Description

1 Pollution of traditional undegradable plastics

1.1 Application Status of Traditional Plastics

The current application status of traditional undegradable plastics mainly includes the following aspects:

1. Packaging industry, e. g. plastic bags, films, bottles and other products
2. Agricultural industry, e. g. agricultural mulch and greenhouse films
3. Construction industry, e. g. insulation materials, cement additives, reinforced concrete additives
4. Automotive industry, e. g. automotive interiors and body components

1.2 Current situation of pollution caused by the application of plastics

Firstly, excessive land occupation leads to serious waste of land resources and soil pollution issues. Landfills are the primary way at present to deal with plastic pollution, but plastic waste takes hundreds of years to degrade, accumulating and harming crop yields

Second, incineration will emit a large number of Carcinogen, which will be mixed into the air with the wind and eventually poison human health. Burning plastic waste releases carcinogens like dioxins, posing a serious health risk as they can enter the human body

Thirdly, polluting water bodies and endangering the ecological environment of rivers and seas. Plastic waste in the ocean transforms into fragments that are eaten by fish and eventually return to humans

2 Advantages of biodegradable bioplastics

2.1 About biodegradable plastics

Biodegradable plastics are plastics made from natural renewable materials whose molecular structures can be decomposed by microorganisms under natural conditions, thus reducing pollution to the environment. These plastics can be produced from renewable resources such as starch, wood or cellulose, which can be taken from agricultural waste and food processing waste, with less impact on environment during production and waste disposal process than conventional plastics.

2.2 Advantages of biodegradable plastics

1. Environmentally friendly: Biodegradable plastics will not pollute the environment during use, because it can be decomposed by nature through biodegradation
2. Renewable resources: Biodegradable plastics are made of natural raw materials such as starch and cellulose, which can be obtained through crop cultivation and forest management, etc
3. Wide range of applications: Biodegradable plastics can be used to be materials in agriculture and packaging, etc., with a wide range of applications
4. Promote sustainable development: Degradable bioplastics research and use can reduce plastic dependence, waste and emissions, promoting sustainability and protection

3 Starch and starch-based biodegradable plastics

3.1 About starch

Starch is a soluble white powdery solid and is the major source of energy stored in plants. It is produced by photosynthesizing plants and serves as a storage compound for glucose. Many crops have high starch yields, including wheat, rice, corn, potato, sweet potato and cassava

Starch is composed of two substances: amylose, which is a linear polysaccharide, and amylopectin, which is a branched polysaccharide. Here are the differences between amylose and amylopectin (Geeksforgeeks, 2023):

Fig. 1 Amylose (Left) and Amylopectin (Right)

Table 1 Difference Between Amylose and Amylopectin

Characteristics	Amylose		Amylopectin
Structure	Linear		Branched
Chain Length	Long		Shorter with branches
Proportion in Starch	20-30%		70-80%
Digestibility	Slower		Faster
Solubility in Water	Lower		Higher
Dissolved in Hot water	Forms a gel		Does not form a gel
Enzyme Activity	More resistant		More easily degraded
Molecular Weight	10^6-10^7 g/mol		10^8-10^9 g/mol

3.2 About Starch-based biodegradable plastics

Starch-based biodegradable plastics are generally produced by blending modified starch with various compounds or monomers. Under specific environmental conditions, processes such as extrusion, injection molding, compression molding, and intensive mixing are used to transform the chemical crystalline structure of the starch into an amorphous state, foaming it into a thermoplastic plastic(Qi YJ et al., 2020).

3.3 Current processing methods and shortcomings of starch-based biodegradable plastics

Based on the differences in materials, starch-based biodegradable plastics are mainly divided into two categories (Anjum F et al., 2018; Zhang Y et Yan S,2016):

(1) Full-Starch-based biodegradable plastics: Full-starch bioplastics are made solely from starch and other biodegradable additives. In the production process, starch is modified using physical or chemical methods to obtain thermoplastic starch (TPS) first, and then plasticizers and other additives are incorporated in to improve its mechanical properties. produce thermoplastic starch by changing the processing method, adding different types and contents of plasticizers, and so on.

Full-starch bioplastics are renewable, biodegradable, and low toxic，but their poor mechanical properties, such as low toughness, elongation, and impact resistance, limit the range of products that can be made from full-starch bioplastics

(2) Blend/composite starch-based biodegradable plastics: Blend/composite starch-based bioplastics are made by blending starch with other biodegradable polymers, such as polyvinyl alcohol(PVA), polylactic acid (PLA), polycaprolactone (PCL), polyhydroxyalkanoate (PHA), and polybutylene succinate (PBS) (Encalada K et al., 2018). Compared to full-starch bioplastics, biodegradable polymers are added firstly, and then plasticizers, compatibilizers, and other additives are incorporated to improve the mechanical and thermal properties

Compared to full-starch-based bioplastics, blend/composite starch-based bioplastics have improved mechanical properties, such as increased toughness, elongation, and impact resistance. However, they may release harmful chemicals during biodegradation and their biodegradation rate may be slowed down depending on the type and ratio of the component polymers, especially when blended with synthetic polymers.

3.3 Advantages of starch-based biodegradable plastics

The outstanding advantages of starch-based biodegradable plastics are low production costs, biodegradable properties, minimal impact on the environment. Compared to other biodegradable plastics, starch-based bioplastics are superior due to several reasons

Firstly, they are made from starch from renewable resources, such as corn, potatoes, and cassava. This means that they reduce the dependency on fossil fuels

Secondly, they have better mechanical properties and can be processed using existing equipment (Lu DR et al., 2009) which reduces production costs

Furthermore, these plastics have a shorter degradation time (Lu DR et al., 2009) and degradation products mainly consisting of CO2 and H2O. They can be considered a healthy and green environmentally friendly material (Xu JH et al., 2023, Emadian SM et al., 2017)

Overall, starch-based bioplastics present a more sustainable and environmentally friendly solution for plastic products

3.5 Influence of raw starch components on starch-based bioplastics

According to [Utami MR et al., 2014; Kamsiati et.al., 2017), amylose and amylopectin produce bioplastics with different characteristics and material compactness was influenced by amylose, while amylopectin affects the stability of the bioplastics.

High amylose tends to form crystals that have more substantial mechanical properties than amylopectin, which is amorphous. Films made from pure amylose have better mechanical properties than films using pure amylopectin as raw material but the crystalline nature of amylose causes starch molecules to become brittle when used as raw material for making bioplastics. (Gabriel AA, et al., 2021). The content of amylose and amylopectin greatly influenced the tensile strength and elongation of biodegradable plastics, the greater the amylopectin content, the higher the value of the tensile strength of biodegradable plastic elongation. (Luqi M et Rahman ED, 2021) .The addition of plasticizers and processing conditions at high humidity increases the crystallinity of bioplastics with high amylopectin starch raw materials and improve their mechanical properties. The addition of plasticizers did not affect the crystallinity of high amylose starch (Kamsiati et.al., 2017)

Thus, it is necessary to separate amylose and amylopectin to obtain bioplastics with better results. We can also obtain starch-based biodegradable plastics with different performance by adjusting the proportion of amylopectin and amylose in the raw materials

4 Biosynthesis of starch

4.1 Partway of starch biosynthesis in plant

Starch biosynthesis in plants is a sophisticated system composed of multiple subunits or variations of five enzyme classes, including ADP-glucose pyrophosphorylase (ADPG), starch synthases (SS), starch branching enzymes (SBEs), starch debranching enzymes (DBEs), and granule-bound starch synthase (GBSS), that are located in the chloroplast or amyloplast (Tetlow IJ, 2006; Tian ZX, et al. 2009). The synthesis of amylose is primarily regulated by GBSS, while the synthesis of amylopectin is not only regulated by SBEs but also influenced by SS and DBEs including isoamylase (ISA) and pullulanase (PUL) (James MG et al., 2003) (Figure 1).

Each enzyme has a distinct function but likely works in conjunction with others as part of a larger network. For instance, the ISA (Isoamylase-type DBE) gene mutation in rice, known as the sugary mutant, not only affects the expression levels of multiple starch synthesis-related genes (SSRGs), but also impacts other components involved. Additionally, genes controlling amylose synthesis, which are integral to this complex biosynthesis pathway, also influence amylopectin formation, whereby amylopectin can subsequently give rise to amylose (Tian ZX et al., 2009)

Fig.2 A simplified starch synthesis system in cereal (Tian ZX et al., 2009)

Eighteen genes are involved in or play distinct roles in different steps of starch synthesis. AGP, ADP-glucose pyrophosphorylase; AGPlar, AGP large subunit; AGPiso, AGP large subunit isoform; AGPsma, AGP small subunit; GBSS, granule-bound starch synthase; SS, soluble starch synthase; SBE, starch branching enzyme; ISA, isoamylase; PUL, pullulanase; ISA and PUL belong to starch debranching enzyme (DBE)

4.2 CRISPR/Cas technology in starch biosynthesis

CRISPR (an acronym for clustered regularly interspaced short palindromic repeats) is a family of DNA sequences found in the genomes of prokaryotic organisms such as bacteria and archaea (Horvath P et Barrangou R., 2010). These sequences are derived from DNA fragments of bacteriophages that had previously infected the prokaryote. They are used to detect and destroy DNA from similar bacteriophages during subsequent infections

Cas9, or CRISPR-associated protein 9, is an enzyme that uses CRISPR sequences as a guide to recognize and open up specific strands of DNA that are complementary to the CRISPR sequence. Cas9 enzymes together with CRISPR sequences form the basis of a technology known as CRISPR-Cas9 that can be used to edit genes within the organisms (Wikipedia, 2023). In the August 2013 issue of Nature Biotechnology three short reports described the first applications of the Cas9/sgRNA system to plant genome engineering (Belhaj, K, et al., 2013).

Engineered CRISPR systems consist of a guide RNA (gRNA) and a CRISPR-associated endonuclease (Cas protein). The gRNA has a scaffold sequence for Cas binding and a user-defined spacer that determines the target to be modified. The gRNA's target sequence can be altered to change the Cas protein's genomic target. Originally used to knock out genes, CRISPR has been modified to activate/repress target genes, purify DNA regions, image DNA in live cells, and edit DNA/RNA precisely. Additionally, generating gRNAs is easy, making CRISPR highly scalable for genome editing and ideal for genome-wide screens. (Addgene, 2023)

Fig.3 How CRISPR works (Ball P, 2016)

Based on the Partway of starch biosynthesis in plant, the proportion of amylopectin or amylose can be increase through CRISPR/Cas technology.An attempt to knock out the sweet potato IbGBSSI gene and IbSBEII gene in 2019 has confirmed the feasibility of this method to a certain extent (Wang HX et al., 2019) in 2022, the CRISPR/Cas9 system was used to knock out the gene GBSS of a potato (Solanum tuberosum) variety, thus producing amylose-free potatoes (Toinga-Villafuerte S et al., 2022). Moreover, a newly improved breeding cycle is used on genetically modified cassava (Manihot esculenta) to fully capitalize on genome editing technology for agriculture (Bull SE et al., 2018)

5 Our project

Due to the wide availability and low cost of plant starch in nature, starch-based bioplastics are particularly important among various biodegradable plastics. However, since plant starch contains both amylopectin and amylose, their varying content ratios have different effects on the performance of starch-based bioplastics. It is difficult to obtain starch-based bioplastics with specific properties using existing plant starches as raw materials. This situation can be remedied by adjusting the ratio of the two starches in the raw starch, which requires the use of high-purity amylopectin or amylose

Sweet potato has the starch content accounting for 36% to 80% of its dry weight in tubers, of which amylopectin accounts for more than 80% of the total starch(Jing Yanping et al., 2013). Additionally, sweet potato is a crop that has wide adaptability to various climates, requires little water and fertilizer, has a short growth period, and produces high yields. Hence, sweet potato is a suitable plant to biosynthesize high-purity amylopectin or amylose using methods of gene editing

In sweet potato, the regulatory protein for amylose synthesis is sweet potato granule-bound starch synthase GBSSI but the synthesis of amylopectin is relatively more complex as it is not only regulated by IbSBEs but also influenced by SSs and DBEs (Lyu RQ et al., 2021). Therefore, we can use CRISPR/cas9 technology to knockout the IbGBSSI gene so that sweet potatoes no longer synthesize amylose, and to knock out or silence IbSBEs and other related genes respectively to stop synthesis of amylopectin. In this way, we can obtain high-purity amylopectin and amylose. This allows us to save a series of complex purification processes and easily obtain pure amylopectin and pure amylose, which can better be used to adjust the content ratio of these two components in raw starch and produce different performance starch-based biodegradable plastic products

Fig.4 What about our project

6 Reference:

Addgene. (2023) CRISPR Guide. https://www.addgene.org/guides/crispr/#PAM

Anjum F, Rehan M et Ullah H. (2018) Starch-based biodegradable polymers: A review. Polymers, 10(11): 1276

Ball P. (2016) CRISPR: Implications for materials science. MRS Bulletin, 41: 832-834.

Belhaj K, Chaparro-Garcia A, Kamoun S, et Nekrasov V. (2013) Plant genome editing made easy: targeted mutagenesis in model and crop plants using the CRISPR/Cas system. Plant methods, 9 (1): 39

Bull SE, Seung D, Chanez C et al. (2018) Accelerated ex situ breeding of GBSS- and PTST1-edited cassava for modified starch. Science advances, 4 (9)

Emadian SM,  Onay TT et Demirel B. (2017) Biodegradation of bioplastics in natural environments. Waste Management, 59: 526-536

Encalada K, Aldás MB, Proaño E et Valle V. (2018) An overview of starch-based biopolymers and their biodegradability. Ciencia e Ingeniería, 39 (3): 245-263

Gabriel AA, Solikhah AF et Rahmawati AY. (2021) Tensile strength and elongation testing for starch-based bioplastics using melt intercalation method: A review. Journal of physics: Conference Series, 1858: 012028

Geeksforgeeks (2023) Difference Between Amylose And Amylopectin. https://www.geeksforgeeks.org/amylose-vs-amylopectin

Horvath P et Barrangou R. (2010) CRISPR/Cas, the immune system of bacteria and archaea. Science. 327 (5962): 167–170

James MG， Denyer K， Myers AM. (2003) Starch synthesis in the cereal endosperm. Curr. Opin. Plant Biol., 6: 215–222.

Jing YP, Li DL, Liu DT et al. (2013) Anatomical structure of the tuberous root growth and its amyloplast development in sweet potato. Acta Bot. Boreal.-Occident. Sin., 33(12)：2415-24222415-2422

Kamsiati E, Herawati H et dan Purwani EY. (2017). Potensi pengembangan platik iodegradable berbasis pati sagu and ubi kayu di Indonesia. Jurnal Litbang Pertanian, 36 (2): 67-76

Lyu RQ, Ahmed S, Fan WJ. (2021) Engineering properties of sweet potato starch for industrial applications by biotechnological techniques including genome editing. Int. J. Mol. Sci., 22(17): 9533.

Lu DR, Xiao CM et Xu SJ. (2009). Starch-based completely biodegradable polymer materials. Express polymer letters, 3 (6): 366–375

Luqi M et Rahman ED. (2021) The effect of amylopectin on plastic quality. ejurnal.bunghatta.ac.id,18 (4): Summary Executive Jurusan Teknik Kimia Wisudawan ke 76

Qi YJ, Wu X, Zhou R, Liu AX. (2021) Application and Development Trend of Starch-based Degradable Plastics. Chemical Enterprise Management, (29): 27-28.

Tian Z, Qian Q, Liu Q et al. (2009) Allelic diversities in rice starch biosynthesis lead to a diverse array of rice eating and cooking qualities. PNAS, 106 (51): 21760-21765

Tetlow IJ. (2006) Understanding storage starch biosynthesis in plants: A means to quality improvement. Can. J. Bot., 84: 1167–1185

Toinga-Villafuerte S, Vales MI, Awika JM et Rathore KS. (2022) CRISPR/Cas9-Mediated Mutagenesis of the Granule-Bound Starch Synthase Gene in the Potato Variety Yukon Gold to Obtain Amylose-Free Starch in Tubers, Int. J. Mol. Sci. 23( 9).

Utami MR, Latifah L et Widiarti N. (2014). Sintesis plastik biodegradable dari kulit pisang dengan penambahan kitosan dan plasticizer gliserol. Indonesian journal of chemical science, vol. 3 (2): 163-167

Wang HX, Wu YL, Zhang YD et al. (2019) CRISPR/Cas9-based mutagenesis of starch biosynthetic genes in sweet potato (Ipomoea Batatas) for the improvement of starch quality. Int. J. Mol. Sci., 20 (19): 4702.

Wikipedia. (2023) CRISPR. https://en.wikipedia.org/wiki/CRISPR

Wikipedia. (2023) CRISPR. https://en.wikipedia.org/wiki/CRISPR

Xu JH. (2023) Study on the Manufacture and Processing Technology of Degradable Starch Plastics. Development & Innovation of Machinery & Electrical Products, 32 (6): 68-70.

Zhang Y et Yan S. (2016) Review on research and development of starch-based biodegradable composites. Carbohydrate polymers, 148: 271-280