Plastic pollution is a global environmental issue, with approximately 460 million metric tonnes (MMt) of plastic produced annually. Of this, only ~9% is recycled, and around 20 MMt pollutes the environment without evidence of significant biodegradation. Tackling this issue requires a multiphasic approach, and one promising area is the exploitation of naturally occurring plastic-degrading enzymes produced by microorganisms. This involves the discovery, characterisation, and engineering of these enzymes to enhance their suitability for industrial use. One notable example is Piscinibacter sakaiensis, a bacterium capable of degrading poly(ethylene) terephthalate (PET) using the enzymes PsPETase and PsMHETase. Recent advancements in sequencing technologies, particularly third-generation methods like Oxford Nanopore Technologies (ONT), have greatly enhanced our ability to investigate the underlying genetic basis for bacterial capabilities such as plastic-degradation in detail. By enabling comprehensive analyses through multi-omics, these technologies have made such studies more feasible and increasingly popular. Such tools, combined with bacteria's ability to rapidly adapt to challenging environments, produce an opportunity for experimentally evolving plastic-degrading bacteria and elucidating their mechanisms of degradation and regulation. This project first benchmarked molecular microbiological techniques including DNA extraction methodologies and the effect of ONT sequencing chemistry updates on 16S rRNA gene taxonomic assignment, metagenomic assembly and adaptive sampling, before applying them to investigate genomic, epigenomic, and transcriptomic changes in P. sakaiensis during adaptive evolution to PET under nutritional stress and/or UV-induced mutagenesis. Along with beginning to elucidate the patterns of methylation and involvement of genes potentially implicated with plastic degradation, this investigation revealed differential expression of paralogs in the PET degradation pathway, suggesting complex degradation mechanisms, and potential involvement of stress-response and epigenetic regulation. For future work, an environmental sample from a contaminated fuel tank was characterised using culture-based and metagenomic techniques, finding potential plastic-degrading properties and highlighting the strengths and weaknesses of each technique, suggesting the depth of metagenomics alone may miss key genes. This research demonstrates the potential of multi-omic investigations to deepen our understanding of bacterial plastic degradation, with future applications aimed at addressing a broader range of man-made pollutants using adaptive evolution techniques.
The Exploration of Plastic-Degrading Bacteria using Multi-Omic Approaches
Herbert, J. (Author). 17 Mar 2025
Student thesis: Doctoral Thesis