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 Biodegradation of low-density polyethylene by fungi Submitted in total fulfilment of the requirements for the degree of Doctor of Philosophy by Sasi Kiran Kumar Kanchi Faculty of Science, Engineering and Technology Swinburne University of Technology October 2015 ii Abstract The accumulation of recalcitrant plastics in the environment, particularly polyethylene, is a major threat to the ecosystem. Among the various types of polyethylene, low-density polyethylene (LDPE) is the most commonly used. Unfortunately, the rate of production and consumption of polyethylene is outweighs the rate of its disposal. Although several approaches such as incineration, landfill treatment and recycling have been proposed, these approaches have been either too costly or only partially effective. An alternative solution, involving the biodegradation of LPDE, has been proposed. Current understanding of the biodegradation of LDPE suggests that it requires a considerable amount of time for the process to be completed. Previous studies on LDPE describe the importance of oxidising the LDPE to make it suitable for microbes to degrade. In this study, an attempt was made to understand the biodegradation process after oxidation, in order to develop strategies for improving the efficiency and reducing the time required for this process to be completed. LDPE samples were treated with fungal isolates that were collected from river and landfill sources. Their intrinsic properties were assessed before and after fungal treatment, and their surface characteristics were determined by atomic force microscopy (AFM) and scanning electron microscopy (SEM). Modifications of functional groups were evidenced by Fourier transform infrared spectroscopy (FT-IR) and Raman spectroscopy. Changes in crystallinity were monitored by X-ray diffraction (XRD). A relatively inexpensive staining technique was proposed to quantify the differences between untreated LDPE and fungal-treated LDPE. Fungal-treated LDPE showed characteristic features that differed significantly from untreated LDPE. Changes in the crystallinity, buoyancy and colour of LDPE were observed following fungal treatment. The fungal strain isolated was identified as Fusarium oxysporum by18S rRNA gene sequencing. The hydrophobicity of this isolate iii and other fungal varieties was measured to estimate their ability to attach to LDPE. These fungi were classified according to their microscopic and macroscopic features. Various factors affecting biodegradation were monitored. Salts, alcohols and sugars were screened for their effects on the biodegradation process. The effects of pH, temperature, oxidation, co-metabolites and biofilm formation were also determined, as well as the weight losses of LDPE samples during the course of biodegradation. This work reports for the first time the ability of Fusarium oxysporum to degrade LDPE. Alcohols and sugars were shown to accelerate the biodegradation process, along with salts, such as MnCl2. The ambiguity regarding the necessity for biofilm formation during biodegradation was clarified. It was demonstrated that formation of biofilm was not necessary for biodegradation to occur. In fact, biofilm formation was shown to slow this process. The effects of co-metabolites, such as monosachcharides, disachccharides and polysachcharides, were described in detail for the first time. Further, the reasons for the enhanced biodegradation of LDPE in the presence of co-metabolites were elaborated. Biodegradation of LDPE with fungal extracts was also performed in order to determine the possible factors affecting the activity of proteins that may participate in this mechanism. The proteins present in fungal extracts were identified by Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). In this study, laccase from Fusarium oxysporum was identified of playing an important role in LDPE biodegradation. In addition, attempts to biodegrade LDPE with enzymes in vitro were described, and the effects of ethanol and sucrose on the oxidation capacity of laccase were also determined. In the final section of this thesis, factors governing the biodegradation rate are discussed. Physical factors, such as surface roughness and crystallinity, are elaborated. Structural factors, such as tertiary carbon atoms and polymer chain length, are also discussed in detail. Various suggestions are made to increase the biodegradability of iv LDPE in its native and oxidised states to encourage biodegradation in natural environments, landfills and laboratory fermenters. v Acknowledgements My first thanks goes to Professor Enzo Palombo, my primary supervisor. He is a kind and highly professional supervisor. He is the inspiration and motivation behind this project. He always had time to discuss about the project. I would also like to thank Dr François Malherbe, my co-supervisor. His vast experience and knowledge about polymer science certainly helped in improvising my thesis. His support and encouragement were vital to develop this thesis. I would like to thank all laboratory technicians namely Chris, Soula, Savi and Ngan. I wish to thank Dr. Peter Mahon for his help regarding Raman spectroscopic analysis. I also wish to convey my thanks to Dr. Hayden Webb for his help regarding atomic force microscopy. My thanks also go to all the colleagues, friends of Swinburne University of technology. I would like to thank my family members for their support and encouragement. vi Declaration I, Sasi Kiran Kumar Kanchi, declare that this thesis is my original work. It does not contain any material that was previously published, except where due reference is made. I also declare that this work was edited by professional editors for its grammatical mistakes