Plastic Biotransformation Technologies: Development of a Novel Environmental QPCR Assay for Polyethylene Terephthalate Hydrolase, and Isolation/Characterization of Polyethylene Degrading Fungi and Bacteria from Environmental Samples

Abstract

Plastic production, use, and accumulation in the environment—including in the bodies of humans and other animals—have been increasing for decades and are a cause of growing global concern. Common plastic waste is generally considered to be non-biodegradable. In recent years, though, a growing assortment of bacteria and fungi capable of degrading a variety of common recalcitrant plastics have been identified. In general, the enzymes capable of depolymerizing long-chain hydrophobic plastic polymers are not well studied. However, Poly(ethylene) Terephthalate (PET) Hydrolase is well described in the literature and is thus a suitable target for molecular identification and quantification technologies. PET is the plastic polymer used in most plastic water bottles and in polyester fabric. The discovery of PET-degrading organisms and PET hydrolases is leading to the generation of biochemical technologies for the recycling and upcycling of PET, as well as the search for PET hydrolases that have greater activity on commercially relevant PET polymers. The incidence of PET hydrolase in metagenomes appears rare, though the quantification of PET hydrolases in environmental samples is unknown. Because plastic-biotransforming organisms are considered rare and slow growing, the process of isolating and characterizing these organisms is long and involved. This thesis presents two distinct, but interrelated, experimental trajectories related to the advancement of the study of plastic biotransformation. The first study focused on the molecular level, and the second study focused on microorganisms. In the first study, novel Quantitative Polymerase Chain Reaction (QPCR) primers were developed and tested for the ability to selectively amplify PET hydrolase genes from environmental samples. The products from these primers, used on eight environmental DNA extracts, were subjected to amplicon sequencing. Multiple sequence analysis methods confirmed the successful amplification of published PET hydrolase sequences, as well as sequences that show a high potential for being PET hydrolases. The on-target hit percentage and on-target hits varied substantially across samples, and this assay will require further optimization for specificity and quantification efficacy before it can be used for absolute quantification (i.e., gene copies/ ng DNA). There is reason to suggest that this assay can measure relative abundance of PET hydrolases, and thus relative genetic PET bioconversion potential. By providing comparative analysis that is both faster and less expensive than traditional techniques, this tool enables the rapid determination of ideal conditions to find and cultivate PET hydrolytic organisms. The core results of this analysis are presented in Figures 26, 28, and 29. In the second study, the focus was to enrich for and isolate (as individual species or consortia), identify, and evaluate microorganisms capable of Polyethylene (PE) biodegradation and biomineralization by culturing microbes in media where PE is the sole carbon source. Although the impact on the environment of PET (the polymer studied in the first study) is substantial, it pales in comparison to the impact of PE, which is used primarily for single-use items and is the most abundant type of plastic manufactured on the planet. Currently, no enzymes capable of degrading PE are well described, though some fungi and bacteria have been shown to degrade and utilize PE as a carbon source. In this set of experiments, preferential focus was given to fungi. Microbes that degrade and live on LDPE powder were enriched from environmental sources. A cogent argument for the confirmation of Low-Density PE (LDPE)-biodegrading organisms is presented from the limited data available (see below for limitations resulting from COVID-19 lockdown). LDPE-biodegradation can be seen in the isolate “Ath” (flask 6, a filamentous fungi that macroscopically appears to be Trichoderma sp.). The macroscopic observation of PE biotransformation for culture “Ath” is documented in Figure 33, where Flask 6 clearly shows modification to the PE powder. Modification increases with longer incubation and is not observed in the otherwise-identical non-inoculated control (Flask 34, Figure 33). Similar results are observed for other cultures, along with the growth of biomass and spore production. Thus, LDPE-biodegradation is also the most likely explanation for at least nine other environmental isolates. And microscopic confirmation of growth in this culture as well as others is presented in conditions where the only carbon source is PE powder. Both bacteria and fungi were shown to degrade the low molecular weight PE powder, though quantitative analysis on commercially relevant PE films was not completed. Tentative taxonomic hypotheses and the exciting possible implications of PE degradation within these taxa are presented, though genetic identification was also unable to be performed due to lockdowns. This research project was cut short prematurely due to mandatory laboratory lockdown in response to the COVID-19 pandemic. While both prongs of the studies described in this thesis were affected, the isolation and PE biodegradation assay was more seriously limited in that all quantitative analyses were unable to be performed. The discussion section reflects the limitations that resulted, as well as the adjustments that were made to compensate for these limitations.