MICROBIAL DEGRADATION OF HDPE AND NYLON 6,6 MICROPLASTICS: A POTENTIAL BIOREMEDIATION STRATEGY FOR CLEANER ECOSYSTEMS

Abstract

An alarming rise of micro-nano plastics (MNPs) in environment is currently causing the biggest threat to biotic and abiotic components around the globe. These pollutants, apart from being formed through fragmentation of larger plastic pieces and are also manufactured for commercial usage. MNPs enter agro-ecosystem, wildlife, and human body through the food chain, ingestion or through inhalation, causing blockage in the blood-brain barrier, lower fertility, and behavioural abnormalities among other problems. Hence, it becomes essential to develop novel procedures for remediation of MNPs. Among the numerous existing methods, microbial remediation promises to degrade/recover MNPs via a green route. Since microbial remediation processes mostly depend upon biotic and abiotic factors such as (temperature, pH, oxidative stress, etc.), it becomes easy to influence changes in the plastic pollutants. Hence, with the help of recent technologies, a complete degradation/removal of MNPs can be expected by utilizing the respective carbon content as energy sources for growth of microorganisms. In our study, considering the urgent environmental need, the degradation of micro-nano plastics with its corresponding degradation mechanisms has been brought out. Finally, the role of enzyme and metagenomics in remediation of MNPs are discussed. Biodegradation is an eco-friendly strategy for removal of contaminants. Present investigation demonstrates bioremediation of nylon 6, 6 microplastics (NMPs) for the first time using a soil isolate called Brevibacillus brevis (B.brevis) by shake flask assay. Interestingly, 22 w/w% weight loss of NMPs were noticed after 35 days of incubation with B.brevis. Upon interaction with microplastics, rod to round shape change along with size reduction of the bacterium and irregular shapes of NMPs with cracks and holes were visualized using SEM and TEM. TGA and FTIR analysis reported the disappearance of intermolecular hydrogen bonding of nylon 6, 6 after microbial interaction. The release of various organic acids and enzyme/enzymatic activities of the bacterium were found to be higher in the presence of NMPs. Interestingly, mass spectrometric analysis confirmed the release of adipic acid and hexamethylenediamine derivatives during the aerobic biodegradation. As a result, the degradation of NMPs by B.brevis as a sole carbon source was proven effectively in the environment. The fragmentation of polyethylene plastics into polyethylene microplastics (PEMPs) due to biotic and abiotic factors affects the environment. Extensive investigations have shown its implications upon accumulation in the living systems. In this study, B.brevis was employed to degrade PEMPs. B.brevis-mediated degradation process has shown a reduction in microplastic's dry weight by 19.8% over 35 days of treatment. The biodegradation was achieved by releasing laccase enzyme and organic acids onto the surface of PEMPs, which were quantified by UP-HPLC and SEM analysis. The biodegradation was achieved by releasing laccase enzyme and organic acids onto the surface of PEMPs, which were quantified by UP-HPLC and SEM analysis. Further, the changes in the structural and functional composition of PEMPs were observed by TEM, DSC, FTIR and TGA analysis. Additionally, after the degradation, by-products were observed to contain a short polymer chain such as 2-hexadecanone, decanone. The products resulting from the biodegradation of PEMPs were further utilized for bacterial metabolism. These outcomes reveal the efficiency of B.brevis in PEMPs degradation. The degradation of polyethylene microplastics (PEMPs) and nylon 6,6 microplastics (NMPs) were demonstrated first time using bacterial culture isolates along with degradation mechanism. Bacterial isolates were isolated from a municipal landfill site and identified through 16S rDNA and metagenomics techniques. The isolates identified as Achromobacter xylosoxidans and mixed culture species in dominance of Pulmonis sp. were used to degrade PEMPs and NMPs. Achromobacter xylosoxidans assisted degradation process has resulted in a decrease in microplastic's dry weight by 26.7% (PEMPs) and 21.3% (NMPs) after 40 days of action. Mixed bacterial culture has shown weight reduction of 19.3% (PEMPs) and 20% (NMPs), respectively. Cell hydrophobicity test, SEM and TEM analysis revealed biodeterioration of microplastics through the attachment of bacterial cells onto the surface of microplastics. The release of enzymes, laccase and peroxidases revealed C-C bond cleavage and reduced polymer chain length. The thermal studies (TGA and DSC) revealed changes in the thermal stability and transition characteristics of microplastics over a period of time. The structural alterations on PEMPs and NMPs were recorded by FTIR analysis. Various byproducts such as alkanes, esters, aromatic compounds and carboxylic acids released were identified by GC-MS. The breakdown of microplastics produced small molecules, which were then employed for bacterial metabolism. These results conclude the effectiveness of isolated bacterial isolates in PEMPs and NMPs degradation and potentially leading to the development of more effective and sustainable solutions for managing plastic waste. The major objectives of the present research investigation include: 1. Understanding the role of Brevibacillus brevis in the degradation of microplastics. 2. Elucidation of mechanisms involved in microbial degradation of microplastics. 3. Isolation and characterization of microbes that degrade microplastics. 4. Assessment of the bacterial cell interaction with plastic and corresponding metabolic products in modulating microplastic degradation. This thesis is summarized in four chapters. Chapter 1 discusses a brief introduction microplastics and its remediation technologies. It also talks about the sources and impacts of microplastics on human health and surrounding ecosystem. Chapter 2 This chapter outlines the process of bioremediation of nylon 6,6 microplastics (NMPs) using a soil-derived strain called Brevibacillus brevis (B.brevis) via a shake flask assay. A novel biodegradation pathway is postulated in this chapter. Objective 1 and 2 covered in this chapter. Chapter 3 The present chapter focuses on B.brevis-mediated degradation of polyethylene microplastics along with the degradation mechanism. Objective 1 and 2 covered in this chapter. Chapter 4 The chapter focuses on the degradation of polyethylene microplastics (PEMPs) and nylon 6,6 microplastics (NMPs) along with degradation mechanism. The microbes were isolated from a municipal landfill site and identified through 16S rDNA and metagenomics techniques. A detailed study of degraded products and microplastics structural alterations were studied. The objectives 3 and 4 covered in this chapter.