Program Book for Sustainability Challenge 2023

REFERENCES [1] Agrawal, A., Kulkarni, S. & Sharma, S.B. (2015). Recent advancements and applications of multiple emulsions. [2] Doan, T.K.Q. & Chiang, K.Y. 2022. Characteristics and kinetics study of spherical cellulose nanocrystal extracted from cotton cloth waste by acid hydrolysis. Sustainable Environment Research 32(1). [3] Trache D, Tarchoun AF, Derradji M, Hamidon TS, Masruchin N, Brosse N and Hussin MH (2020) Nanocellulose: From Fundamentals to Advanced Applications. Front. Chem. 8:392. [4] Emam, H.E. & Ahmed, H.B. 2018. Carboxymethyl cellulose macromolecules as generator of anisotropic nanogold for catalytic performance. International Journal of Biological Macromolecules 111: 999–1009. [5] Goni, Feybi & as, Syaimak & Mukhtar, Muriati & Sahran, Shahnorbanun. (2015). Environmental Sustainability: Research Growth and Trends. Advanced Science Letters. 21. [6] M. Ishizawa, K. Akiyama & S. Kawaguchi. 2003. The 10th FCDIC Fuel Cell Symposium Proc., pp. 271-276. [7] Ioelovich, M. 2021, December 1. Preparation, characterization, and application of amorphized cellulose—a review. Polymers. [8] Shaheen, T.I.& Emam, H.E. 2018. Sono-chemical Synthesis of Cellulose Nanocrystals from Wood Sawdust Using Acid Hydrolysis. International Journal of Biological Macromolecules 107: 1599-1606. [9] Zhao, X.F. & Winter, W.T. 2015. Cellulose/Cellulose-Based Nanospheres: Perspectives and Prospective. Industrial Biotechnology.11(1):34-43. [10] Nishiyama, Y. 2014. Chapter 2 Structure and Physical Properties of Cellulose: Micro-to Nanos.

CHARACTERIZATION OF PHAGE-ENDOLYSIN AS BIOCONTROL IN RICE BLB: AN APPROACH FOR SUSTAINABLE AGRICULTURE Rafidah Saaduna , Hazri Ab. Rashida , Ishak Mohd Yusuffa , Tan Geok Hunb,c a Industrial Biotechnology Research Centre, SIRIM Berhad, 40700 Shah Alam, Selangor, Malaysia and [email protected], +60355446957; [email protected], +60355446960; [email protected], +60355446982 b Microbial Culture Collection Unit, Institute of Bioscience, Universiti Putra Malaysia, 43400 Serdang, Malaysia and [email protected], +60397694893 cDepartment of Land Management, Faculty of Agriculture, Universiti Putra Malaysia, 43400 Serdang, Malaysia. The relationship between phages and bacteria forms the foundational idea for developing biocontrol to protect rice plants against Xanthomonas oryzae pv. oryzae (Xoo). Xoo is a gram-negative bacterium responsible for bacterial leaf blight (BLB), a devastating disease in rice plants. The disease causes water-soaked lesions, necrotic growth, and reduced yield. Xoo colonizes the vascular system, spreading through xylem vessels and influencing plant defense mechanisms. Effective management strategies are crucial for sustainable agricultural productivity. Phages play an important role in maintaining ecological balance in Earth’s ecosystems by targeting and infecting bacterial hosts [1, 2 & 3]. This balance is vital for the overall health of ecosystems, as it prevents bacterial overgrowth and associated problems that promote sustainability in various environments. These natural viruses have coexisted with bacteria for millions of years, forming an integral part of the microbial ecosystem. This predator-prey dynamic regulates bacterial populations, preventing rampant growth and promoting sustainability. Phages are viruses that target specific bacteria by exploiting their unique surface molecules for infection. They multiply within their bacterial hosts and produce multiple copies of themselves. At the end of the replication cycle, phages synthesize endolysins specialized enzymes that target and cleave bonds within the peptidoglycan layer of the bacterial cell wall, weakening its stability and eventually causing cell lysis and death. The endolysins’ role in the natural life cycle of phages highlights their function and demonstrating their potential as biocontrol agents in combatting bacterial pathogen. Endolysin, derived from phages, exhibits a unique biological mechanism that contributes to their role as biocontrol agents. It is a prime example in utilizing natural resources for biocontrol in a sustainable manner. Phages, as living organisms, abundantly exist in various environments and specifically target bacterial pathogens. Endolysin, which is a product of natural mechanism is unlimited abundance in nature, reducing the reliance on synthetic or limited resources. Utilizing endolysins from phages as biocontrol is like using an easily accessible and renewable biological

materials and tool. This approach aligns with sustainability goals by promoting the responsible and efficient use of natural resources in biocontrol strategies. The implementation of phage endolysin as a biocontrol for Xoo caused to BLB in rice begins with the isolation and characterization of phages specific to this pathogenic bacterium. The characterization of the phage involved conducting tests on a double layer agar, resulting in the formation of clear plaques against the target Xoo. These plaques signify the lytic activity of the endolysin against the bacterial cell walls, revealing its potential as a biocontrol agent. The clear zones formed on the agar demonstrated the ability of the endolysin to degrade the Xoo cell wall efficiently. This characterization procedure provides essential information on the efficacy and specificity of endolysin, paving the way for further development of this biocontrol strategy against Xoo. In the subsequent stages of developing biocontrol using phage endolysin, the gene encoding the endolysin was further constructed as recombinant endolysin. Through a process of molecular cloning, the gene was isolated and ligated into an expression vector pETite, which was then introduced into E.coli BL21 (DE3) cells. The recombinant endolysin was expressed by an Isopropyl β-D-1-thiogalactopyranoside (IPTG) inducer. The controlled and scalable of recombinant endolysin was then subjected to further assessment for its efficacy against BLB in greenhouse trial. The final step in deploying phage endolysin as a biocontrol measure against BLB in rice involves a greenhouse trial. This assessment aimed to evaluate the effectiveness of recombinant endolysin and its ability to control BLB in a greenhouse environment. The key objectives include measuring BLB disease reduction and assessing overall rice plant health when recombinant endolysin is applied as a treatment against Xoo. Monitoring will be carried out on the rice plants by recording the development of the BLB disease, the severity of the symptoms and their growth progression. This greenhouse experiment will provide critical insight into the potential use of phage endolysin as a targeted and sustainable biocontrol solution to effectively manage BLB in rice. The utilization of phage endolysin to control BLB has profound implications for society, communities, and countries. It presents a safer and more eco-friendly method for plant disease management, effectively reducing the exposure of both farmers and consumers to harmful chemicals. Traditional chemical controls pose significant health risks and the chemical residues that persist on crops can be transferred to consumers which lead to long term health problems. Moreover, the chemical controls also contribute to the development of antibiotic or chemical resistant organisms [4] which necessitating an increase in chemicals applications resulted to the potential harm. In addition, the application of chemicals in plant disease management often leads to the unintended killing of beneficial soil bacteria along with the targeted pathogen. This disruption affects soil health, reducing its overall resilience. In contrast, phage endolysin biocontrol offers a targeted approach, specifically

attacking harmful bacteria without interfere other bacteria [5] and eliminating them in the soil, thereby maintaining biological balance, ecology and promote more sustainable agricultural ecosystems. In addition, phage endolysin, as biodegradable proteins, offer an acceptable alternative to conventional chemical control methods. They naturally degrade into harmless substances, minimizing their environmental footprint, benefiting the environment and public health compared to chemical control that often leads to soil and water contamination. Phage endolysin also offers a substantial sustainable impact on a country’s food security. As biocontrol agent, it strengthens agricultural productivity crop yield, directly contributing to a more secure and stable food supply. The increase in domestic rice production further minimizes the need for rice imports, consequently reducing country expenses thus fostering economic stability. In addition, the use of phage endolysin as biocontrol could also increase the production of high-quality rice for the local consumption further controlling safer rice stock. The development and application of phage endolysins as biocontrol agents represents a significant advancement in sustainable agriculture. Their specificity, environmental friendliness and potential to reduce antibiotic usage make them a potential valuable tool for plant disease management. However, challenges in the development of phage endolysin as a biocontrol agent remain for improvement in achieving a more sustainable agriculture contributing to a greener and more resilient future. Optimization of production processes to ensure scalability, stability and cost-effectiveness is essential for widespread adoption. By accepting biocontrol strategies, the country moves towards a more resilient and sustainable agricultural future, ensuring food security and a healthier planet for subsequent generations. [1] L. Jain, V. Kumar, S. K. Jain, P. Kaushal, &P. K. Ghosh. 2023. Frontiers in Microbiology, 14, 1- 21. [2] J. Liu, S. L. Chia, & G. H. Tan. 2021. PHAGE, 2(3), 142–151. [3] Z. Dong, S. Xing, J. Liu, X. Tang, L. Ruan, M. Sun, Y. Tong, & D. Peng. 2018. Journal of General Virology, 99(10), 1453–1462. [4] C. Sieiro, L. Areal-Hermida, Á. Pichardo-Gallardo, R. Almuiña-González, T. De Miguel, S. Sánchez, A. Sánchez-Pérez, & T. G. Villa. 2020. Antibiotics, 9(8), 493. [5] D. Holtappels, K. Fortuna, R. Lavigne, & J. Wagemans. 2021. Current Opinion in Biotechnology, 68, 60–71.

CONVERSION OF PLASTIC WASTE TO FILTER MEMBRANE FOR WATER TREATMENT Thurgashiny A/P Veeramani, Muhammad Ilham Bin Bacho, Nur ‘Dayana Batrisya Binti Zainal, Vishnu A/L I. Sureasha , Teow Yeit Haanb a Department of Chemical and Process Engineering, Faculty of Engineering & Built Environment,Universiti Kebangsaan Malaysia,43600 UKM Bangi,Selangor Darul Ehsan,0194406008,[email protected] bResearch Centre for Sustainable Process Technology (CESPRO),Universiti Kebangsaan Malaysia,43600 UKM Bangi,Selangor Darul Ehsan, Malaysia,0389217095,[email protected] This research aims to show that plastic waste, especially polystyrene, has the potential to produce synthetic polymer membranes in the separation process. Population growth and stable demand for plastic products lead to increased plastic production, resulting in more plastic waste and environmental pollution. This experiment seeks to reduce plastic waste and utilize plastic waste in the separation process, such as wastewater treatment. Environmental pollution by plastic waste has been widely recognized as a major environmental burden, especially in aquatic environments, which has a negative impact on wildlife and restrictions on plastic disposal. The objective of this study is to determine the most effective solvent to dissolve plastic waste, study the ratio of plastic waste to the optimal solvent, and determine the best filler concentration to increase the hydrophilic effect on the polymer membrane. Plastic waste can be reused as a raw material to produce new products of high value, such as synthetic polymer membranes, as an alternative to reduce plastic waste. The use of solid waste in the development of polymer membranes can reduce the use of conventional raw materials and minimize the negative environmental impact from uncontrolled plastic management. Studies show that membranes produced from polystyrene waste have the same separation performance as membranes commonly used in industry. The production of this synthetic polymer membrane provides benefits in many ways, especially through the sustainable production of membranes through recycling and replanting of waste resources. This study discusses the use of plastic waste, especially polystyrene (PS), to produce polymer membranes that are competitive in separating pollutants from polluted water. The results of the study show that the PS polymer membrane successfully removes humic acid from polluted water with high efficiency. In conclusion, polystyrene can produce a synthetic polymer membrane as a filter membrane for water treatment. The following are the chemical substances and laboratory equipment used in this experiment. Polystyrene (PS) was obtained from discarded foam food containers, while N-Methyl-2-Pyrrolidone (NMP) and N,N-Dimethylacetamide (DMAc), were procured as solvents for dissolving the waste

plastic. The hydrophilic additive, Titanium (IV) oxide (TiO2) with an assay of ≥ 99%, was sourced from Sigma-Aldrich Co., USA. The model solution for membrane performance study, HA, was purchased from Sigma-Aldrich Co., USA. Sodium hydroxide (NaOH) from Classic Chemicals Sdn. Bhd., Malaysia, was used to dissolve HA. This test is conducted using two different solvents, namely N-Methyl-2-Pyrrolidone (NMP) and N,N-Dimethylacetamide (DMAc). Subsequently, the small polystyrene pieces are mixed with the solvent according to the polymer-solvent ratio of 16:84 and continuously stirred overnight at a speed of 250 rpm to achieve a homogeneous solution. Meanwhile, 0.025 g/L of TiO2 is weighed and added to the polymer solution to enhance the hydrophilic properties of the membrane. Once a homogeneous solution is obtained, the polymer membrane solution is left at room temperature overnight to remove any bubbles present. The above steps are then repeated using different TiO2 concentrations: 0.050 g/L and 0.100 g/L. After completing the test for the 16:84 polymer-solvent ratio, the test is repeated with different polymer-solvent ratios of 17:83 and 18:82, with the same three TiO2 concentrations as mentioned above: 0.025 g/L, 0.050 g/L, and 0.100 g/L. Subsequently, after completing all test sets for NMP, the testing process is repeated using the second solvent, DMAc. The DMAc test sets are conducted with the same polymer-solvent ratio and TiO2 concentration sets as those used for NMP. Therefore, a total of 18 test sets are required, considering three different polymer-solvent ratios and three different TiO2 concentration levels. The synthetic polymer membrane produced will be placed in a Dead-end Filtration unit to determine the permeation flux and removal of Humic Acid (HA). This unit causes HA to accumulate either on the membrane's surface or within the membrane over time, resulting in an increase in layer thickness and a decrease in permeation flux. The membrane integrity test begins with the synthetic polymer membrane being cut into disk shapes with an effective surface area of 10.75 cm2 and inserted into the base of the unit. Rubber 'O' rings are used to secure the membrane in place. (Khaled et al., 2020).The unit is then filled with 200mL of ultra-pure water as the feed solution and operated at 4 bar with a stirring speed of 170 rpm for 1 hour with a 60-second interval. Permeate flows out of the unit and is collected in a 500 mL beaker. The permeation yield is then weighed using a digital balance and recorded on a computer. This process is repeated by filling the unit with 50mL of HA solution as the feed and operating it at 3 bar for 15 minutes (time interval: 15 seconds). As HA is passed through, any color change to orange on the membrane is observed and recorded as evidence of the presence of humic acid in untreated water. Samples of the HA solution feed and the permeation yield from the HA solution are poured into cuvettes, and changes in optical density at 254 nm are observed using a spectrophotometer.(Karimi et al., 2020).

Figure 1 Graph of the removal percentage of humic acid against the concentration of Titanium Dioxide Figure above shows that the highest percentage of humic acid is shown by the membrane with TiO2 concentration 0.1 g/L and 18:82 ratio of polymer: solvent. This shows that the higher the flux value, the more humic acid can be removed. As a conclusion, ultrafiltration membrane efficiently operates at a pressure of 3 bars in processing a combination of titanium dioxide concentration and polymer ratio. The effective concentration for plastic waste is 0.1 g/L, with a favorable plastic waste to solvent ratio of 18:82. N,NDimethylacetamide is more effective than N-methyl-2-pyrrolidone in humic acid separation, yielding superior and efficient separation outcomes. We can conclude that our study also achieves the sustainable development goals which are clean water and sanitation, responsible consumption and production and life below water. Lastly, we express our gratitude to God for enabling us to complete this Sustainability Challenge 2023. We would like to take this opportunity to extend our heartfelt thanks and show our appreciation to all parties who assisted in the completion of this abstract. We would like to dedicate this appreciation to our laboratory staff, Mr. Mohamad Hisyam Abdul Rahman, who granted us permission to conduct our experiments in the lab. Additionally, we would like to dedicate our gratitude to our supervisor, Associate Professor Dr. Teow Yeit Haan, who greatly aided in completing this study. We would also show our gratitude to Prof. Madya Ir. Dr. Hassimi Bin Abu Hasan who sponsored us for this Sustainability Challenge 2023.

REFERENCES Banoub, J., Ghada , A., Mikhael, A., Christopher, P. C., & T. Le, T.-A. (2021). Environmental impact of bioplastic use. Heliyon, 9. Karimi, A., Khataee, A., Vatanpour, V., & Safarpour, M. (2020). The effect of different solvents on the morphology and performance of the ZIF-8 modified PVDF ultrafiltration membranes. Separation and Purification Technology, 253(April), 117548. https://doi.org/10.1016/j.seppur.2020.117548 Khaled, M., Noby, H., Mansor, E., El-Shazly, A., & Aissa, W. (2020). Water Purification using Recycled Polymeric Microfiltration Membranes. International Journal of Applied Energy Systems, 2(2), 122–126. https://doi.org/10.21608/ijaes.2020.169930 Teow, Y. H., Ooi, B. S., Ahmad, A. L., & Lim, J. K. (2021). Investigation of anti-fouling and uvcleaning properties of PVDF/TiO2 mixed-matrix membrane for humic acid removal. Membranes, 11(1), 1–22. https://doi.org/10.3390/membranes11010016

1 INVESTIGATION OF EGGSHELL, PEANUT HUSK AND POMELO PEEL AS NON-TOXIC ADSORBENTS FOR METHYLENE BLUE DYE TREATMENT FROM WASTEWATER Farah Syakinah Md Sokor1 , Noorashikin Md Saleh 1*Chuah You Quan1 ,Ahmad Luqman Hakim1 , Nur Zulaikha Mat Kamil1 , Tanusha Devi 1Department of Chemical and Process Engineering, Faculty of Engineering and Built Environment, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia. *Corresponding author: [email protected] Abstract This study focused on treating wastewater containing dye content using non-toxic natural materials as absorbents, namely eggshells, peanut husk, and pomelo peel. These materials underwent preparation involving washing, drying, and grinding. A standard solution of methylene blue represented the dye present in the wastewater. The adsorption study investigated various parameters such as pH value, absorbent dosage, and extraction time. Peanut husk displayed the highest efficiency for all three parameters, with 78.44% at pH, 73.81% for absorbent dosage, and 73.25% for extraction time. FTIR analysis confirmed the composition of eggshells (95% calcium carbonate) and the presence of protein and cellulose in peanut husk. XRD analysis revealed that eggshells primarily consist of calcite (26.6%), while peanut husk contains cellulose (31.2%) and pomelo peel includes polyethylene (30.1%). FESEM analysis showcased different adsorbent structures. In conclusion, this study highlights the potential of using non-toxic natural materials for effective wastewater treatment, with peanut husk exhibiting the highest efficiency for dye removal and pomelo peel as the most efficient absorbent for various wastewater types. Keywords: Dye residue; absorbent; wastewater; methylene blue; natural adsorbent; eggshell: peanut husk: pomelo peel.

2 INTRODUCTION Dyes are essentially natural or synthetic organic compounds that can adhere to surfaces or fabrics to produce bright and long-lasting colours. They are used in various industries, including the food, leather, textile, paper, rubber, cosmetics, and plastic sectors. Most dyes are complex organic compounds that are resistant to various forces, including detergent action (Chincholi et al., 2014). The disposal of dyes from industrial wastewater poses a significant threat to the environment. Although the discharge of dyes into water may be minimal, it can have a major impact on water pollution due to their visibility and vibrant colors. Environmental concerns regarding dyes stem from their ability to hinder the absorption and reflection of sunlight entering the water, leading to a reduction in bacterial growth and biological purification processes (Pierce, 1994). More than 700,000 tons of commercial dyes are produced each year, with over 100,000 of them being recognized. It is estimated that 2% of dyes are released into wastewater during production, and 10% are released in the textile and related sectors (Sen et al., 2011). Therefore, various methods have been employed to treat dye wastewater, including adsorption, membrane separation, and coagulation. In this study, the treatment of dye residues was carried out through adsorption. Based on previous studies, adsorption has yielded the best results, as it can remove various types of dyes. Activated carbon has been favoured as an absorbent for colour removal due to its numerous benefits, but it is an expensive method due to the cost of the absorbent material used (Kinoshita, 1988). Adsorption is a process that extracts soluble substances from a liquid by using an absorbent material, where the combination of multiple liquid components (gas or liquid) is bound to the surface of a solid absorbent through physical or chemical bonding (Sasaki, 2014). Eggshells were selected as an absorbent due to their abundant availability from food producers and restaurants, where chicken eggs are consumed in large quantities, resulting in eggshells being discarded as waste. Many studies have been conducted to investigate the utility of eggshells. Eggshells are suitable absorbents due to their porous nature, with an estimated 7,000-17,000 pores per eggshell (William and Owen, 1995).

3 Additionally, peanut shells were chosen as an absorbent due to their potential as an absorbent material. They are readily available agricultural byproducts with low cost and high cellulose content, allowing them to adsorb waste materials. However, cellulose content alone does not determine the efficiency of the adsorption process (Wardalia et al., 2019). The final absorbent material in this study was pomelo peels. A pomelo is a citrus fruit from the Rutaceae family, has a diameter of 30 cm and weighs 10 kg, with its peel accounting for approximately 30% of its weight. Pomelo peels, a natural bioabsorbent, exhibit good adsorption capacity for certain pollutants due to their micron-sized pores (Zhang et al., 2018). The objective of this study is to investigate the potential of eggshells, peanut shells, and pomelo peels as non-toxic absorbents for extracting methylene blue from wastewater in Selangor, by identifying the optimum parameters for each absorbent, including the optimum pH, extraction time, and absorbent dosage. Furthermore, the study aims to obtain the mechanism of interaction between the non-toxic adsorbents with the methylene blue. MATERIALS AND METHODS Chemicals and Reagents Methylene blue powder purchased from R&M Chemicals, Sodium Hydroxide powder purchased from Emplura (Germany), Hydrochloric Acid solution purchased from R&M Chemicals (Malaysia), Deionised water, Eggshells, Peanut husks, Pomelo peels. Instrumentation Spectrum SP-UV 300SRB Spectrophotometer that was bought from Spectrum Instruments (Shanghai, China) was utilized to determine the absorbance of methylene blue in the wastewater in the Selangor. Procedure of Solid Absorbent and Methylene Blue Solution Preparation First of all, a variety of absorbent materials have been selected: eggshells, peanut husks, and also pomelo peels. All three skins were washed using deionized water and placed on top of an

4 aluminium sheet. After that, the aluminium sheets were placed in a 80°C oven and let for 24 hours so that the moisture in the skin completely disappeared. Next, these skins have been crushed and scratched using a grinder. These powders have been quoted and stored in different containers for use in future procedures such as optimization and wastewater analysis. 1L of methylene blue stock solution is prepared by dissolving 0.03 g in volumetric shrimp using deionized water. This will give a methylene blue solution to have a concentration of 30 ppm or 30 mg L-1. Methylene blue will be used in various subsequent analytical uses such as data formulation and also optimization for various parameters. Figure 1. Schematic diagram of procedure for methylene blue adsorption from wastewater. Data Calibration For the data calibration, the dissolved methylene blue has been again dissolved to several concentrations, namely 5 ppm, 10 ppm, 15 ppm, 20 ppm, 25 ppm and finally 30 ppm. These solutions have been dissolved into 100 mL each. The concentrations of all solutions and as well as the equivalents can be calculated using the following equations.

5 ଵଵ = ଶଶ After the dissolving process, the sample of each solvent was taken and filled in a different cuvette as well as the absorbance readings were measured using the UV-vis Spectrophotometer. The wavelength that has been used during the measurement of absorption readings is the range of 500 nm to 700 nm. The wavelength to absorption reading chart has been plot first for each concentration. After that, a wavelength to absorption reading graph containing six-six concentrations was combined and painted onto a single graph. Finally, by using the highest absorption reading or top of the graph that has been plot for the respective concentration, a fixed wavelength has been determined. A absorption reading to concentration graph has been plotted according to the previously selected fixed wavelength. Optimization of Parameters for Methylene Blue Absorption Sample pH First, for different pH values, the pH value for each sample of methylene blue with a density of 30 ppm has been set at a pH value from 2 to 12, namely pH 2, 4, 6, 8, 10, 12. The discharge for each sample is 150 mL and all the samples are placed in different conical flask. After that, 1 g of the peanut husk absorbent was mixed into each conical flask containing different pH of methylene blue solution. The six of the cones were placed into the shutter in a parallel position and the shaking rate was set at 200 rpm. Parafilm have been used to close the opening of the flask to prevent the spill from occurring. A time period of 20 minutes has been set and the temperature used is also at room temperature of 25°C. After 20 minutes of shaking, the absorbance readings have been collected before and after the shaking occurs to determine the efficiency of different absorbent materials using the UV-vis spectrophotometer device. The calculation can be made using the following equation. = ௜ − ௙ ௜ × 100%

6 ௜ = ௙ = This step is repeated with the use of different absorbent materials, namely eggsehells, and also pomelo peels. The efficiency graph against pH values has been plot and the optimal pH value has been determined. Absorbent Dosage First, the calculated and found optimum pH value has been used in this second analysis. For the absorbent dose, the dose for each sample of methylene blue solution with a density of 30 ppm has been set at doses ranging from 0.5 g to 3 g, namely 0.5 g, 1 g, 1.5 g, 2 g, 2.5 g and 3 g. The discharge for each sample is 150 mL and all the samples are placed in the conical flask. The six of the conical flasks were placed into the shaker in a parallel position and the shaking rate was set at 200 rpm. Parafilm have been used to close the opening of the shell to prevent the spill from occurring. A time period of 20 minutes has been set and the temperature used is also at room temperature of 25°C. After 20 minutes of shaking, the absorbance readings have been collected before and after the shaking occurs to determine the efficiency of different shaking materials using the UV-vis spectrophotometer device. The calculation was done using the equation above. After that, these steps are repeated using different absorbent, namely eggshells, and also pomelo peels. The anti-dose absorbent material efficiency chart has been plotted and the optimal absorbent material dose has been determined. Contact Time First, the calculated and optimally found pH values and absorbent doses have been used in this third parameter analysis. For the contact time analysis, the time period used for each 30 ppm methylene blue sample was set at different times ranging from 20 minutes to 60 minutes, namely 20, 30, 40, 50 and 60 minutes. The discharge for each sample is 150 mL and all the

7 samples are placed in the conical flask. The five conical flasks have been placed into the shaker in a parallel position and the shaking rate has been set at 200 rpm. Parafilm have been used to close the opening of the shell to prevent the spill from occurring. The temperature used is also at a room temperature of 25°C. After the shake is completed, the absorbance readings have been collected before and after the shaking occurs to determine the efficiency of different shaking materials using the UV-vis spectrophotometer device. The calculation was done using the equation 2.2 above. After that, these steps are repeated using different absorber materials, namely eggshells, and also pomelo peels. The efficiency graph against the touch time period has been plot and the optimal contact time has been determined. Wastewater Analysis For wastewater analysis, 10 samples of collected wastewater will be supplemented with a 30 ppm blue methylene dye to be used as a spike solution. 10 samples of wastewater are taken from different locations but close to textile-producing factories. 10 mL of the sample has been mixed with dye with optimal parameters following analysis previously conducted in ten test tubes. After that, the test tube will be placed on top of the shaker and then added with the absorbent whose dosage is also determined by the optimal analysis. The temperature has been set at a room temperature of 25°C and the shaking rate has been established at 200 rpm. The time period and pH values have also been set at the optimal time period. After preparation, the absorption readings have been collected before and after the absorption occurred to determine the efficiency of the adhesion of different absorptive materials using the UV-vis Spectrophotometer device. The calculation was done using the equation above. After that, these steps are repeated using different absorber materials, namely eggshells, and also pomelo peels. RESULTS AND DISCUSSION FESEM Analysis

8 FESEM, also known as Field-Emission Scanning Electron Microscopy, is crucial in providing high-resolution imaging and surface characterization of absorbent materials. It aids in analyzing surface morphology and visualizing adsorption sites, enabling a better understanding of the adsorption mechanism and optimization of the adsorption process. Figure 1 (a) FESEM Analysis for Eggshell (b) FESEM Analysis for Peanut Husk (c) FESEM Analysis for Pomelo Peel Figure 1 (a) depicts the morphology of the eggshell adsorbent. It is found that the eggshell adsorbent has rod-shaped structures that are unevenly arranged and porous. This differs significantly from the study by Fahad et al. (2017), which found that the morphology of the eggshell adsorbent is irregularly shaped and uniformly dispersed (Fahad et al., 2017). Additionally, the study by Faiz et al. (2021) found that the adsorbent accumulates in irregular cloud-like structures with porosity (Faiz et al., 2021).

9 Figure 1 (b) refers to the morphology of the peanut shell adsorbent, revealing that it has uneven and closely packed elongated shapes. This also differs significantly from previous studies, such as the study by Aryee et al. (2020), which found a fibrous arrangement with a porous and rough surface (Aryee et al., 2020). Furthermore, the study by Srisamai et al. (2021) found that the adsorbent is in the form of layered fibers with multiple interconnections forming pores of different sizes (Srisamai et al., 2021). Figure 1 (c) shows the morphology of the pomelo peel absorbent. The FESEM analysis results indicate that the absorbent has uneven, porous, and rough surface characteristics. This aligns with previous analyses, such as the study by Ren et al. (2018), which found clearly visible pores in the pomelo peel absorbent but with a slightly different structure resembling a honeycomb (Ren et al., 2018). Additionally, the study by Zheng et al. (2020) supports the findings of the FESEM analysis and found that the pomelo peel absorbent has a porous and rough surface (Zheng et al., 2020). FTIR Analysis FTIR, known as "Fourier Transform Infrared," is one of the important analytical techniques for researchers. This is because FTIR can be utilized to characterize samples in various forms such as liquids, solutions, powders, films, and gases. FTIR is also capable of analyzing substances present on the surface of substrates (Nandiyanto et al., 2019). Through FTIR analysis, the sample interacts with infrared (IR) radiation, which affects the molecular vibrations of atoms in the sample, leading to the absorption or transmission of specific energy. Therefore, FTIR is useful for determining specific molecular vibrations present in the sample (Nandiyanto et al., 2019). The FTIR analysis for each absorbent after the absorption process is discussed as follows.

10 Figure 2 (a) FTIR Analysis for Eggshell (b) FTIR Analysis for Peanut Husk (c) FTIR Analysis for Pomelo Peel Figure 2 (a) shows the FTIR analysis of the eggshell absorbent sample. Eggshell is composed of 95% calcium carbonate, as indicated by the peak at 1422.7 cm-1, which corresponds to the carbonate group (-CO3), and the peak at 872.42 cm-1, indicating the presence of alkane groups (C-H). Eggshell also contains other components such as proteins and other organic substances that carry a negative charge. However, functional groups such as hydroxyl groups (-OH) and carboxyl groups (-COOH) that should have in the molecular structure of eggshell cannot be determined. This is because there is an overlap in the absorption bands of different functional groups, making it difficult to interpret the peaks. Figure 2 (b) shows the FTIR analysis of the peanut husk absorbent. Peanut husk contains cellulose and protein molecules. The peak at 3331.32 cm-1 indicates the presence of hydroxyl (-OH) functional groups, the peak at 1634.73 cm-1 corresponds to amine groups (- NH), the peak at 1025.23 cm-1 represents carboxyl groups (-CO), and the peak at 2925.15 cm-

11 1 also range of peaks from 1421.38 cm-1 to 1245.84 cm-1 signifies alkane (C-H) groups. This indicates the presence of peanut husk in the sample. Hydrogen bonding occurs between the positively charged methylene blue, such as amino NH2 or phenolic hydroxyl OH groups, and the NH and OH groups in the peanut husk. This interaction happens when the electronegative nitrogen atom in the NH group attracts the partially positive hydrogen atom in another molecule. This interaction enhances the absorption of methylene blue on the surface of the peanut husk. Hydrogen bonds can also form between the oxygen atom in the OH group and hydrogen atoms in other molecules, leading to the absorption of methylene blue on the surface of the peanut husk (Ossman et al., 2014). Figure 2 (c) shows the FTIR analysis of the pomelo peel absorbent. Pomelo peel contains molecules such as pectin and naringin (a flavonoid). The peak at 3338.91 cm-1 indicates the presence of hydroxyl functional groups, the peak at 1633.19 cm-1 corresponds to carbonyl groups, the peaks at 1149.98 cm-1, 1101.75 cm-1, 1052.69 cm-1, and 1017.43 cm-1 represent carboxyl groups, and the peak at 2934.9 cm-1 signifies alkane groups. These peaks indicate the presence of pomelo peel in the sample. The electrostatic interaction between the negative charges of the pomelo peel (carboxyl groups) and the positive charges of methylene blue facilitates the absorption process. The stretching of C-H bonds in both pomelo peel and methylene blue indicates the presence of hydrocarbons. These hydrocarbon groups can contribute to hydrophobic interactions between the pomelo peel and methylene blue molecules, facilitating the absorption of methylene blue onto the surface of the pomelo peel (Dinh et al., 2019). XRD Analysis The three samples that have been adsorbed from this research were analysed using x-ray diffraction (XRD) analysis method. In materials research, X-ray diffraction analysis (XRD) is a method to identify the crystallographic structure of a material. When using XRD, the material will be exposed to incoming X-rays, and subsequently, the intensity of X-rays and the scattering angle are measured. Furthermore, the identification of materials based on their diffraction patterns which is one of the main applications of XRD analysis. Besides that, XRD also provides information on how internal pressures and weaknesses causing the actual

12 structure to differ from the ideal structure (TWI 2020). In this research, XRD was used to identify the materials found in all the samples. Figure 3 XRD Analysis Based on the graph shown in Figure 3 (a) which is an eggshell sample, the observation that can be explained is that there are 6 high diffraction peaks at 2θ (29.4˚, 36.0˚, 40.0˚, 43.1˚, 47.5˚, and 48.5˚) on the base (104), (110), and (113) and other peaks at sites (202), (018) and (116). Each peak has the highest calcite composition. This is because there are several chemical compositions in the eggshell, which are calcium carbonate, phosphorus, magnesium, and sodium. In this XRD analysis, the percentage of calcium carbonate (CaCO3, Calcite) was chosen in the eggshell after adsorption because calcite contains the highest percentage of 95% compared to other chemical composition (Butcher and Miles 2021). The overall percentage of this graph has been analysed that calcite has a percentage of 26.6% and calcium thiocyanate hydrate as a representative for methylene blue is 73.4%. The percentage of calcite is lower than that of methylene blue and this indicates that the eggshell has adsorbed methylene blue with a very high concentration. Based on the graph shown in Figure 3 (b) which is a peanut husk sample, the observation that can be explained is that there are 5 high diffraction peaks at 2θ (14.8˚, 15.8˚,

13 16.4˚, 20.8˚, and 22.7˚). At the 2θ peaks (14.8˚, 16.4˚, and 22.7˚) which are based on (101), (101) and (002), there is the highest cellulose composition. This is because the lignocellulosic material in the peanut shell has a complex fibre structure and consists of cellulose as much as 44.8%, hemicellulose as much as 5.6%, and lignin as much as 36.1% (Rubin 2008). Due to the highest percentage of cellulose in the peanut shell, we chose cellulose as the material analysed to represent the peanut shell. Based on the XRD graph above, the percentage of cellulose is 31.2% and the percentage of heptadecylcyclohexane which represents methylene blue has a percentage of 68.8%. This shows that the adsorption rate of peanut husk is also high because the percentage of methylene blue is higher compared to cellulose. Based on the graph shown in Figure 3 (c), which is the XRD analysis of a sample of pomelo peel, the observation that can be explained is that there are 3 high diffraction peaks at 2θ (15.8˚, 21.2˚, and 21.5˚). At the 2θ peak (21.5˚) which is based on (200), there is the highest polyethylene composition. This is because pomelo peel accounts for approximately 30% of the total weight of the fruit and contains phytochemicals, including aroma active volatiles, pectin, flavonoids, phenolic acids, carotenoids, coumarins and polysaccharides which are polyethylene (Tocmo et al 2020). There is polyethylene in the pomelo peel because the surface of the peel does not absorb water. Based on the XRD graph above, the percentage of polyethylene is 30.1% and the percentage of heptadecylcyclohexane which represents methylene blue has a percentage of 69.9%. This shows that the adsorption rate of the pomelo peel is also high because the percentage of methylene blue is higher than that of polyethylene. Quantitative Analysis For data calibration, all data has been measured using the UV-vis spectrophotometer. The data that will be used when making the calculation will be referenced to the data calibration graph. The graph that has been plot will be shown as in Figure 4 below. Table 1. Regression statistic Regression Statistics Multiple R 0.9985

14 R Square 0.9969 Adjusted R Square 0.9954 Standard Error 0.0730 Observations 4 Coefficients Intercept 0.072 Conc. Variable 0.097 LOD (mg/L) 2.444 LOQ (mg/L) 7.405 Figure 4 Calibration Curve of Absorbance Against Concentration The graph that has been plot shows absorption increasing as concentration increases. This graph can be used to calculate the absorption efficiency as well as the efficiency rate for y = 0.097x + 0.1317 R² = 0.9969 0 0.5 1 1.5 2 2.5 3 3.5 0 5 10 15 20 25 30 35Absorbance Concentration (mg per L)

15 different parameters such as pH, absorbent dose and contact time by using the equation sateted above. Parameter of Sample pH In this study, the efficiency of various absorbents derived from pomelo peels, peanut shells, and eggshells will be tested under different pH conditions. The pH levels tested include 2, 4, 6, 8, 10, and 12. To evaluate the efficiency of these absorbents, a UV-vis spectrophotometer will be used to measure data and plot efficiency graphs based on the absorbent dosage. Figure 5 presents a specific graph related to the analysis of eggshell absorbent. Figure 5 Graph of Percentage Efficiency (%) Against pH Based on the plotted graph, the efficiency of eggshell absorbent increases with increasing pH. It can be observed that higher pH values correspond to higher efficiency of eggshells as an absorbent. Therefore, the maximum efficiency for eggshells is achieved at pH 12, with an efficiency of 40.34%, while pH 2 to pH 10 shows no adsorption taking place with eggshells. For peanut shell, the efficiency of the absorbent increases from pH 2 to pH 4, decreases from pH 4 to pH 6, increases from pH 6 to pH 10, and decreases from pH 10 to pH 12. For pH 2, pH 4, pH 6, pH 8, pH 10, and pH 12, the efficiencies are 65.68%, 74.40%, 73.18%, 76.00%, 78.44%, and 72.98%, respectively. The optimal pH condition for peanut shell 0 10 20 30 40 50 60 70 80 90 100 2 4 6 8 10 12Efficiency (%) pH Eggshell Peanut Husk Pomelo Peel

16 absorbent is at pH 10 with an efficiency of 78.44%. For pomelo peel, the efficiency of the absorbent increases from pH 2 to pH 6 and decreases from pH 6 to pH 12. For pH 2, pH 4, pH 6, pH 8, pH 10, and pH 12, the efficiencies are 53.93%, 61.78%, 76.01%, 63.63%, 61.60%, and 61.26%, respectively. The optimal pH condition for Bali lemon peel absorbent is at pH 6 with an efficiency of 76.01%. The different pH conditions play a significant role in the adsorption efficiency of methylene blue by the different absorbents. In this study, pH values ranging from 2 to 12 were tested, allowing for a comprehensive assessment of the performance of the three different absorbents: pomelo peel, peanut shell, and eggshell. The results indicate that the efficiency of methylene blue adsorption varies significantly with changes in pH for all three absorbents. Starting with eggshells, it exhibits negligible adsorption efficiency at pH 2, 4, 6, and 8 but shows a significant increase in efficiency at pH 10 (40.34%), which is the optimum pH, consistent with previous studies (Ngadi et. al. 2013). This suggests that eggshells are not effective absorbents for methylene blue at low to neutral pH levels but exhibit optimal adsorption characteristics at pH 10. At pH 10, the surface properties of the eggshell material appear to promote adsorption, possibly due to increased surface ionization or charge. In the case of peanut shells, the adsorption efficiency shows an increasing trend with increasing pH, reaching maximum efficiency of 78.44% at pH 10, which significantly differs from previous studies where the optimum pH was pH 8 (Castalino & Aravinda, 2020). This indicates that peanut shells are more effective as absorbents under alkaline conditions. The enhanced adsorption efficiency at higher pH levels can be attributed to the increased availability of negatively charged sites on the absorbent surface, facilitating electrostatic interactions with the positively charged methylene blue molecules. Next, pomelo peel exhibits a different adsorption behaviour compared to the other two absorbents. It shows the highest efficiency at pH 6 (76.01%) and relatively lower efficiencies at pH 2 (53.93%), pH 4 (61.78%), pH 8 (63.63%), pH 10 (61.60%), and pH 12 (61.26%), which significantly differs from previous studies where the optimum pH was pH 5 (Tanzim & Abedin, 2015). The observed pH dependency for pomelo peel indicates that surface chemistry and ionization characteristics of the absorbent material are important factors governing the adsorption process. The maximum adsorption at pH 6 can be attributed to the optimal

17 combination of surface charge and active site availability, creating favourable conditions for methylene blue adsorption. Overall, pH parameters greatly influence the adsorption efficiency of methylene blue by the three absorbents. The observed variations in efficiency at different pH values can be linked to changes in surface charge, ionization, and active site availability on the absorbents. These findings highlight the importance of pH optimization when employing adsorption processes for methylene blue removal using pomelo peel, peanut shell, or eggshell as potential absorbents. Further studies are warranted to investigate the underlying mechanisms controlling the pH-dependent adsorption process and optimize process conditions for maximum efficiency. Parameter of Absorbent Dosage The quantities of absorbent are crucial factors in determining the absorbent's capacity that need to be used. They were investigated using different amounts of absorbents, added to a constant initial dye concentration and an optimized pH that had been analyzed. The mixtures were then shaken for a set period of time. The dosing levels used were 0.5g, 1.0g, 1.5g, 2.0g, 2.5g, and 3.0g. UV-vis spectrophotometer was used to measure the data and plot the graph efficiency against the dosage of the absorbent material.

18 Figure 6 Graph of Percentage Efficiency (%) against Dosage (g) Based on Figure 6, the efficiency of eggshell increases with an increase in the dosage of the absorbent material. It can be observed that higher dosages result in higher efficiency. Therefore, the maximum efficiency for eggshell is achieved at a dosage of 3.0g, with an efficiency of 67.65%, while the minimum efficiency is obtained at a dosage of 0.5g, which corresponds to 28.20% efficiency. On the other hand, the efficiency of peanut husk shows an inconsistent pattern with varying dosages of the absorbent material. It exhibits a wave-like graph that oscillates periodically. Therefore, the maximum efficiency for peanut husk is attained at a dosage of 1g, with an efficiency of 73.81%, while the minimum efficiencies are observed at dosages of 1.5g and 3g, corresponding to 70.60% efficiency. Meanwhile, the efficiency of pomelo peel decreases with an increase in the dosage of the absorbent material. It can be observed that higher dosages result in lower efficiency. Therefore, the maximum efficiency for pomelo peel is achieved at a dosage of 0.5g, with an efficiency of 65.61%, while the minimum efficiency is obtained at a dosage of 3g, corresponding to 38.07% efficiency. 0 10 20 30 40 50 60 70 80 90 100 0.5 1 1.5 2 2.5 3Efficiency (%) Dosage (g) Eggshell Peanut Husk Pomelo Peel

19 These results indicate that the three absorbent materials exhibit different absorption efficiencies. For eggshell, a dosage of 3.0g is optimal for the absorption of methylene blue. This is due to the availability of more absorption sites at higher dosages. With a higher amount of eggshell absorbent, more molecules of methylene blue can be absorbed and bound from the solutio n (Tark et al., n.d.). Thus, higher absorption efficiency is achieved at higher dosages. For peanut husk and pomelo peel, the optimal dosages for methylene blue absorption are 1.0g and 0.5g, respectively. At these dosages, the quantity of absorbent material used is sufficient to achieve maximum absorption of methylene blue. The particle density of the absorbent reaches a point where the available absorption sites for methylene blue are optimal, allowing for efficient absorption to occur. The subsequent decline in efficiency after reaching the optimum is attributed to the density or aggregation of the absorbent particles. Particle aggregation leads to a decrease in absorption efficiency (Kayranli et al., 2019). Among the three absorbents, peanut husk exhibits the highest efficiency at 73.81%, followed by eggshell at 67.65%, and pomelo peel at 65.61%, based on their respective optimal dosages. Comparison with other Study Parameter of Contact Time A graph of the percentage efficiency (%) of the absorbent against the contact time (minute) shows the effect of time contact on adsorption for all three absorbents.

20 Figure 7 Graph of Percentage Efficiency (%) against Contact Time (min) Figure 7 shows the absorbents’ efficiency against the contact time for all three samples which are eggshell, peanut husk and pomelo peel. For the eggshell graph (blue) shows that at the 20th minute the efficiency was 67.65%, then increased to 68.84% at the 30th minute, and reached a maximum efficiency of 70.30% at the 40th minute, the efficiency of the absorbent eggshell to absorb methylene blue is increasing. However, the graph shows a decrease in efficiency after this point. In the 50th minute, efficiency dropped to 67.99%, and at the 60th minute, it rose again to 69.29%. Based on the peanut husk graph (orange) shows that at the 20th minute, the efficiency of the peanut husk absorbent reached the maximum of 73.25% and shows decrease after 20 minutes. At the 30th minute, the absorbent efficiency decreases to 58.82%, then decreases again to 54.88% at the 40th minute. But at the 50th minute, efficiency increases slightly to 59.34% and decreases again at the 60th minute which was 55.71%. Based on the graph above, the pomelo peel graph (grey) shows that at 20th minute, the adsorption efficiency of the pomelo peel reached the maximum of 72.13% and shows a sharp decrease after 20 minutes. At 30th minute, the absorbent efficiency decreased to 68.87%, then decreased further to 68.79% for 40th minute. At the 50th minute, the absorbent efficiency decreases to 67.57% but increases at the 60th minute with the efficiency is 68.82%. 0 10 20 30 40 50 60 70 80 90 100 20 30 40 50 60Efficiency (%) Contact Time (min) Eggshell Peanut Husk Pomelo Peel

21 The results for the optimal contact time for eggshell is at the 40th minute (70.30%) which is pH 12 and the use of a dose of 3.0g while the results for the time the optimal contact for peanut husk is at the 20th minute (73.25%) which is pH 10 and the use of a dose of 1.0g and the results for the optimal contact time for the pomelo peel is at the 20th minute (72.13%) which is pH 6 and the use of a dose of 0.5g. A rapid increase in the rate of adsorption in the first 40 minutes for eggshell, 20 minutes for peanut husk and pomelo peel can be attributed to the presence of a few large empty site, occupied by time. Increase contact time though up to 150 minutes cannot produce a useful increase in efficiency adsorption because at a certain minute, it has reached the equilibrium time for the absorbents. At equilibrium, there is a decrease in the number of active sites and the force induces adsorption, which results in adsorption not being able to continue (Wu et al 2013) resulting in fewer accessible sites, and the absorbent moves toward crowding resulting pores, which restrict the mobility of the absorbent. Wastewater Analysis For wastewater analysis, we have used 10 different types of wastewaters and used different shell types to analyse the shell’s efficiency in adsorption. For the eggshell, we will start the analysis with 10 types of wastewaters that have been collected from 10 different rivers but near to the textile industry. For all types of wastewaters collected can be categorized into wastewater A to J. Wastewater A to J is collected from Sungai Klang, Sungai Pinang, Sungai Damansara, Sungai Gombak, Sungai Langat, Sungai Kluang, Sungai Perai, Sungai Juru, Sungai Jawi, and Sungai Kuyoh respectively.

22 Figure 8 Graph of Percentage Efficiency for Eggshell against Wastewater Based on the graph shown in Figure 8 which is a graph of the adsorption efficiency against 10 wastewaters river by using eggshell. For the eggshell, the results obtained can conclude that the eggshell is not very effective for wastewater treatment for all river water samples. All of them samples have shown an efficiency of no more than 40%. Figure 9 Graph of Percentage Efficiency for Peanut Husk against Wastewater Based on the graph shown in Figure 9 which is a graph of the adsorption efficiency against 10 wastewaters river by using peanut husk. As for peanut husk, wastewater A is the most efficiently absorbed wastewater which is close to 100% compared to other wastewater while D wastewater is the lowest efficiency wastewater by using peanut husk which is close to 0 10 20 30 40 50 60 70 80 90 100 A B C D E F G H I J Efficiency (%) Wastewater 0 10 20 30 40 50 60 70 80 90 100 A B C D E F G H I J Efficiency (%) Wastewater

23 10%. On average, the efficiency is higher than half that is only 50% occurred in 5 out of 10 wastewater samples and the other 5 were less than 50 %. Figure 10 Graph of Percentage Efficiency for Pomelo Peel against Wastewater Based on the graph shown in Figure 10 which is a graph of the adsorption efficiency against 10 wastewaters river by using pomelo peel. As for the pomelo peel, wastewater A is still the most efficient wastewater adsorption which is close to 90%. Wastewater C is the least efficient wastewater which is less than 10%. Most wastewater has achieved an efficiency of more than 50%. The conclusion is pomelo peel is the absorbent with the highest efficiency for all types of wastewater collected from various rivers. Conclusion Overall, every objective in this test has been achieved. Data calibration is carried out by plotting the absorption graph against concentration and is used to determine the concentration of methylene blue having a R2 value of 0.9969 and showing the data measurement graph is reliable. For the pH parameter of the solution, it was found that the optimum pH for eggshell absorbent is at pH 10 that is, with an efficiency of 40.34%, the optimal pH for peanut husk absorbent is at the pH 10 which is as much as 78.44% and the ideal pH for pomelo peel 0 10 20 30 40 50 60 70 80 90 100 A B C D E F G H I J Efficiency (%) Wastewater

24 absorbent is on the pH 6 that is as many as 76.01%. For the absorbent dosage parameter, for eggshell, it was found that the optimal dose is at 3.0 g with an efficiency of 67.65%, for peanut husk the found optimum dosage is at 1 g with an effectiveness of 73.81% and for pomelo peels, the found optimal dose was at 0.5 g with efficiency of 65.61%. For the contact time parameter, for the eggshell, we found contact time is at the 40th minute with an efficiency of 70.30%, for the peanut husk, found the optimum contact time was at the 20th minutes with an efficiency of 73.25%, and for the pomelo peels, was found the optimal time is in the 20th minute with a efficiency of 72.13%. Acknowledgements First of all, we would like to express our gratitude to the Lord because with his grace and grace, we can complete this report perfectly within the specified time frame. I would also like to thank Dr. Noorashikin Md Saleh is the supervisor of the group, who has given us a lot of teaching and guidance and shared information in helping us complete this report. In addition, we would also like to express the highest recognition on the part of the Department of Chemical and Process Engineering, Faculty of Engineering and Built Environment, Universiti Kebangsaan Malaysia, Bangi, Selangor for the facilities that have been provided to the students such as the equipment facilities for obtaining tools related to the tasks and facilities to facilitate us to obtain information related to this laboratory work. We would also like to express millions of thanks to our group members for their cooperation and commitment as well as during the preparation of this report. Finally, we would like to express thousands of thanks to classmates for showing teaching and encouragement as well as sharing information and not hesitating in channelling the views and information that we have completed this course. Comparison of Efficiency Absorbance

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CINNAFRESH: INNOVATIVE ACTIVE PACKAGING FOR FRESH-CUT FRUITS AND VEGETABLES Zhi Zhou Siewa , Eric Wei Chiang Chanb , Chen Wai Wongc aDepartment of Food Science with Nutrition, Faculty of Applied Sciences, UCSI University, Kuala Lumpur, Malaysia. ([email protected], +60164115843) bDepartment of Food Science with Nutrition, Faculty of Applied Sciences, UCSI University, Kuala Lumpur, Malaysia. ([email protected], +60163610678) cDepartment of Biotechnology, Faculty of Applied Sciences, UCSI University, Kuala Lumpur, Malaysia. ([email protected], +60179851286) Background Fresh-cut fruits and vegetables (FFV) are popular among consumers because they are convenient, minimally processed and rich in vitamins and minerals. The spoilage of FFV is a major contributor to global food waste, accounting for an estimated 40 to 50% of such waste, equivalent to 520 to 650 million tonnes [1]. Maintaining the freshness of FFV presents a significant challenge, largely due to enzymatic browning (EB) and microbial spoilage [2]. Presently FFV are kept fresh with refrigeration which is costly and chilling damage can exacerbate EB [3,4]. Coupled with this, the prevalent use of conventional single-use plastic food packaging poses a severe environmental challenge due to its non-biodegradable and nonrenewable nature, resulting in extensive environmental pollution including landfilling, incineration and marine ecosystem disruption. Moreover, biodegradable and biorenewable plastics, although superior alternatives to petroleum-based plastics, can contaminate recycling streams, making them particularly apt for single-use food packaging which often isn't recycled due to the complexities in separating plastic from food residues. Innovative product CinnaFresh, an innovative bio-renewable and sustainable active packaging, offers a solution to the dual challenge of FFV spoilage and environmental pollution from plastic waste. By utilizing bio-renewable materials like cellulose acetate, cinnamon bark extract and polyethylene glycol (PEG) 1450, CinnaFresh not only maintains and extends the quality and shelf life of FFV, but also positions itself as an environmentally friendly alternative that promotes sustainable consumption and reduced plastic waste. This breakthrough in active

packaging stands to redefine industry standards by enhancing food supply chains, potentially altering the infrastructure of fresh produce storage. Novelty Breaking away from conventional preservation methods, CinnaFresh pioneers a new frontier in active packaging, amalgamating both environmental integrity and food preservation efficacy. By harnessing the bio-renewable properties of cellulose acetate, the antimicrobial prowess of cinnamon bark extract and the stabilization offered by polyethylene glycol (PEG) 1450, CinnaFresh establishes itself as an ecologically responsible alternative to traditional single-use plastics. Cellulose acetate is an ideal as a food packaging material as it biodegrade within 1 to 10 years, which allows it to maintain its integrity on the shelf [5,6]. Its demonstrated capability to inhibit enzymatic browning and retard fungal growth across a diverse range of FFV samples [7,8], all without the need for refrigeration, is a testament to its groundbreaking nature. Moreover, its consumer-friendly transparent design with a subtle yellowish tint ensures visual quality assurance, while its distinction of reaching Technology Readiness Level 5 (TRL 5) signifies its readiness for real-world application and its potential to revolutionize the food packaging industry. Efficacy The efficacy of CinnaFresh as a novel, sustainable active packaging solution has been rigorously tested on six diverse types of FFV at room temperature. In specific tests on apple, banana, celery, lettuce, mung bean sprout, and potato, CinnaFresh showcased its prowess in effectively preventing browning in fresh-cut produce with low phenolic content (below 54.39 ± 2.71 mg GAE/100 g FW), such as celery, lettuce, and mung bean sprout for a commendable duration of up to 4 days. The underlying science reveals that the cinnamon bark extract's bioactive compound, (E)-cinnamaldehyde, acts as a competitive PPO inhibitor. However, its effect can be compromised by the presence of high concentrations of phenolic substrates in the produce. Impressively, CinnaFresh also extended the yeast and mold resistance of all tested FFV samples by a week. A noteworthy added benefit is its ability to deter shrinkage in freshcut lettuce samples. In direct comparison, produce packaged in just cellulose acetate bioplastic, without the CinnaFresh treatment, was found to degrade in quality and become unsalable in a mere two days.

Commercialization plan To capitalize on CinnaFresh's proven efficacy at TRL 5 and its unique proposition of extending shelf-life without refrigeration, our immediate commercialization strategy is to secure investment for a pilot-scale production line, targeting a capacity of 5,000 units/day with an initial investment of RM 122,000 for the necessary machinery. This setup offers a swift capital recovery, projected at just 175 days. In parallel, our team will engage with supermarkets and retailers, customizing CinnaFresh to seamlessly integrate with their fresh-cut produce lines. As production ramps up, we'll collaborate with OEMs to ensure consistent and quality packaging material supply. Initial success and market validation will pave the way for a broader roll-out, positioning CinnaFresh as the go-to sustainable packaging solution across an expansive network of supermarkets and retailers, heralding a new era in eco-friendly and efficient produce packaging. Societal impact CinnaFresh food packaging presents a transformative solution addressing key societal challenges, primarily targeting the Sustainable Development Goals (SDGs) related to good health and well-being (SDG 3), industry, innovation and infrastructure (SDG 9), and responsible consumption and production (SDG 12) [9]. By substantially extending the shelf life of fresh-cut produce without the need for refrigeration, CinnaFresh directly combats food waste, contributing to improved food security and reduced health risks from contaminated produce. Furthermore, its innovative biodegradable design provides an eco-friendly alternative to singleuse plastics, championing sustainable consumption patterns and reducing environmental pollution. As such, CinnaFresh not only promotes healthier and sustainable living but also encourages industries to adopt and invest in groundbreaking, environmentally-conscious innovations, thus accelerating our journey towards achieving the United Nations' Sustainable Development Goals. References: [1] M.C. Boliko. 2019. Journal of Nutritional Science and Vitaminology, 65, S4-S8. [2] I. Ioannou, & M. Ghoul, M. 2013. European Scientific Journal, 9(30), 310-341. [3] N.J.H. Se Hoo, Z.Z. Siew, W.Y. Lim & C.W. Wong. 2022. Journal of Food Processing and Preservation, 46(11), e16963. [4] Z.Z. Siew, E.W.C Chan, & C.W. Wong. 2022. Journal of Food Processing and Preservation, 46(12), e17216.

[5] B. Rohmawati, F. Atikah Nata Sya’idah, R. Rhismayanti, D. Alighiri, & W. Tirza Eden. 2018. Oriental Journal of Chemistry, 34(4), 1810–1816. [6] H. Kurmus, & A. Mohajerani, A. 2020. Waste Management, 104, 104–118. [7] M.B.S. Weerawardana, G. Thiripuranathar, & P.A. Paranagama, P. 2020. Journal of Chemistry, 2020, 1-6. [8] S. Andrade-Ochoa, K.F. Chacón-Vargas, L.E. Sánchez-Torres, B.E. Rivera-Chavira, B. Nogueda-Torres, & G.V. Nevárez-Moorillón. 2021. Membranes, 11, 405. [9] United Nations Sustainable Development Goals, 2023. Available at: https://sdgs.un.org/goals [Accessed 17 August 2023].

Active and Smart Packaging Based on Gelatin with the Addition of Anthocyanin from Purple Cabbage (Brassica oleracea var. capitata L) and Essential Oil from Lemongrass (Cymbopogon citratus) Tika Mayang Saria , Sri Muliyawatib , Ifa Puspasaric , Muvika Putri Puspitasarid , Mita Dian Cahyanie aUniversitas Islam Indonesia, Indonesia ([email protected], +62 822-6926-3270) bUniversitas Islam Indonesia, Indonesia ([email protected], +62 877-2836-7565) cUniversitas Islam Indonesia, Indonesia ([email protected], +62 858-1052-0363) dUniversitas Islam Indonesia, Indonesia ([email protected], +62 822-4176-9656) eUniversitas Islam Indonesia, Indonesia ([email protected], +62 813-2569-1562) Along with the development of the food industry, food packaging is expected to be able to create new innovations that can meet and follow current trends. In the food industry, packaging is very important where the quality of the food ingredients in the packaging must be well maintained. There are several factors that cause a decrease in the quality of packaged food products. External factors include exposure to light, dust, temperature, carbon dioxide, oxygen and also internal factors including chemical substances that come from within the food itself and microorganisms that cause spoilage. Many food packages are still produced using synthetic polymers. This raises some concerns for the environment due to its non-biodegradable nature. Therefore, packaging that can detect a decrease in food quality by just looking at the sensory devices on the packaging to be implemented in society is needed, making it easier to assess the suitability of a product for consumption. Active packaging is an innovative strategy designed to increase product safety and maintain the quality aspects of a packaging. Active packaging focuses on antimicrobial and antioxidant activity so that it can extend the shelf life of food. This active packaging can be obtained from materials containing antimicrobials which are considered to inhibit the appearance of spoilage bacteria such as essential oils. Meanwhile, smart packaging is designed to provide information about the condition or quality of the product in the packaging. This intelligent packaging system will utilize biopolymer compounds combined with dyes contained in anthocyanins which have sensitivity to pH so that they can show color changes that occur when there is a change in product quality.

This research aims to produce intelligent active packaging from gelatin by adding purple cabbage extract and lemongrass leaf essential oil. Purple cabbage contains anthocyanin which can be an indicator in smart packaging, while the essential oil of lemongrass leaves has antimicrobial capabilities so it functions as active packaging. There are three stages conducted in this research. The first stage is the extraction of anthocyanins from purple cabbage, the second stage is making a gelatin film-purple cabbage extract-essential oil, and the third stage is testing the effectiveness of the film to analyze the freshness of fish filets. Different content of purple cabbage extract was added in the film of 0%, 2%, and 4% w/v. Variations of lemongrass essential oil were added in the range of 0 ml, 0.75 ml, and 1.5 ml. The results of the research show that purple cabbage extract changes color at different pH, namely pink at pH 2-4, purplish pink at pH 5-10, blue at pH 11-12, and green at pH 13. This shows the sensitivity of the purple cabbage extract sensitive to the pH. The addition of purple cabbage extract to the film causes the film to turn purplish, while the addition of lemongrass leaf oil extract causes the film to turn yellowish. However, all the films produced have transparent properties. The films produced in this research have a thickness of between 0.025 - 0.105 mm, which still meets the standard thickness of edible film. The results of testing the mechanical properties of the film show that the film has a tensile strength value between 0.75 - 5.97 MPa while the film elongation value is in the range 46 - 176%. In this study, the average water content of the film obtained was 2.18%, which meets the standards of the Food and Agriculture Organization [1]. Water vapor permeability (WVP) is a critical measurement that is important for food packaging, because the main function of food packaging is to prevent or reduce the transfer of moisture between food and the surrounding environment. Based on this research, the average WVP value is 0.23, which meets the Japanese Industrial Standard [2] with the maximum WVP value for food packaging being 10 WVP. The biodegradability test showed that the film could degrade about 85% within 15 days. The application of the film as fish filet packaging for 48 hours results in the film containing purple cabbage extract and lemongrass leaf oil having a lower pH compared to film without additional extracts and essential oils. This shows that films with the addition of purple cabbage extract and lemongrass oil can extend the shelf life of fish filets. Apart from that, the film also

shows a change in color to brownish green with the increasing shelf life of the fish. This shows that film can be used as an indicator of food freshness. Based on the research results obtained, it can be concluded that anthocyanin produced from purple cabbage extract can be used as a pH indicator by changing color at several different pH values. The pH-sensitive properties of gelatin-anthocyanin-essential films can be used as active and intelligent packaging because they can extend the shelf life of food while also having the ability to monitor the freshness of fish filets. References [1] Food and Agriculture Organization of the United Nations Sub Regional Office for the Pacific Islands, 2012. Manual on Food Packaging for Small and Medium Size Enterprises in Samoa [2] Japanese Industrial Standard. 1975. Japanese Standards Association, Vol. 2: 1707

UTILIZATION OF WASTE RICE FLOUR AS A BIOPLASTIC WITH THE ADDITION OF UWI YAM STARCH Syafa Atika Widya Wati1, Aldi Widya Suwandi2 , Ifa Puspasari3 , Sri Endah Fitriani4 , Rahmatan Amartya Ula5 Department of Chemical Engineering, Universitas Islam Indonesia, Yogyakarta 55584, Indonesia ([email protected], +62 858-1052-0363) The use of plastic packaging required by society has a very high amount, because plastic is a lightweight and easy to use item.According to data from the National Waste Management Information System (SIPSN) of the Ministry of Environment and Forestry (KLHK), the monthly volume of garbage in Indonesia by 2022 reached 19.45 million tons. Excessive use of plastic can have a negative impact on the environment, because plastic is difficult to degrade and can lead to the accumulation of garbage. One type of plastic that is beneficial to the environment is biodegradable or also called bioplastic.This type of plastic is entirely made of renewable raw materials and can be degraded by microorganisms.In this research, we have created an innovation that uses organic garbage in the form of rice jams (aking) and added kanji flour Dioscorea spp. (uwi yam). Studies have shown that bioplastics produced from the waste rice alone have weaker mechanical strengths, are more rigid and are easy to break, while bioplasts made from uwi yam bulbs alone have good elasticity but are less mechanical. Therefore, this research was carried out by combining straw rice and uwi onions in bioplastic production with the aim of obtaining bioplasty that has better characteristics and meets standards. In this study there are five stages, namely, the first stage of the preparation of the research material, the second stage of extraction of the purple uwi bubble soot which in this stage uses sodium metabisulfite with the aim of inhibiting the bloating reaction on Uwi yam bubbles, the third stage of waste rice flour with a meal size of 100 mesh, the fourth phase of making a solution of soot, the fifth stage of bioplastic making with variations of the composition of rice and uwi yam bubbles. The results of the research were several tests: elongation test, pull test, thickness test, biodegradation test and water absorption test. Then on the bioplastic characteristic test is the elongation test that aims to find out the extension value that results in the maximum change that occurs during the intersection until the Bioplastics sample is cut off. Elongation test results of each composition obtained a score of 84.79% - 136.42%. The elongation values at the highest were achieved on bioplastics with a composition of 30% waste rice and 70% of uwi yam oatmeal is of 136,42%, where the values have met the National Standard of Indonesia (SNI) 718:2016 which is ranging from 21% - 220%. Traction tests are carried out to determine the maximum value of traction that the bioplastic is capable of holding. In the bioplastic with a composition of 30% waste rice and 70% yam uwi, a test of the film thickness of 0.181 mm - 0.298 mm was obtained from the results of each composition. The test results showed the average value of 0.24 mm, which meets the standard of JIS (Japanese Industrial Standard) in Krochta et al., (1994) which is a maximum of 0.25 mm, and from the thickening tests carried out it can be found that the lower the concentration of rice, the higher the value of the bio-plastic thicker. The average time it takes for a bioplastic to be perfectly degraded is 3 days. In each composition, the time range is 1-3 days until the biodegradation is perfectly achieved using soil media because the soil contains various types of microorganisms such as bacteria, fungi and mushrooms in large quantities that can support the degradation process. It already meets the standard of biodegradable plastics based on the ASTM D6400 standard and the European standard EN 13432. Then the water absorption test is performed with the aim to find out the occurrence of binding in the polymer as well as the degree or regularity of the binding within a polymer determined by the presence of the addition of the weight of polymer after undergoing development. The water absorption test was carried out by inserting the bioplastic into 100 ml of aquadest for

5 minutes, which showed an average value of 88.75% and at each variation of the composition the value was obtained of 87.75% - 89.82% where the more mass of the purple uwi bubble starch added will obtain a lower water absorbtion value the better. Based on the results of the research carried out, it can be concluded that bioplastics that have a composition of 30% waste rice and 70% purple uwi pepper may have better characteristics compared to other compositions. The results of this research give the potential to utilize the waste of rice so that it has a greater added value. References [1] ANIK, K. (2019). Pemanfaatan Nasi Aking Sebagai Bahan Baku Pembuatan Bioplastik dengan Campuran Kitosan dan Gliserol (Doctoral dissertation, Universitas Gadjah Mada). [2] Ulfah, Z. (2016). Karakteristik Fisik Dan Mekanis Bioplastik Terhadap Variasi Temperatur Dan Lama Pengeringan Berbahan Dasar Nasi Aking (Doctoral dissertation, Universitas Brawijaya). [3] Albar, A., Rahmaniah, R., & Ihsan, I. (2021). PEMBUATAN DAN KARAKTERISASI BIOPLASTIK BERBAHAN DASAR PATI UMBI UWI UNGU, PLASTICIZER GLISEROL DAN KITOSAN. Teknosains: Media Informasi Sains dan Teknologi, 15(3), 253-257. [4] E. Indrastuti. Harijono. B. Susilo. 2012.Jurnal Teknologi Pertanian. 13. 169-170. [5] H. Haryanto. F. Retno., T. 2017. Jurnal Fakultas Teknik. 18.

SULFUR BEAD-MILLING PROCESS FOR RUBBER ARTICLES Mohamad Firdaus Omara , a Department of Chemical Engineering and Sustainability, Kulliyyah of Engineering, IIUM, P.O. Box 10, 50728 Kuala Lumpur. Email: [email protected] Phone num.: +6011-2076 3772 In the world of modern materials science and engineering, there is a big focus on finding ways to be kind to the Earth. This is because we are facing some big challenges worldwide – like the changing climate and using up important resources. It is becoming equally important to figure out how to do things in a way that does not “hurt” our planet or the people living on it. Production of goods by factories has been becoming necessary where there’s demand from us (people) as various product consumers. Understanding parts of the manufacturing processes are essential as in to reduce wastages that may arise from both suppliers and consumers. Essentially, most of manufacturing of materials or products will employ bead-milling process in one of their processes for various purposes. Bead-milling is an eco-friendly method known for modifying materials via friction, collision, impact, and shear from milling beads and the chamber wall. This process generally alters particle size, surface properties, morphology, and functional characteristics including solubility, water absorption, swelling, pasting, and gelation of materials [1]– [3]. Numerous researchers have employed the bead-milling process to alter their products or materials and achieve desired attributes as in [4]–[6] – where in short, these studies reflected bead-milling’s wide application scope across industries like chemicals, nanomaterials, food processing, and pharmaceuticals, impacting particle size, surface morphology, stability, and overall product performance. A curing agent, also known as a curative, is a chemical used to initiate reactions, particularly for cross-linking elastomer molecules and it is a common curative for rubber. Colloidal or dispersed sulfur, created through mixing/milling, yields fine particles that suspend well, suitable for rubber production. Insoluble sulfur is preferred over soluble to prevent “sulfur blooms”, which can hinder rubber adhesion and product performance [7]–[9]. Extensive research aims to improve sulfur’s dispersion, involving ball-milling for dispersion [10], modified sulfur for compatibility [11], and nano-additives for mechanical properties [11], enhancing elastomeric materials {Citation}. While various milling parameters’ effects on products are studied, relatively lack of studies or exploration on sulfur dispersion, particularly on its dispersion performance after bead-milling. This study investigates how flow rate and rotational speed affect stability and its performance in the rubber matrix – optimizing bead-milling for sulfur to yield elastomeric items with superior properties.

In principle, “sustainability” encompasses the philosophy of responsible stewardship towards the current resources – focusing on leveraging aspects such as economic growth, social equity, and environmental protection and preservation. In other words, it is also an approach that seeks to meet the present needs without negatively impacting the ability of future generations to meet their needs. The bead-milling study on sulfur dispersion conforms with the very importance of sustainability – employing integration of multidisciplinary understanding of complex systems and interactions. The concept of sustainability in the bead-milling study is that it addresses the necessity for more efficient and environmentally conscious methods of material modification. Hence, by leveraging the capabilities of current resources on production of materials or goods – especially on unit operations such as the bead-milling – the study promotes essentially to reduce waste generation, enhance resource efficiency, and improve product performance while may as well minimize the impact towards ecological footprint. The implementation of sustainable concepts on this bead-milling study process parameters towards sulfur for rubber articles application aligns significantly with the United Nations’ Sustainable Development Goals (SDGs). SDG is a comprehensive framework that go towards tackling the world’s most pressing challenges – sustainability. The bead-milling study on sulfur dispersion compromises particularly with Goal 9 (Industry, Innovation, and Infrastructure) as it brings about innovative methods for materials processing, as well as it correlates with Goal 12 (Responsible Consumption and Production) via its potentiality to optimize utilization of available resources and minimize wastes. Additionally, by potentially leading to processes with lower energy consumption and emissions, the study indirectly supports Goal 13 (Climate Action). The realization of sustainable goals necessitates the integration of a multiple range of plans and strategies that encompasses technical, economic, and social dimensions. In the context of the beadmilling study, these strategies can include technical essentials such as advanced process optimization techniques, exploration of available resources in conjunction to running the bead-milling process, as well as the establishment of robust manufacturing systems to minimize waste generation. In this point, collaborative efforts between academia, industries experts and regulatory bodies may be necessary to realize and drive these strategies effectively. The impact of adopting a proper process studies such as the bead-milling on sustainability aspects can contribute not only towards safer and more efficient methods of material production and modification, but it also may cultivate continuous improvement and innovation, promote economic growth, and decrease the burden on waste management systems. In the aspect of environment, this may also contribute to the reduction of greenhouse gas emissions, energy consumption, and overall ecological footprint.

Let’s look into the tangible impact of the bead-milling study on two most obvious scenarios – the reduction of sulfur waste in the process and the mitigation of rubber waste in the rubber industry. Consider a hypothetical case where an industry (such as the ‘supplier’ for rubber producer) employs bead-milling for sulfur dispersion product to reduce particle size and enhance stability. The conventional parameters or methods in the bead-milling of sulfur may result in a certain percentage of sulfur waste due to inefficient processes. By optimizing the bead-milling parameters, the waste generated can be minimized significantly. For instance, if the conventional process generates 20% waste during sulfur dispersion, the application of bead-milling might reduce it to a mere 5%, thereby achieving a significant 15% reduction in waste. This reduction not only conserves resources but also translates to cost savings for the industry. Secondly is from the rubber manufacturing industry – where sulfur addition is a critical step in vulcanization – a process that imparts strength and elasticity to rubber products. The sulfur from conventional bead-milling process may often lead to sulfur blooming on the rubber surface, necessitating additional steps and resources for reworking or additional ‘treatment’ need to carry out. This not only consumes energy but also may generates more wastes and time consuming. By adopting the optimized bead-milling approach, the potential for sulfur blooming can be significantly reduced. For example, let’s consider a case where 30% of rubber products require reworking or additional treatments due to sulphur blooming. With the new and optimized approach, this figure might drop to 10%, resulting in a 20% reduction in the need for reworking or additional treatments. Thus, the optimized approach for bead-milling not only conserves resources but also reduces energy consumption and waste generation. With fewer rubber articles requiring disposal, the overall environmental impact is reduced – contributing to the preservation of natural ecosystems and resulting in the reduction of pollution. In short, by advancing material modification processes while minimizing environmental and societal impacts, this study comprises and reflects a comprehensive approach to addressing the current trend in achieving sustainability. As we tread this path toward sustainable materials, the bead-milling study may serve as a benchmark in moving towards a more balanced and resilient future for generations to come.

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