriously threatened the environment and creatures. Here, we designed a burying yard with concentrated PET , using the modified Bacillus subtilis carried by earthworms to initially degrade PET, accelerating the degradation of PET plastics in it. At the same time, the activities of earthworms are used to improve the distribution of engineering bacteria and enzymes, solving the difficulty of frequent mixing in the dump site.
The modified engineering bacteria release PETase and MHETase to hydrolyze PET into ethylene glycol and terephthalic acid, finally metabolizing it into carbon dioxide and water.
What's more, we also inserted GFP gene into the plasmid to further track the distribution of engineering bacteria in the soil. And by detecting the fluorescence in the body or feces of the earthworms, we can know whether the engineered bacteria have been swallowed by the earthworms. The expected result is that the engineered bacteria can be carried elsewhere through the activities of earthworms, which will promote the distribution of engineered bacteria in the soil.
. (Plastic Pollution Knowledge Handbook and Action Guide)—Earth Day
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. Cao Yonglin, Yan Yurong, Xing Yujing, Wu Songping, Guo Xitao, Ma Yizhong, Ma Junbin. Progress in the recycling of waste PET[J] Aging and application of synthetic materials:2021,50(2):128-131