Isolation and characterization of potential PHB-degrading microorganisms from a Southern Ontario landfill

Abstract

Plastic is essential to day-to-day life. Most plastics create large amounts of waste and emit greenhouse gasses at every life cycle stage from production to degradation, which can have harmful impacts on the environment and cause permanent damage to ecosystems. Polyhydroxybutyrate (PHB) is a proposed ecofriendly alternative to petroleum-based plastics with promising applications as a plastic packaging alternative. It is expected that PHB plastics will be disposed of similarly to petroleum plastics, and thus with increased production and use, the amount of PHB plastic waste in landfills will increase. This research explored the ability of landfill-derived microorganisms to biodegrade PHB through culture-based and genomic methods. The objectives of this research were to confirm the function of and identify PHB-degrading microorganisms from the landfill and to identify and explore their PHB degradation mechanisms. Landfill leachate was spread on PHB-containing selective plates to identify PHB-degrading colonies. From this, colonies were isolated confirming the presence of microorganisms capable of degrading PHB. Isolates from this assay were sequenced using Nanopore MinION sequencing and genomes were assembled using a long-read consensus assembler, autocycler, and annotated using bakta, creating high-quality long-read genome assemblies. Isolates were then taxonomically classified using GTDB-tk and genus/family trees created using GtoTree. Streptomyces, a genus containing many PHB-degrading species, was the most frequently isolated genus. Other taxa isolated include members of the genera Comamonas, Afipia, and Pseudomonas as well as a member of the family Micrococcaceae. All these lineages already contain known and predicted PHB degrading species. Two isolates from genera with no currently described PHB degrading species, Rahnella and Leifsonia, were cultured. Following gene annotation, the majority of isolates’ genomes encoded a likely PHB depolymerase. The Comamonas isolates’ genomes encoded both extracellular and intracellular depolymerases. Four isolates, the three Comamonas and the Afipia, had the full pathways for PHB degradation and synthesis encoded on their genomes. The Pseudomonas isolate genome encoded potential proteins for most of the PHB degradation and synthesis pathways, however, it appears to encode an intracellular medium chain length PHA depolymerase, and no PHB depolymerases were identified in this genome. The Leifsonia, Micrococcaceae, and Rahnella isolates’ genomes encoded probable Blastp predicted PHA depolymerases. The Rahnella isolate’s genome encoded a Bakta predicted PHB depolymerase. For all four, no matches to PHB depolymerases were found in the NCBI protein database. These isolates may have novel, more divergent PHB depolymerases or novel PHB degrading mechanisms, representing potential new pathways and enzymes for biotechnologists looking to industrialize PHB production and degradation. This work identifies microorganisms from landfill communities that can degrade PHB. This indicates that there is the potential for this process to occur in landfills and identifies the members of the microbial community that may be involved. The identification and analysis of the isolates expand the known diversity of PHB-degrading microorganisms, while identifying both known PHB degrading mechanisms and new areas of uncertainty for further research.