Sugarcane waste-based biovinyl and method for manufacturing same

Abstract

Disclosed is sugarcane waste-based biovinyl according to an embodiment of the present invention. The sugar cane waste-based biovinyl may comprise distilled water, cornstarch, agar, sugar cane bark (SCB), and a mixed bacterial solution in which two or more types of bacteria are mixed. When the sugar cane waste-based biovinyl is provided to soil, beneficial bacteria are supplied to damaged soil, thereby improving and strengthening the structure of the soil. Accordingly, it is possible to reduce the negative impact of sugar cane agriculture on the environment and improve the growth environment of crops.

Description

Biovinyl based on sugarcane waste and method for producing the same

[0001] The present invention relates to the development of environmentally friendly materials, and more specifically, to a method for producing biovinyl having soil strengthening and pollution prevention functions by utilizing sugarcane waste.

[0002]

[0003] Sugarcane agriculture is a vital global industry, serving as a major source of income, particularly in tropical and subtropical regions such as Latin America, Africa, and Asia. However, the environmental impacts associated with the expansion of sugarcane agriculture cannot be ignored, and the disposal of sugarcane waste, in particular, poses significant environmental problems.

[0004] Burning sugarcane waste is a major cause of increased soil porosity and infiltration, increasing vulnerability to flooding. Furthermore, deforestation for sugarcane plantation expansion increases soil porosity, reduces water retention capacity, and alters soil physical, chemical, and biological properties, exacerbating the region's vulnerability to natural disasters.

[0005] For example, countries like Honduras experience dozens of floods, storms, and droughts annually, and these natural disasters are often linked to widespread deforestation. Sugarcane is the region's primary industrial crop, and deforestation for sugarcane plantations further increases the region's ecological vulnerability.

[0006] In particular, global sugarcane production increased by approximately 345% from 1961 to 2020, and this growth trend is expected to continue, further exacerbating the environmental problems caused by sugarcane cultivation.

[0007] Moreover, the environmental impacts of sugarcane farming extend beyond increased soil porosity and deforestation. Improper disposal of sugarcane waste can lead to soil acidification and water eutrophication, which can severely damage ecosystems. In other words, improper disposal of sugarcane waste leads to soil acidification and water eutrophication. The damage to the ecosystem can be mitigated by appropriately recycling sugarcane waste.

[0008] However, conventional recycling methods based on sugarcane waste can be somewhat inefficient. For example, conventional bioethanol production using sugarcane waste can be inefficient due to high production costs, increased emissions of hazardous air pollutants, and uncertainty about the effectiveness of reducing carbon dioxide emissions.

[0009] Therefore, there may be an increasing need for efficient recycling of sugarcane waste, along with solutions to the problem of soil degradation caused by sugarcane farming.

[0010] [Prior Art Document] Republic of Korea Patent Publication No. 10-0863158

[0011]

[0012] The present invention aims to solve the problem of soil devastation caused by sugarcane farming and to provide environmentally friendly biovinyl by effectively recycling sugarcane waste.

[0013] The problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.

[0014]

[0015] In order to solve the above-described problem, a sugarcane waste-based biovinyl according to one embodiment of the present invention is disclosed. The sugarcane waste-based biovinyl may include distilled water (DW), cornstarch, agar, sugarcane bark (SCB), and a mixed bacterial solution containing two or more types of bacteria.

[0016] In an alternative embodiment, the corn starch may be provided in an amount of 0.5 parts by weight per 100 parts by weight of the distilled water, the agar may be provided in an amount of 1 to 1.5 parts by weight per 100 parts by weight of the distilled water, and the sugarcane husk may be provided in an amount of 1 part by weight per 100 parts by weight of the distilled water.

[0017] In an alternative embodiment, the mixed bacterial solution may comprise one species of bacteria selected from the group of Calcite Producing Bacteria and another species of bacteria selected from the group of Lignin Decomposing Bacteria.

[0018] In an alternative embodiment, the mixed bacterial solution may comprise Glutamicibacter halophytocola and Priestia megaterium.

[0019] In an alternative embodiment, the mixed bacterial solution may be a solution in which two or more bacteria are diluted to 0.001 ABS.

[0020] In an alternative embodiment, the biovinyl may further comprise 1 part by weight of molasses per 100 parts by weight of the distilled water.

[0021] A method for producing bio-vinyl based on sugarcane waste according to another embodiment of the present invention is disclosed. The method may include the steps of adding corn starch, agar, and sugarcane husk to distilled water to produce a mixture, heating the mixture, heat-treating the mixture, adding a mixed bacterial solution containing two or more types of bacteria to the mixture, and coagulating the mixture to which the mixed bacterial solution has been added to produce bio-vinyl based on sugarcane waste.

[0022] In an alternative embodiment, the step of generating the mixture may further comprise the step of adding molasses.

[0023] In an alternative embodiment, the mixed bacterial solution may comprise one species of bacteria selected from the group of calcite-forming bacteria and another species of bacteria selected from the group of lignin-degrading bacteria.

[0024] In an alternative embodiment, the mixed bacterial solution may comprise Glutamibacter halopytocola and Pristeria megaterium.

[0025]

[0026] The bioplastic made from sugarcane waste produced by the present invention can improve and strengthen soil structure by supplying beneficial bacteria to damaged soil. This reduces the negative environmental impact of sugarcane farming and improves the growing environment for crops.

[0027] Furthermore, recycling waste generated from the sugarcane industry can support a circular economy and provide additional environmental benefits, such as reducing greenhouse gas emissions. This can address waste issues that arise during sugarcane cultivation and processing, and reduce environmental pollution.

[0028]

[0029] Various aspects are now described with reference to the drawings, wherein like reference numerals are used to refer to similar components. In the following examples, for purposes of explanation, numerous specific details are set forth to provide a comprehensive understanding of one or more aspects. However, it will be apparent that such aspects may be practiced without these specific details.

[0030] Figure 1 illustrates an exemplary flow chart for a manufacturing method for manufacturing biovinyl based on sugarcane waste of the present invention.

[0031] Figure 2 is an example diagram to explain soil quality according to soil pores.

[0032] Figure 3 is a drawing showing a process for preparing sugarcane waste related to one embodiment of the present invention.

[0033] Figure 4 is a drawing for explaining a soil collection process related to one embodiment of the present invention.

[0034] FIG. 5 is a drawing for explaining the process of generating UA and UB badges related to one embodiment of the present invention.

[0035] FIG. 6 is a drawing for explaining a process of culturing bacteria on a UA medium related to one embodiment of the present invention.

[0036] FIG. 7 is a drawing related to one embodiment of the present invention. FIG. 7 is a drawing related to the observation results of farm soil bacteria cultured in NA and UA media.

[0037] FIG. 8 is a drawing related to the observation results of farm soil bacterial colonies cultured in UA medium related to one embodiment of the present invention.

[0038] FIG. 9 is a drawing for explaining preparation and procedures for a moisture retention capacity test related to one embodiment of the present invention.

[0039] FIG. 10 is a drawing for explaining a process of inoculating each bacterial solution into three types of agar media using a streaking method according to one embodiment of the present invention.

[0040] Figure 11 is a drawing of a medium observed through a microscope to explain the lignin decomposition ability related to one embodiment of the present invention.

[0041] Figures 12 to 14 are exemplary diagrams showing the germination rates of soil samples related to one embodiment of the present invention.

[0042] FIG. 15 is a drawing showing a process for preparing a bacterial dispersion rate test of SCB biovinyl related to one embodiment of the present invention.

[0043] FIG. 16 is a drawing for explaining a bacterial mobility test process related to one embodiment of the present invention.

[0044] FIG. 17 is a drawing of a microscope image of SCB biovinyl placed on soil after 24 hours in accordance with one embodiment of the present invention.

[0045] Figure 18 is an exemplary diagram illustrating the reproduction rate of each bacterium related to one embodiment of the present invention.

[0046] FIG. 19 is an exemplary diagram illustrating the reproduction rate according to a change in the composition ratio of calcite-forming bacteria compared to lignin-decomposing bacteria related to one embodiment of the present invention.

[0047] Figure 20 shows experimental results showing improvement in soil structure through SCB biovinyl related to one embodiment of the present invention.

[0048]

[0049] According to one embodiment of the present invention, sugarcane waste-based biovinyl (SugarCane Bark Biovinyl) can improve and strengthen soil structure by supplying beneficial bacteria to damaged soil. The sugarcane waste-based biovinyl of the present invention can be manufactured in the form of a degradable matrix and utilized by being inserted into soil. When inserted into soil, the sugarcane waste-based biovinyl can release beneficial bacteria to improve soil structure.

[0050] In an embodiment, sugarcane waste-based bioplastics are incorporated with a mixed bacterial solution comprising calcite-forming bacteria and lignin-degrading bacteria to enhance the physical structure of the soil and promote the decomposition of organic matter, thereby improving soil nutrition.

[0051] Sugarcane waste-based bioplastics can naturally decompose, releasing selected bacteria into the soil, which can provide a variety of benefits, including reducing soil erosion, increasing water retention capacity, and enhancing soil microbial diversity.

[0052] Sugarcane waste-based bioplastics can be manufactured using sugarcane waste (i.e., sugarcane husks). Sugarcane waste-based bioplastics are biodegradable and, when incorporated into soil, release beneficial bacteria, improving soil health and preventing soil degradation. By utilizing waste materials like sugarcane husks, they promote the recycling of agricultural byproducts, reduce waste disposal costs, and contribute to environmental protection. The use of sugarcane waste-based bioplastics offers a sustainable and environmentally friendly solution to soil degradation caused by sugarcane farming. Furthermore, the recycling of sugarcane waste provides economic value.

[0053] The application of biovinyl produced by the present invention is particularly useful in solving soil degradation problems not only in sugarcane-growing areas but also in other agricultural areas, and provides a new way for sustainable agricultural practices and environmental protection.

[0054]

[0055] Hereinafter, with reference to FIGS. 1 to 20, the sugarcane waste-based biovinyl, its manufacturing method, and the effects generated thereby will be described in more detail.

[0056] FIG. 1 is an exemplary flowchart illustrating a manufacturing method for producing sugarcane waste-based biovinyl of the present invention. FIG. 2 is an exemplary diagram illustrating soil quality according to soil pores. FIG. 3 is a diagram illustrating a process for preparing sugarcane waste according to an embodiment of the present invention. FIG. 4 is a diagram illustrating a process for collecting soil according to an embodiment of the present invention. FIG. 5 is a diagram illustrating a process for producing UA and UB media according to an embodiment of the present invention. FIG. 6 is a diagram illustrating a process for culturing bacteria in a UA medium according to an embodiment of the present invention. FIG. 7 is a diagram relating to the observation results of farm soil bacteria cultured in NA and UA media according to an embodiment of the present invention. FIG. 8 is a diagram relating to the observation results of farm soil bacterial colonies cultured in a UA medium according to an embodiment of the present invention. FIG. 9 is a diagram relating to the preparation and procedure for a water retention capacity test according to an embodiment of the present invention. FIG. 10 is a diagram illustrating a process of inoculating each bacterial solution into three types of agar media by a streaking method according to an embodiment of the present invention. FIG. 11 is a diagram illustrating a medium observed through a microscope to illustrate lignin decomposition ability according to an embodiment of the present invention. FIGS. 12 to 14 are exemplary diagrams illustrating germination rates of soil samples according to an embodiment of the present invention. FIG. 15 is a diagram illustrating a process for preparing a bacterial dispersion rate test of SCB biovinyl according to an embodiment of the present invention. FIG. 16 is a diagram illustrating a process for testing bacterial mobility according to an embodiment of the present invention. FIG. 17 is a diagram illustrating a microscope image of SCB biovinyl placed on soil according to an embodiment of the present invention after 24 hours. FIG. 18 is an exemplary diagram illustrating the reproduction rate of each bacterium according to an embodiment of the present invention.Figure 19 is an exemplary diagram illustrating the reproduction rate according to changes in the composition ratio of calcite-forming bacteria compared to lignin-decomposing bacteria in accordance with one embodiment of the present invention. Figure 20 illustrates experimental results demonstrating that soil structure is improved through SCB biovinyl in accordance with one embodiment of the present invention.

[0057]

[0058] Figure 1 illustrates an exemplary flowchart of a manufacturing method for producing sugarcane waste-based biovinyl (hereinafter referred to as "SCB biovinyl") of the present invention. The steps illustrated in Figure 1 may be rearranged as needed, and at least one step may be omitted or added. In other words, the following sequences are merely exemplary embodiments of the present invention, and the scope of the present invention is not limited thereto.

[0059] According to one embodiment of the present invention, a method for manufacturing SCB biovinyl may include a step (S100) of producing a mixture by adding corn starch, agar, and sugarcane husk (SCB) to distilled water.

[0060] In one embodiment, distilled water may be used as a solvent to ensure that the components of the mixture are uniformly dispersed.

[0061] Cornstarch provides the structural backbone of biovinyl and, as it decomposes, contributes beneficial nutrients to the soil, promoting biological activity.

[0062] Agar forms a gel-like matrix, enhancing the physical stability of biovinyl and maintaining the product's shape. This helps biovinyl maintain its shape and function for a certain period of time when applied to the environment.

[0063] Sugarcane husks, a renewable resource, provide the organic components necessary for bioplastics. Sugarcane husks can increase the overall strength of the final bioplastic. The utilization of sugarcane husks effectively recycles agricultural waste generated from sugarcane cultivation, thereby promoting environmental sustainability.

[0064] According to an embodiment, sugarcane husk can be prepared in powder form through the following process for effective use.

[0065] First, the sugarcane husk is removed and then dried in a fume hood for seven days to ensure complete drying. This step removes moisture from the sugarcane husk, making the grinding process more efficient.

[0066] Completely dried sugarcane husks (SCB) can be ground into a fine powder in a food processor. This process refines the physical structure of the sugarcane husks, improving their mixing efficiency during bioplastic production.

[0067] Sieving the crushed SCB through a sieve yields fine SCB powder. This fine powder is stored in a container and used later in the biovinyl manufacturing process. The prepared SCB powder is sterilized in an autoclave at 121 degrees Celsius and 1.5 atm for 15 minutes. Sterilization may be used to prevent microbial contamination and ensure product safety before the sugarcane husk powder is used in the biovinyl manufacturing process.

[0068] According to an embodiment, corn starch may be provided in an amount of 0.5 parts by weight per 100 parts by weight of distilled water, agar may be provided in an amount of 1 to 1.5 parts by weight per 100 parts by weight of distilled water, and sugarcane hull may be provided in an amount of 1 part by weight per 100 parts by weight of the distilled water. For example, when 50 mL of distilled water is provided, corn starch may be provided in an amount of 0.25 g, agar may be provided in an amount of 0.5 to 0.75 g, and sugarcane hull may be provided in an amount of 0.5 g.

[0069] In one embodiment, the step of generating the mixture may further comprise the step of adding molasses.

[0070] In this example, the addition of molasses can have several positive effects on the performance of the final product, SCB biovinyl. The addition of molasses acts to increase the growth rate and dispersion rate of bacteria, particularly calcite-forming bacteria, within the SCB biovinyl, providing two key benefits:

[0071] First, by increasing bacterial growth rates, SCB biovinyl, when applied to soil, enhances bacterial activity for soil enrichment and improvement. Molasses provides additional nutrients to bacteria as the SCB biovinyl decomposes, creating an environment where more bacteria can thrive and thrive.

[0072] Second, the rapid bacterial dispersal rate resulting from the addition of molasses can help the bioplastic work more evenly within the soil. This allows the soil improvement and strengthening effects to be more evenly distributed throughout the soil to which the SCB bioplastic has been applied, thereby improving overall soil health and structure.

[0073] In this way, SCB biovinyl produced by adding molasses can contribute to promoting and strengthening soil health through increased bacterial activity and efficient dispersion.

[0074] Additionally, in the embodiment, molasses increases the viscosity of the biovinyl mixture and enhances the flexibility and strength of the final product. Furthermore, molasses, along with sugarcane husks, acts as a natural binder during the biovinyl manufacturing process, ensuring the consistency of the mixture. Preferably, 1 part by weight of molasses is used per 100 parts by weight of distilled water. For example, for 50 ml of distilled water, 500 μL of molasses is preferably used. This ratio is determined to provide the mixture with sufficient viscosity and flexibility necessary to manufacture biovinyl with optimal properties.

[0075] According to one embodiment of the present invention, the method for manufacturing SCB biovinyl may include a step (S200) of heating a mixture.

[0076] For example, the mixture can be heated to a temperature between 80°C and 150°C on a hot plate until boiling, and then cooled for approximately 10 minutes to lower the temperature. The heating of the mixture ensures that the cornstarch, agar, molasses, and sugarcane husk powder are effectively mixed and dissolved, forming a uniform and stable mixture. The mixture formed through this process serves as the basis for biovinyl. Heating and cooling at appropriate temperatures can optimize the physical properties and biodegradability of the final product.

[0077] According to one embodiment of the present invention, the method for manufacturing SCB biovinyl may include a step (S300) of adding a mixed bacterial solution containing two or more types of bacteria to the mixture after heat-treating the mixture.

[0078] According to an embodiment, the mixed bacterial solution may be a solution in which two or more bacteria are diluted with 0.001 ABS.

[0079] The mixed bacterial solution may include one species of bacteria selected from the group of calcite-forming bacteria and another species of bacteria selected from the group of lignin-degrading bacteria. The introduction of the selected bacteria activates the biovinyl in the soil and allows it to exert beneficial effects, which may contribute to improving the soil structure and nutritional status.

[0080] In an embodiment, the mixed bacterial solution may be a solution in which the bacteria are diluted to 0.001 ABS (absorbance or optical density). This represents a very low bacterial concentration, which may be a condition for optimizing bacterial activity in a particular environment.

[0081] In one embodiment, for soil improvement and biovinyl activation, it is preferable to extract and combine selected bacteria from each of the calcite-forming bacteria group and the lignin-decomposing bacteria group. The selected bacteria can interact with each other to promote calcite formation and decompose lignin in the soil, thereby improving the physical and chemical properties of the soil.

[0082] In other words, the combination of bacteria described above may be designed to optimize the function of biovinyl. Calcite-forming bacteria strengthen soil structure when biovinyl is applied to soil, and lignin-decomposing bacteria enhance the nutritional status of the soil by promoting the decomposition of organic matter such as sugarcane husks.

[0083] In a more specific embodiment, the mixed bacterial solution is preferably provided comprising Glutamibacter halopytocola, which promotes calcite formation, and Pristeria megaterium, which promotes lignin decomposition.

[0084] Glutamibacter halopytocola and Pristeria megaterium may be the most calcite-forming and lignin-decomposing bacteria in their respective groups. Glutamibacter halopytocola contributes to soil strengthening and structural stability, while Pristeria megaterium accelerates the decomposition of organic residues, such as sugarcane husks, thereby providing valuable nutrients to the soil.

[0085] In the present invention, the selected calcite-forming bacterium, Glutamibacter halopytocola, is a Gram-positive, aerobic, non-motile bacterial species belonging to the genus Glutamicibacter, the family Microbacteriaceae, and the class Actinobacteria. Glutamibacter halopytocola is found in various environmental sources and can produce plant growth-promoting bioactive compounds, possess chitinolytic activity, and maintain antifungal efficacy. The optimal temperature for Glutamibacter halopytocola is 25-28°C, and the optimal pH is 7-7.5.

[0086] In the present invention, the selected lignin-degrading bacterium, Pristeria megaterium, formerly known as Bacillus megaterium, is a Gram-positive bacterium. Pristeria megaterium is primarily known for its beneficial effects on plant growth. Pristeria megaterium can act as a phosphate biofertilizer and promote nitrogen fixation. Pristeria megaterium can enhance plant growth and drought stress tolerance. Additionally, Pristeria megaterium can act as a biological pesticide due to its antipathogenic mechanism.

[0087] According to one embodiment of the present invention, the method for producing SCB biovinyl may include a step (S400) of coagulating a mixture to which a mixed bacterial solution is added to produce sugarcane waste-based biovinyl.

[0088] This step involves the interaction of organic and inorganic substances formed through bacterial activity to form a robust biovinyl material. This process can be carried out under specific conditions, with optimized environmental conditions such as temperature, pH, and humidity.

[0089] For example, the SCB biovinyl of the present invention can be produced by pouring a mixture with a mixed bacterial solution into a 150 mm diameter petri dish and solidifying it in a fume hood.

[0090] SCB biovinyl, produced through the aforementioned manufacturing steps, plays a vital role in improving and strengthening soil structure by supplying beneficial bacteria to damaged soil. For example, referring to Figure 2, if soil has high porosity, its water retention capacity may be reduced, limiting air and water access for plant roots.

[0091] Meanwhile, bacteria contained in SCB biovinyl (i.e., calcite-forming bacteria) produce calcium carbonate (CaCO3) within the micropores of the soil. This process occurs through a reaction between calcium ions and carbon dioxide in the soil. The resulting calcium carbonate partially fills the soil pores and strengthens the bonds between soil particles. Consequently, overall soil porosity increases and permeability decreases, which reduces water runoff and allows water to remain in the soil longer, allowing plant root systems to more effectively absorb the moisture and nutrients they need.

[0092] The effectiveness of SCB biovinyl can contribute to reducing the negative environmental impacts of agricultural activities, including sugarcane farming. Improving and stabilizing soil structure enhances water retention capacity, prevents erosion, and promotes root development, improving overall crop growth conditions. Furthermore, the use of SCB biovinyl provides a sustainable method for recycling sugarcane waste, promoting waste management and efficient resource use, and contributing to the realization of a circular economy model within the agricultural ecosystem.

[0093] In conclusion, the development and utilization of SCB bioplastics presents an innovative approach for sustainable management and improvement of the agricultural environment. This holds significant value as a strategy that simultaneously promotes soil health, protects water resources, and enhances agricultural productivity. Furthermore, it supports a circular economy through the recycling of waste generated from the sugarcane industry, providing additional environmental benefits such as reducing greenhouse gas emissions. This will address the waste issues that can arise during sugarcane cultivation and processing, and reduce environmental pollution.

[0094] Below, the various experimental methods and results performed to verify the effectiveness of the SCB biovinyl of the present invention are described in detail.

[0095]

[0096] Preparation of sugarcane waste (sugarcane husk)

[0097] In the case of sugarcane husk, it was prepared through a process related to Fig. 3. In Fig. 3, A represents sugarcane, B represents sugarcane waste, i.e., sugarcane husk, C and D represent the process of collecting SCB powder through sieving, and E represents sterilized SCB powder.

[0098] More specifically, sugarcane husk was prepared into powder form through the following process for effective use.

[0099] First, the sugarcane husks were removed and dried in a fume hood for 7 days. The completely dried sugarcane husks were finely ground in a food processor into a powder. The ground SCB was then sieved to collect the fine SCB powder and stored in a beaker. To prevent microbial contamination and ensure product safety, the stored SCB powder was sterilized in an autoclave at 121 degrees Celsius and 1.5 atm for 15 minutes.

[0100]

[0101] Soil collection for experiments

[0102] Two types of soil were collected from sheep farms and donkey farms in Seorak-myeon, Gapyeong-gun, Gyeonggi-do, and placed in 400g zipper bags. The collected soil was transported to the laboratory within 24 hours and stored at 4℃ until use.

[0103] Figure 4A is an example diagram showing a donkey farm located in Gapyeong-gun, and Figure 4B is an example diagram showing soil collected from each of a sheep farm and a donkey farm.

[0104]

[0105] Preparation of bacterial culture medium

[0106] 1) NA badge and NB badge

[0107] 250 mL of distilled water (DW) and 2 g of Nutrient Broth (NB) powder were added to each of two 500 mL Eplenmeyer flasks. That is, two flasks containing distilled water and NB powder were prepared, and 3.75 g of agar powder was added to one of the two flasks to create NA (Nutrient Agar) medium, and the other flask was created with NB medium without agar.

[0108] Afterwards, the generated NA and NB mixture was sterilized using an autoclave, and after sterilization, NB was poured into a tube and NA was poured into a petri dish and stored in a refrigerator at 4°C until solidification.

[0109] Through this process, NA medium with added agar powder and NB medium without added agar powder were created, respectively.

[0110]

[0111] 2) UA badge and UB badge

[0112] 250 mL of distilled water (DW), 5 g of yeast extract, 2.5 g of ammonium chloride, 0.75 g of sodium chloride, and 5 g of urea were added to each of two flasks and combined.

[0113] UA (Urea Agar) medium was created by adding 3.75 g of agar powder to one of the two flasks, and UB (Urea Broth) medium without agar was created in the other flask.

[0114] Thereafter, the resulting UA and UB mixture was sterilized using an autoclave. After sterilization, UB was poured into a tube and UA was poured into a petri dish and stored in a refrigerator at 4°C until solidification. Figure 5A is an exemplary diagram showing the process of pouring a UA solution into a petri dish to create a UA medium, and Figure 5B is an exemplary diagram showing a UB medium in a conical tube.

[0115]

[0116] calcite-forming bacteria

[0117] For each soil type, two 1:5 dilution mixtures were prepared. For each soil type, 1 g of soil was added to a 50-mL tube with 4 mL of distilled water. The mixtures were mixed using a vortex mixer and allowed to stand until the supernatant and sediment separated. A 10-μL loop was immersed in the supernatant and inoculated onto two UA plates per soil sample by streaking. The agar-containing medium was placed in an incubator at 28°C for 48 h.

[0118] The above process is as illustrated in Fig. 6. Fig. 6 shows the process of culturing farm soil bacteria in UA medium, where Fig. 6 A represents a diluted mixture of farm soil, and Fig. 6 B represents farm soil inoculated onto UA medium. In addition, Fig. 6 C shows UA medium inoculated inside an incubator.

[0119] Figure 7 shows farm soil bacteria cultured on NA and UA media, where Figure 7A shows bacteria from donkey farm soil cultured on UA ​​media, and Figure 7B shows bacteria from sheep farm soil cultured on UA ​​media. Also, Figure 7C shows bacteria from donkey farm soil cultured on NA media, and Figure 7D shows bacteria from sheep farm soil cultured on NA media.

[0120] More specifically, the bacterial culture medium was observed as shown in Figure 7, and each distinct bacterial colony was identified. Each bacterial colony had a different morphology, and each colony was annotated to distinguish it. Based on the annotation indicated on each colony, each bacterial group was scraped using a 10 μL loop and mixed into a tube containing UB medium in a corresponding conical tube. In this case, each tube was made to contain only one bacterial colony. This process was repeated until all distinct bacterial colonies were isolated in each tube, and then cultured for 48 hours and mixed in a vortex mixer.

[0121] Afterwards, 200 μL of each bacterial solution was inserted into each well of a 96-well plate using a micropipette, and the ABS absorbance of the cultured bacteria was measured at a wavelength of 630 nm using a microplate reader.

[0122] That is, calcite-forming bacteria were cultured on UA ​​medium from soils of donkey and sheep farms, respectively, and the cultured bacterial colonies were classified into six categories: size, edge, color, texture, height, and transparency. Figure 8 is a diagram related to the observation results of farm soil bacterial colonies cultured on urea agar medium.

[0123] As a result of the isolation, 24 individual colonies were isolated and cultured on UB medium. Ten bacterial colonies from the donkey farm were labeled DA, DB*DJ, and 14 bacterial colonies from the sheep farm were labeled SA, SB*SO.

[0124]

[0125] Water retention capacity test

[0126] As illustrated in Fig. 9, preparations and procedures for a moisture retention capacity test were performed. Fig. 9 A shows soil bacteria samples in paper cups, B shows the process of drilling holes in the paper cups, C shows paper cups with holes (i.e., drainage holes) drilled in them, D shows the completed moisture drainage system, E shows the process of adding distilled water (DW) to the soil, and F shows the paper cups being placed on a rocker. The paper cups are used as containers for the bacteria samples, and holes are drilled in the cups to allow water to pass through. The water drainage system helps the added water to flow through the soil and be removed. The step of adding DW to the soil controls the moisture conditions of the bacteria samples, and finally, the cups are placed on the rocker to ensure even processing of the samples.

[0127] More specifically, a drainage system was constructed by stacking six-hole paper cups on top of each other. In this case, one drainage system was provided for each bacterial solution. 250 g of sterilized soil was mixed with 250 ml of distilled water at a 1:1 ratio, and 20 g of soil was added to each perforated paper cup.

[0128] A 2 mL bacterial solution was prepared by mixing 0.1 ABS of each bacterial solution with distilled water, and 2 mL of the bacterial solution was added to each soil-filled cup using a micropipette. After 72 hours at room temperature, 10 mL of distilled water was added to the soil inside the drainage cup, waited for 10 minutes, and after 10 minutes, another 10 mL of distilled water was added. To ensure uniform treatment of the sample and improve the accuracy of the experimental results, the cup was placed on a rocker after 50 minutes to reduce the adhesive force of the water and allow the water molecules within the sample to move more freely.

[0129]

[0130] Moisture retention capacity test results

[0131] Water Holding Capacity (WHC) can refer to the ability of a given soil to retain water within its pore spaces. This test is intended to identify the presence of calcite within the soil. A high WHC may indicate that calcite has formed and compacted the soil.

[0132] WHC is measured by observing the release of moisture from the soil. A small incision is made in the bottom of a paper cup containing bacteria, and after 72 hours of bacterial growth, 20 ml of distilled water (DW) is added to the soil. The release of moisture from the soil is observed after 10 minutes, 1 hour, and 24 hours.

[0133] Of the 24 bacterial soil samples, the following 9 were selected due to the absence of water release (i.e., showing calcite formation): SA, SH, SK, SL, SR, DG, DH, DK, and DL.

[0134]

[0135] lignin-decomposing bacteria

[0136] For each soil type, a 10 μL loop was dipped into the supernatant of a 1:5 diluted mixture of farm soil and distilled water (DW), and streaked onto two NA agar plates per soil sample. The agar plates were incubated at 28°C for 48 h. Each distinct bacterial colony that appeared on the culture medium was observed and labeled with a different annotation. Each bacterial colony was scraped using a 10 μL loop and mixed with NB agar into the corresponding conical tube. This process was repeated until all distinct bacterial colonies were isolated in each tube, and the tubes were then incubated for 48 h and mixed on a vortex mixer.

[0137]

[0138] Lignin decomposition test

[0139] Three types of agar media were produced as shown in [Table 1] below.

[0140] Agar Medium TypeMaterialsFine SCB1 g fine SCB powder, 3 g agar powderRough SCB1 g rough SCB powder, 3 g agar powderFine SCB+NA powder, 0.4 g nutrient broth powder1 g fine SCB powder, 3 g agar

[0141] Fine SCB and Rough SCB provide differences in texture, while Fine SCB+NA refers to media with added NA nutrients to meet specific nutritional needs.

[0142] The ingredients listed in Table 1 were added to three beakers, each containing 200 mL of distilled water (DW). A magnetic bar was added to each beaker, and the mixture was mixed using a magnetic stirrer. The mixtures were then sterilized in an autoclave.

[0143] After cooling the sterilized mixture for a certain period of time, it was continuously mixed on a magnetic stirrer. The mixture was poured into a 60x15mm petri dish on a clean bench and left until solidified.

[0144] Afterwards, each bacterial solution was taken using a 10 μL loop and inoculated onto three types of agar media using the streaking method. The inoculated media were cultured at room temperature for 72 hours, as shown in Figure 10. Figure 10A shows the process of preparing the three mixtures, and Figure 10B shows farm soil cultured on SCB agar media.

[0145] A nutrient-free medium and a medium containing NA were compared, and the medium that showed sufficient bacterial growth even without nutrients was given priority.

[0146] The lignin-decomposing ability of the selected bacteria was confirmed under a microscope.

[0147] The decomposition ability was observed by comparing two bacterial candidates with E. coli, which does not decompose lignin, and a control group without bacteria.

[0148] As shown in Figure 11, the SCB broth medium containing the two bacterial candidates demonstrated greater degradation compared to E. coli and the control group. While the morphology was clearly visible in the SCB broth medium, the E. coli and control groups demonstrated a structureless, amorphous state. Through this process, the lignin-degrading capabilities of the selected bacterial candidates were confirmed.

[0149]

[0150] Biological toxicity testing of selected bacteria

[0151] Toxicity testing of selected bacteria is conducted to determine whether they have any harmful effects on the ecosystem or human health.

[0152] For toxicity testing, lettuce seeds were cultured under two different conditions and the results observed.

[0153] First, for soils containing calcite-forming bacteria, after removing any remaining water, nine lettuce seeds were evenly arranged three by three on the selected soils containing calcite-forming bacteria. In this case, the cups were insulated with plastic dome covers measuring 8.5 cm x 5.5 cm.

[0154] For soil containing lignin-decomposing bacteria, sterilized soil and distilled water were mixed in a 1:1 ratio. In addition, half of a plate (5.5 cm x 1.5 cm) was filled with the soil mixture, and nine lettuce seeds were placed on top of the soil.

[0155] Afterwards, 1 mL of a bacterial solution with a concentration of 0.1 ABS was sprayed onto each sample, and germination was observed to determine the effects of calcite-forming bacteria and lignin-decomposing bacteria on lettuce seed germination. The number of germinated seeds was recorded daily for 4 days, and the effect of bacteria was evaluated based on the germination rate of the seeds.

[0156] Among the soil samples containing calcite-forming bacteria, the bacterial soil samples with the highest germination rates were identified as samples containing DG and DH bacteria, as shown in Fig. 12.

[0157] In addition, soil samples with lignin-forming bacteria were also recorded daily for 4 days, and as can be seen in Figures 13 and 14, SB, SC, DD, DE, and DF bacterial soil samples showed better growth rates than the control. Figure 13 is a graph showing the number of shoots grown in lignin-decomposing bacteria soil samples for 4 days. Figure 14 is a diagram showing shoots germinated from the selected candidate bacteria soil samples on the 4th day. Considering the lignin-decomposing ability and seed germination rate, SB and SC were finally selected.

[0158]

[0159] SCB biovinyl containing selected bacteria

[0160] 1) Creation of two types of biovinyl without bacteria

[0161] (1) First, 50 mL of distilled water (DW), 0.25 g of corn starch, 0.75 g of agar, and 0.5 g of SCB were mixed in a beaker.

[0162] (2) Boil the mixture on a hot plate and then let it cool for about 10 minutes.

[0163] (3) Then, pour the mixture into a 150 mm diameter petri dish and let it harden in a fume hood.

[0164] Two types of biovinyl were produced through the above procedure, but one biovinyl production process was performed by adding 500 μL of molasses. In other words, two types of biovinyl (without bacteria) were produced, with or without molasses added.

[0165]

[0166] 2) Production of SCB biovinyl containing selected bacteria

[0167] (1) First, 50 mL of distilled water (DW), 0.25 g of corn starch, 0.75 g of agar, and 0.5 g of SCB were mixed in a beaker.

[0168] (2) Boil the mixture on a hot plate and then let it cool for about 10 minutes.

[0169] (3) After the mixture has cooled, add 0.001 ABS mixed bacteria solution.

[0170] (4) Then, the mixture with the mixed bacterial solution added is poured into a 150 mm diameter petri dish and allowed to harden in a fume hood.

[0171] Two types of biovinyl were produced through the above procedure, but one biovinyl production process was performed by adding 500 μL of molasses. In other words, two types of biovinyl (including bacteria) were produced, with and without molasses added.

[0172]

[0173] Dispersion rate of bacteria dispersed by SCB biovinyl

[0174] Figure 15 illustrates the preparation process for a bacterial dispersal rate test of SCB biovinyl. Figure 15 A illustrates the process of adding DE to soil, B illustrates the process of cutting biovinyl pieces, C illustrates the cut biovinyl, and D illustrates the process of placing the biovinyl on soil.

[0175] First, spread sterilized soil thinly on each of eight petri dishes with a diameter of 100 mm, and moisten with 20 mL of distilled water.

[0176] Cut out 0.5 cm x 0.5 cm pieces from biovinyl containing calcite-forming bacteria and lignin-decomposing bacteria.

[0177] Place each piece of bioplastic in the center of the soil, cover with a petri dish lid, and leave for 48 hours. Mark three spots equally spaced from the center of the petri dish lid.

[0178] For biovinyl containing calcite-forming bacteria, a 10 μL loop was first immersed in the UB medium, then immersed at each marked point, and then mixed in a microtube containing 1000 μL of UB medium. For lignin-degrading bacteria, the same procedure was performed using NB medium.

[0179] After culturing at room temperature for 24 hours, 200 μL was taken from each cultured bacterial medium and transferred to each well of a 96-well culture plate.

[0180] Next, to quantitatively evaluate the growth amount or density of the cultured bacteria, the absorbance (ABS) of the bacterial medium was measured using a microplate reader. This process confirmed how well the bacteria contained in the biovinyl were dispersed into the surrounding soil. Figure 16 is a diagram of the bacterial mobility test. Figure 16 A shows the specific location where the soil was collected, Figure 16 B shows the process of recovering bacteria from the soil, and Figure 16 C shows the process of measuring ABS.

[0181] According to an embodiment of the present invention, the SCB biovinyl was manufactured based on cornstarch and agar, with SCB added to increase the strength of the vinyl. To address potential problems caused by SCB not decomposing in soil, lignin-decomposing bacteria were incorporated into the biovinyl. The effectiveness of the SCB biovinyl, which included calcite-forming bacteria DG and DH and lignin-decomposing bacteria SC and SB, was tested by observing the rate of bacterial dispersal from the biovinyl.

[0182] A piece of bio-vinyl containing the selected bacteria was placed on a petri dish containing sterilized soil, and after 48 hours, bacterial growth was observed in the soil near, in the middle, and far from the bio-vinyl.

[0183] Figure 17 shows microscopic images of SCB biovinyl placed on soil after 24 hours. Here, comparing the dry control and the normal control, it can be confirmed that the biovinyl maintains its structure and discolors when wet, regardless of the presence of bacteria. This demonstrates that the biovinyl provides sufficient time and nutrients for bacteria to reproduce. Notably, the biovinyl containing lignin-degrading bacteria decomposed SCB to a greater extent than the other samples. This demonstrates that the SCB biovinyl promotes bacterial activity, leading to faster degradation.

[0184] In an example, a bacterial dispersion test was performed on a sterilized soil containing SCB bioplastic, and it was found that bacteria on a 0.5 cm x 0.5 cm piece of bioplastic were dispersed over a radius of 2.5 cm. This could mean, for example, that if a bioplastic with a diameter of 15 cm is placed on soil, bacteria will be dispersed over a soil area with a diameter of 1.5 m.

[0185] BacteriaNearMiddleFarCalciteformingDG1.2911.0680.891DH0.7410.6750.297LigninDecomposingSC0.1690.2170.143SB0.2040.1190.130

[0186] For calcite-forming bacteria, the absorbance of DG and DH was verified by observing bacteria at all three locations, as shown in [Table 2]. DG showed much higher growth than DH, but as shown in Figure 18, DG showed slower movement and a slower dispersal rate, as the bacterial dispersion at the intermediate location was less than that at the closest location. On the other hand, for DH, the dispersion at the intermediate location was higher than that at the closest location, showing rapid dispersal, especially on biovinyl containing molasses. Therefore, although DH showed a faster dispersal rate, DG was selected because it showed a significantly higher reproduction rate. For lignin-degrading bacteria, SB and SC had similar reproduction rates, as shown in [Table 2]. Referring to Figure 18, SC showed a faster dispersal rate than SB. While SC showed an increase in dispersion as the distance increased, SB showed a decrease. Therefore, SC was selected because it showed a faster dispersal rate while maintaining a similar reproduction rate.

[0187]

[0188] Final screening of bacteria

[0189] The final selected calcite-forming and lignin-degrading bacteria were cultured on agar media and then sent to MACROGEN in Korea for DNA sequencing using 16s rRNA and BLASTN analysis to identify their species.

[0190] The selected DG was identified as 99.58% identical to Glutamicibacter halophytocola, and the SC was identified as 92% identical to Priestia megaterium. These results indicate that neither bacteria is pathogenic, ensuring their safety for humans and the ecosystem.

[0191] That is, through the experimental content and results described above, it can be confirmed that Glatamibacter halopytocola has lime-forming properties that improve soil porosity, and Pristeria megaterium has lignin-decomposing properties that can effectively decompose cellulose.

[0192] These experimental results confirm that SCB biovinyl effectively disperses calcite-forming bacteria, improves soil strength, and promotes the decomposition of remaining SCB.

[0193] Utilizing the SCB bioplastic of the present invention can improve and strengthen soil structure by supplying beneficial bacteria to damaged soil. This can reduce the negative environmental impact of sugarcane farming and improve the growing environment for crops.

[0194] Furthermore, recycling waste generated from the sugarcane industry can support a circular economy and provide additional environmental benefits, such as reducing greenhouse gas emissions. This can address waste issues that arise during sugarcane cultivation and processing, and reduce environmental pollution.

[0195]

[0196] In an embodiment, when calcite-forming bacteria are contained in the SCB biovinyl in an amount greater than that of lignin-decomposing bacteria, bacterial reproduction becomes active and dispersion can be improved.

[0197] Specifically, it is preferable that calcite-forming bacteria be provided in an amount of 100 parts by weight or more per 100 parts by weight of lignin-decomposing bacteria. That is, when Glatamibacter halopytocola is provided in an amount of 100 parts by weight or more per 100 parts by weight of Pristeria megaterium, bacterial reproduction becomes active and dispersion can be improved.

[0198] [Table 3] below shows the dispersion results for SCB biovinyls manufactured by changing the ratio of Glatamibacter halopytocola to Pristeria megaterium. Specifically, the results of a bacterial dispersion test performed on SCB biovinyls manufactured based on mixed bacterial solutions corresponding to 50 parts by weight of Glatamibacter halopytocola to 100 parts by weight of Pristeria megaterium, 80 parts by weight of Glatamibacter halopytocola to 100 parts by weight of Pristeria megaterium, 100 parts by weight of Glatamibacter halopytocola to 100 parts by weight of Pristeria megaterium, 120 parts by weight of Glatamibacter halopytocola to 100 parts by weight of Pristeria megaterium, and 150 parts by weight of Glatamibacter halopytocola to 100 parts by weight of Pristeria megaterium are shown on sterilized soil. Each SCB biovinyl was placed on the soil and the absorbance measured at each location after 24 hours is shown.

[0199] Bacterial ratio (Pristeria megaterium: Glatamibacter halopytocola) NearMiddleFar1: 0.50.8460.8240.7561: 0.80.9220.9890.8191: 11.2821.1120.8961: 1.21.3211.1180.9451: 1.51.3871.1320.986

[0200] Referring to [Table 3], it can be confirmed that as the ratio of Glatamibacter halopytocola to Pristeria megaterium in the mixed bacterial solution increases, a higher reproduction rate is observed. In particular, when Glatamibacter halopytocola is less than 100 parts by weight per 100 parts by weight of Pristeria megaterium (i.e., 50 parts by weight of Glatamibacter halopytocola to 100 parts by weight of Pristeria megaterium or 80 parts by weight of Glatamibacter halopytocola to 100 parts by weight of Pristeria megaterium), the growth rate decreases, but when Glatamibacter halopytocola is 100 parts by weight or more per 100 parts by weight of Pristeria megaterium, the growth rate is confirmed to be significantly improved.

[0201] Figure 19 is an example diagram showing the dispersal speed according to the change in the stocking ratio of Glatamibacter halopytocola compared to Pristeria megaterium. In the graph of FIG. 19, a relates to a biovinyl manufactured based on a mixed bacterial solution having a composition ratio of Pristeria megaterium and Glatamibacter halopytocola of 1:0.5, b relates to a biovinyl manufactured based on a mixed bacterial solution having a composition ratio of Pristeria megaterium and Glatamibacter halopytocola of 1:0.8, c relates to a biovinyl manufactured based on a mixed bacterial solution having a composition ratio of Pristeria megaterium and Glatamibacter halopytocola of 1:1, d relates to a biovinyl manufactured based on a mixed bacterial solution having a composition ratio of Pristeria megaterium and Glatamibacter halopytocola of 1:1.2, and e relates to a biovinyl manufactured based on a mixed bacterial solution having a composition ratio of Pristeria megaterium and Glatamibacter halopytocola of 1:1.5.

[0202] As shown in Fig. 19, when Glatamibacter halopytocola is less than 100 parts by weight relative to 100 parts by weight of Pristeria megaterium, it can be confirmed that the dispersion of bacteria in the middle position is significantly less than that in the near position, as in a and b, and thus movement is slow. On the other hand, when Glatamibacter halopytocola is included in a quantity greater than that of Pristeria megaterium (i.e., Glatamibacter halopytocola is more than 100 parts by weight relative to 100 parts by weight of Pristeria megaterium), the dispersion of bacteria in the middle position is higher than that in the nearest position, and thus, it can be confirmed that rapid dispersion is observed.

[0203] As shown in [Table 3] and Fig. 19, when the mixed bacterial solution is composed of a calcite-forming bacterium, Glatamibacter halopytocola, in a larger amount than the lignin-decomposing bacterium, Pristeria megaterium, and the sugarcane waste-based biovinyl of the present invention is manufactured based on the mixed bacterial solution, it can be confirmed that the growth rate and dispersion rate are significantly increased. This shows that as the composition ratio of calcite-forming bacteria increases, more calcite is formed, strengthening the soil strength and improving porosity, thereby improving the growth rate and dispersion of lignin-decomposing bacteria.

[0204] In one embodiment, when the SCB biovinyl of the present invention is utilized, beneficial bacteria can be supplied to damaged soil to improve and strengthen the soil structure. Referring to the experimental results of Fig. 20, it can be confirmed that the TOC (Total Organic Carbon) and MBC (Microbial Biomass Carboc) measured in the soil in which the SCB biovinyl of the present invention was placed (i.e., 6 experimental groups) were improved compared to the 6 control groups in which the SCB biovinyl was not placed, and the P value was less than 0.05, confirming that the result was highly reliable. TOC represents the total amount of organic carbon, and MBC represents the carbon content of microbial biomass, and can be an important indicator for evaluating the health and productivity of soil. That is, the experimental results showing an improvement in TOC and MBC can mean that the structure of the soil is improved and strengthened by utilizing the SCB biovinyl of the present invention.

Claims

1. In bio-vinyl based on sugarcane waste, Distilled Water (DW); Cornstarch; Agar; Sugarcane bark (SCB); and A mixed bacterial solution comprising two or more species of bacteria; Bioplastic based on sugarcane waste.

2. In paragraph 1, The above corn starch is provided in an amount of 0.5 parts by weight per 100 parts by weight of the distilled water, The above agar is provided in an amount of 1 to 1.5 parts by weight relative to 100 parts by weight of the distilled water, The above sugarcane husk is provided in an amount of 1 part by weight per 100 parts by weight of the distilled water. Bioplastic based on sugarcane waste.

3. In paragraph 1, The above mixed bacterial solution is, Comprising one species of bacteria selected from the group of Calcite Producing Bacteria and another species of bacteria selected from the group of Lignin Decomposing Bacteria, Bioplastic based on sugarcane waste.

4. In paragraph 1, The above mixed bacterial solution is, including Glutamicibacter halophytocola and Priestia megaterium. Bioplastic based on sugarcane waste.

5. In paragraph 1, The above mixed bacterial solution is, A solution of two or more bacteria diluted to 0.001 ABS, Bioplastic based on sugarcane waste.

6. In paragraph 1, The above biovinyl is, Further comprising 1 part by weight of molasses per 100 parts by weight of the distilled water, Bioplastic based on sugarcane waste.

7. In a method for manufacturing bio-vinyl based on sugarcane waste, A step of creating a mixture by adding corn starch, agar, and sugarcane husk to distilled water; A step of heating the above mixture; After heat-treating the above mixture, a step of adding a mixed bacterial solution containing two or more types of bacteria to the mixture; and A step of coagulating a mixture to which the above mixed bacterial solution is added to produce a sugarcane waste-based biovinyl; comprising; Method for producing bioplastics based on sugarcane waste.

8. In paragraph 7, The step of creating the above mixture is: a step of adding molasses; further comprising; Method for producing bioplastics based on sugarcane waste.

9. In paragraph 7, The above mixed bacterial solution is, Comprising one species of bacteria selected from the group of calcite-forming bacteria and another species of bacteria selected from the group of lignin-decomposing bacteria, Method for producing bioplastics based on sugarcane waste.

10. In paragraph 7, The above mixed bacterial solution is, Containing Glutamibacter halopytocola and Pristeria megaterium, Method for producing bioplastics based on sugarcane waste.

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