Composition of mixed culture PHA biopolymers and implications for downstream processing

Abstract

Polyhydroxyalkanoates (PHAs) are biopolymers synthesised and accumulated by most bacteria for carbon and energy storage. They have properties and applications comparable to petrochemical thermoplastics. Although PHAs are produced at high yields using pure biological cultures, the use of mixed cultures can significantly reduce the production costs and make use of waste streams to produce environmentally sustainable materials. In order to produce mixed culture PHAs of relevance for broader industry applications it is necessary to develop the ability to tailor polymers with diversified mechanical properties through understanding and controlling the monomer composition and compositional distribution. Diverse and complex PHA polymer structures have been achieved in mixed microbial cultures using time-based feeding strategies. However, PHA monomer composition and compositional distribution in PHA random and blocky copolymers are sensitive to substrate feeding history, making more complex the prediction of final PHA content and composition. In this sense, a better understanding of the changing cell physiologies that develop in response to different feeding strategies and substrates is necessary to design optimised feeding strategies and process control algorithms for PHA production. This thesis presents for the first time a comprehensive flux characterisation of monomer development during mixed culture PHA accumulation concurrent with biomass growth using Metabolic Flux Analysis (MFA). A dynamic trend in active biomass growth and in polymer composition was observed and was consistent over replicate accumulations. Monomer (3-hydroxybutyrate and 3-hydroxyvalerate) incorporation into poly(3-hydroxybutyate-co-3-hydroxyvalerate) copolymers in a pilot scale production system was evaluated based on published models that describe polymer production during PHA accumulation. However, it was found that these existing models could not describe the fluctuations in the proportion of 3HV monomer units that were observed through some of the PHA accumulations in this work. Most models of PHA accumulation in mixed cultures use condensed reactions for active biomass formation, neglecting the associated pathways for generation of reducing equivalents which control the concentration of available precursors for cell growth and monomer development. In this sense, a new model describing biomass growth concurrent with PHA accumulation, sufficiently detailed to consider 3HB and 3HV production as a function of metabolic state, was proposed. The metabolic network developed was decomposed by Elementary Flux Analysis (EMA), providing insights into the activities of pathways for simultaneous PHA storage and active biomass synthesis. Another factor impacting PHA composition in mixed cultures is the possible fluctuation in microbial population. This fluctuation was characterised in the same series of accumulations as was used for the metabolic modelling, through the use of 16S rRNA gene amplicon pyrosequencing. The PHA accumulation capacity of the community was found to be robust to population flux during enrichment and even PHA accumulation. This community adaptation suggests that mixed culture PHA production is a robust process. Diverse final polymer compositions and microstructures were achieved with the different feeding compositions and strategies used. Determination of chemical and thermal properties of the as-produced polymers confirmed that the product was a mixture of copolymers. Additionally, thermal degradation of mixed culture PHAs during melt processing was assessed by Near-Infrared (NIR) spectroscopy coupled to Multivariate Data Analysis. It was shown that, with correct pretreatment, a copolymeric product that was much more stable to extrusion processing than commercially available PHA was produced. Overall, this thesis explores both the fundamental and applied aspects of PHA production by mixed microbial cultures with concurrent active biomass growth. The use of computational tools to explore and help understand the underlying metabolic processes was explored. Polymer composition seems to follow a very complex regulation processes which can be described through the incorporation of more detailed reactions in current metabolic models. Furthermore, this thesis gives further insight into the fundamental properties of the materials produced and an assessment of their potential for degradation during processing.