The Extraction of Polyhydroxybutyrate from Methanotrophs using Switchable Solvents
Creators: Lawley, Mark D
Publisher: name: University of Alberta Library
Year: 2024

<p>One of the major challenges that humanity faces is the buildup of plastic waste in the environment. As a biodegradable plastic with similar physical properties to consumer plastics, poly(3-hydroxybutyrate) (PHB) is an excellent option for their replacement. PHB is produced inside bacterial cells, typically induced by periods of nutrient limitation, and can be produced from a variety of low-cost feedstocks. However, the challenges associated with the extraction of PHB from bacterial cells remain significant. In this thesis, switchable hydrophilicity solvents (SHSs) – which can be toggled between a hydrophobic and hydrophilic form – were examined as recyclable solvents for the extraction and processing of PHB. <br>
    Through a theoretical screening using group-contribution parameters followed by experimental validation, it was found that two SHSs were good for recovering PHB: N,N-dimethylbenzylamine (DMBA) and N,N-dimethylcyclohexylamine (DMCHA). Both of these showed gelation of porous PHB at room temperature and dissolution at high temperatures.  It was shown that both DMBA and DMCHA could dissolve PHB and precipitate it through the addition of water and carbon dioxide (switching the solvent into its hydrophilic form), and the solvent could afterwards be recovered by switching back to the hydrophobic form and recycled to perform subsequent dissolutions and recoveries. However, the molecular weights of the PHB were reduced due to the high temperatures and long exposure times needed, and the molecular weight of the final product was further affected by longer PHB chains being more prone to gelation while filtering the PHB-rich solvent.<br>
    When PHB was dissolved in DMBA to high concentrations at elevated temperatures, a gel was formed upon cooling.  It was observed that the solvent in these gels could still be switched through the addition of carbon dioxide and water. This “switched gel” still had a large portion of its mass as liquid, and could be formed into shapes and dried. Similar to the precipitation of dissolved PHB, it was shown that DMBA could be switched back from its hydrophilic form and recovered for subsequent dissolution-gelation cycles, although with lower recycle rates due to the amount of solvent retained in the gel.<br>
    Using the knowledge gained from previous experiments, extraction procedures from PHB-containing methanotrophic bacteria were developed using the SHSs DMBA and DMCHA. The focus of extraction experiments was to determine whether certain steps (dewatering, mechanical lysis before treatment, separation by centrifugation, heating) were necessary for PHB extraction, with the aim of informing a simple and efficient extraction protocol. Two primary methods carried out at room temperature were found to be effective separations. In the first, concentrated PHB-containing biomass was vortexed with water and SHS, the mixture was centrifuged, the interface layer was separated, and the solvent form was switched through the addition of water and carbon dioxide. In a second method, unconcentrated PHB-containing bioreactor effluent was stirred in the presence of a SHS, separated by pipet, and the solvent was then switched to recover PHB. A third method was tested where dried or concentrated PHB-containing biomass was held at high temperature, as in the dissolution experiments, but it was found that this did not effectively separate PHB from biomass. For the room-temperature protocol from unconcentrated biomass, it was demonstrated that the SHS could be recovered and recycled for subsequent extractions.<br>
    The two successful PHB separations have significant advantages over many published extraction protocols in that they were performed at room temperature with minimal process steps and a recyclable extraction agent, which gives them the potential as low-cost PHB extraction solvents with minimal material and energy use.</p>