FINAL RP LUMANOG RAGADIO RELOS RESEARCH PROPOSAL PDF

RESEARCH PROPOSAL OUTLINE Project title: Microbial Degradation of Polyethylene (PE) using Thalassospira lucentensis and Ultraviolet light Proponents: Lumanog, Rui Aaron Ragadio, Ramie...

RESEARCH PROPOSAL OUTLINE Project title: Microbial Degradation of Polyethylene (PE) using Thalassospira lucentensis and Ultraviolet light Proponents: Lumanog, Rui Aaron Ragadio, Ramiel Daine S. Relos, Jerameeh Rae M. Project Cost: Table 1. List of Expenses Items Amount (Php) Quantity Total Amount (Php) 1. Sterile gloves 30 Php 1 30 Php 2. Specimen 10 Php 5 50 Php container 3. 70% ethanol 250 Php 1 250 Php (laboratory grade) 4. Plastic bags 0 Php 1 0 Php 5. Laboratory tests ~6000 Php 1 ~6000 Php (Bioreactor, FTIR, Analytical balance, Incubator) TOTAL: ~6330 Php RATIONALE The amount of plastic waste being improperly discarded into natural bodies has become a significant problem globally. Due to the ease of manufacturing plastic and its importance in many industries, the rate of plastics produced and discarded into the environment is increasing every year. In 2010, the world saw the production of close to 275 million metric tons of plastic waste. In 2023, production of plastic waste grew to more than 400 million metric tons and is expected to gradually increase over the following years. According to UNDP (2023), scientists estimate that only about 9% of all plastic garbage produced worldwide is recycled. The vast majority, specifically, 79% of our plastic waste, ends up in landfills or scattered in nature, and around 12% is incinerated. Every year, our oceans receive 12 million tons of discarded plastic. These plastics slowly break down into smaller pieces known as microplastics. Due to their non-biodegradable nature, these plastics are difficult to discard. As they remain in our environment, they pose multiple threats to marine life. Life on water is more vulnerable to suffocation, entanglement in plastic pollution, and ingestion of plastic. Common types of discarded plastic materials include polyethylene (PE), polypropylene (PP), polyvinyl chloride (PVC), polystyrene (PS), polyethylene terephthalate (PET), acrylonitrile butadiene styrene (ABS), and many more. Specifically, this research will be using polyethylene (PE), as it is the most common type of plastic produced in the world. PE is a plastic polymer that is made of repeating units of ethylene monomers, and these ethylene monomers have a chemical formula of C₂H₄ and a structure of CH2=CH2. Enzymes are special proteins that are capable of speeding up chemical reactions in living cells. Plants, animals, and microorganisms produce different enzymes that possess different functions and purposes. In 2016, a group of researchers led by Kohei Oda of Kyoto Institute of Technology and Kenji Miyamoto of Keio University discovered Ideonella sakaiensis 201-F6. It is a microbacterium that can use polyethylene terephthalate (PET) as its major source of energy and carbon (Shosuke Yoshida et al., 2016). This discovery led to other similar research regarding the ability of enzymes produced by microorganisms to break down certain plastics. Such enzymes include oxidative enzymes, which are capable of breaking down ethylene monomers. These enzymes catalyze an oxidation reaction, a process that involves the addition of oxygen atoms to the polymer chain or structure. The researchers will be using Thalassospira lucentensis, which has potential in degrading plastics due to the enzyme that it produces. Additionally, there are many studies regarding Thalassospira and its ability to degrade polyethylene terephthalate (PET) with positive results. If this research is conducted successfully, it can help contribute to SDG-14 (Life Below Water) by reducing the amount of microplastics that can and will be found in bodies of water. Statement of the problem This study aims to investigate the potential of Thalassospira lucentensis and ultraviolet (UV) radiation in the degradation and breakdown of polyethylene (PE). Research Questions 1. Can Thalassospira lucentensis degrade polyethylene (PE)? 2. What is the effect of ultraviolet (UV) radiation on the degradation of polyethylene (PE) by Thalassospira lucentensis? 3. What byproducts are produced during the degradation of polyethylene by Thalassospira lucentensis under UV radiation? Research Hypothesis 1. Thalassospira lucentensis can not degrade polyethylene (PE). 2. There are no significant byproducts produced during the degradation of polyethylene by Thalassospira lucentensis under UV radiation. Expected Outcomes The researchers are highly expecting this experimentation to work since Thalassospira lucinensis has already been tested for polyethylene terephthalate (PET) and the outcomes are positive. This study will also broaden our current understanding of microbial degradation of plastics and serve as a step towards cleaning our environment, more specifically, our bodies of waters from plastics and the harmful effects they induce upon surrounding marine life. Significance of the Research Proposal Marine Environment. Plastics have caused a massive catastrophe in the marine environment since they harm many different parts of our planet. Plastics, which are not biodegradable, can only break down to microplastics after centuries or decades. If they remain present, they will continue to contaminate our world and may possibly pose further threats to living life. Students and Future Researchers. This study will provide new information and further open doors to many new studies and discoveries in the field of environmental science. Review of Related Literature: Introduction to Polyethylene (PE) and its environmental impacts Polyethylene is a polymer composed of ethylene C₂H₄, a gas with a molecular weight of 28. Its chemical formula is -(C₂H₄)n-, where n represents the degree of polymerization (Steven M. Kurtz, 2016). Polyethylene waste is a significant problem in our environment. Discarded plastic waste eventually oxidizes due to natural sunlight. Some of the C-H bonds in the polymer structure will break under the sun's warmth and UV radiation, releasing the plasticizers and additives into the atmosphere and producing greenhouse gases. Polyethylene is also common in marine environments. Due to its scent and appearance, polyethylene waste appeals to aquatic organisms, often mistaken as prey. Entanglement or consumption of polyethylene by marine life can result in organ damage, apoptosis, genotoxicity, and death (Zhuang Yao et al., 2022). Microbial Degradation of Polyethylene (PE) Polyethylene degradation can be classified as either biotic or abiotic. The former is characterized as degradation by environmental factors like temperature and UV radiation, while the latter is characterized as biodegradation by microorganisms that can alter and consume the polymer, changing its characteristics (Juan-Manuel Restrepo-Flórez et al., 2014). Microbes depolymerize MPs, releasing free radicals and extracellular enzymes that catalyze the breakdown of biodegradable polymers into smaller components. Microbiological degradation of plastics often involves breaking down the polymer into shorter chains or smaller molecules (such as oligomers, dimers, and monomers). Polymers that are small enough are depolymerized into monomers, which can be used for microbial absorption and growth across semipermeable membranes and, eventually, mineralization in cells. The monomers in the cells are then mineralized into CO₂, H₂O (under aerobic conditions) or CO₂, H₂O, and CH₄ (under anaerobic conditions), producing biomass for energy (Zeming Cai et al., 2023). Ultraviolet rays degradation of plastics UV light induces photooxidative degradation, which breaks down polymer chains, creates free radicals, and lowers molecular weight, causing mechanical characteristics to deteriorate and materials to become worthless after an unknown amount of time. Polystyrene (PS), one of the most significant materials in the modern plastic industry, is widely employed due to its superior physical qualities and inexpensive cost. When polystyrene is exposed to UV irradiation in the presence of air, it experiences fast yellowing and slow embrittlement (Emad Yousif, Raghad Haddad, 2013). Research gaps Currently, there are many studies that assess the effectiveness of using different microorganisms in degrading different types of plastics. Many studies have narrowed down our understanding of the enzymes involved in these processes and their functions. Most studies on Thalassospira focus on its ability to break down polyethylene terephthalate (PET); however, research concerning the potential capabilities of Thalassospira lucentensis on degrading polyethylene (PE) is limited. This research aims to provide new information regarding microbial degradation of plastics and to better understand the potential of Thalassospira lucentensis in these areas. METHODOLOGY Polyethylene (PE) Sample Preparation The researchers will obtain grocery polyethylene (PE) plastic bags found at home, 70% ethanol and specimen containers on e-commerce platforms, and sterile gloves at grocery stores. Next, the researchers will prepare the polyethylene (PE) by cutting pieces of plastic bags, ideally 1 cm² each. The researchers will then submerge the small pieces of cut plastic bags and specimen container into 70% ethanol for an estimated 15-20 minutes. Afterward, the plastics will be dried for 1 hour in a clean and sterile area, ensuring that they are not touched by unsterile surfaces. This step is done to guarantee that all the ethanol evaporates. Pre-UV Light Exposure of Polyethylene (PE)  The researchers will then place the dried pieces of plastic into a tray, spreading each one of them apart. The tray will now be positioned below the UV lamp and position the lamp around 10–30 cm above the tray. Evaluation of Thalassospira lucentensis for Degradation of UV-Exposed Polyethylene Plastics The researchers will place the polyethylene (PE) sample into a sterile container and seal it tightly, ensuring it does not get contaminated. The sample will then be shipped to De La Salle University Laguna for testing. The Thalassospira lucentensis will be separately acquired from PNCM; this will be sent to the institute, and along with the shipping, documents with test requests will be sent alongside the polyethylene (PE) sample. The requests for the Thalassospira lucentensis will include the following: Culture Preparation: The university lab will prepare the Thalassospira lucentensis bacterial culture by inoculating it into a sterile growth medium and incubating it under optimal conditions for bacterial growth. Enzyme Collection: After the bacterial culture has reached sufficient growth, the enzymatic supernatant will be extracted through centrifugation. This process separates the liquid fraction containing the enzymes secreted by the bacteria. The resulting supernatant will subsequently be utilized for the treatment of polyethylene (PE) plastic in the bioreactor. Enzymatic Degradation: After everything is prepared, the university will now conduct the experiment using the bioreactor, inserting the (PE) plastic the researchers sent and the prepared enzymatic supernatant from the Thalassospira lucentensis. The bioreactor will be used to create a suitable environment for cell growth and microbial degradation of Thalassospira lucentensis for the experiment. Microbial Degradation of PE in Bioreactor  After everything is prepared, De La Salle University will now conduct the experiment using the bioreactor, inserting the (PE) plastic the researchers sent and the prepared enzymatic supernatant from the Thalassospira lucentensis. The bioreactor will be used to create a suitable environment for cell growth and microbial degradation of Thalassospira lucentensis for the experiment. Data Analysis The researchers will be using the paired t-test, whereas the researchers will compare the plastic’s condition before and after treatment. The following factors shall be observed before and after: a. Weight difference: How much the (PE) plastic weighs. An analytical balance will be used for this test. b. chemical changes: May use an FTIR, or Fourier Transform Infrared Spectroscopy, a tool that is used to look and check at the chemical structure of the plastic (PE). And to check if the (PE) is successfully being oxidized by the enzymatic supernatant that contains the oxidative enzymes. Risk and Safety: The risks of this research are very limited due to the fact this experiment will mostly be conducted by the institute lab. Despite this, there may be some dangers the researchers need to be wary of. UV light exposure may be harmful for human skin, and it may damage the eyes. For protection, laboratory coats, protective glasses, and gloves must be worn at all times to ensure the safety of the researchers. Duration and Schedule of Activities: January February March April W1 W2 W3 W4 W5 W6 W7 W8 W9 W10 W11 W12 W13 W14 Material Experimentation done Analysis of Data Writing of Finalized Gathering by the Institute Research Paper Additional Information Gathering