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Composition of Mixed Culture PHA Biopolymers and Implications For Composition of Mixed Culture PHA Biopolymers and Implications for Downstream Processing Liliana Montaño Herrera BEng (Hons) Biotechnological Engineering, MSc Environmental Engineering A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2015 School of Chemical Engineering 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 i 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 ii 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. iii Declaration by author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis. iv Publications during candidature Peer-reviewed papers Montaño-Herrera, L., Pratt, S., Arcos-Hernández, M. V., Halley, P. J., Lant, P. A., Werker, A., Laycock, B. In-line monitoring of thermal degradation of PHA during melt-processing by Near-Infrared spectroscopy. New Biotechnology . 2014; 31(4): 357-363. Arcos-Hernández, M., Montaño-Herrera, L., Murugan Janarthanan, O, Quadri, L., Anterrieu S., Hjort, M., Alexandersson, T., Karlsson, A., Karabegovic, L., Magnusson, P., Johansson, P., Bengtsson, S., Morgan-Sagastume, F., de Vegt, O., Laycock, B., Pratt, S., Halley, P., Lant, P. and Werker, A. Value-added Bioplastics from Services of Wastewater Treatment. Water Practice & Technology . 2015; 10(3): 546–555. Janarthanan, O. M., B. Laycock, L. Montaño-Herrera, Y. Lu, M. V. Arcos-Hernández, A. Werker and S. Pratt. Fluxes in PHA-storing microbial communities during enrichment and biopolymer accumulation processes. Accepted for publication in New Biotechnology . Conference abstracts Montaño-Herrera, L., Werker, A., Laycock, B., Pratt, S. Polyhydroxyalkanoate thermal degradation monitored by Near-Infrared spectroscopy. (2012). ISBP International Symposium on Biopolymers 7–10 October, Cairns, Australia. Montaño-Herrera, L. (2013). Polymerisation mechanisms of mixed culture PHA. RACI (The Royal Australian Chemical Institute) Student Symposium, 8th November 2013, Queensland University of Technology Gardens Point, Brisbane, Australia. v Garvey, C., Montaño-Herrera, L, Pratt S., Werker, A., Laycock, B. (2013). SAXS/WAXS studies of structure in poly( β-hydroxybutyrate-co -β-hydroxyvalerate) produced by mixed bacterial cultures. Australian Synchrotron User Meeting 2013 held across 21–22 November at the National Centre of Synchrotron Science, Melbourne, Australia. Montaño-Herrera, L. (2014). Microbial synthesis dynamics of PHA biopolymers from waste streams. The University of Queensland EAIT Postgraduate Conference, 11 June 2014, Brisbane, Australia. Montaño-Herrera, L. (2015). Metabolic carbon fluxes during production of PHA biopolymers from waste streams. The University of Queensland EAIT Postgraduate Conference, 10 June 2015, Brisbane, Australia. Publications included in this thesis Janarthanan, O. M., B. Laycock, L. Montaño-Herrera, Y. Lu, M. V. Arcos-Hernández, A. Werker and S. Pratt. Fluxes in PHA-storing microbial communities during enrichment and biopolymer accumulation processes. Accepted for publication in New Biotechnology. Incorporated wholly as Chapter 7 Contributor Statement of contribution Montano Herrera, Liliana (Candidate) Conceived the research (20%) Designed and performed experiments (60%) Janarthanan, Om Murugan Conceived the research (20%) Analysed and interpreted data (20%) Wrote the paper (70%) vi Contributor Statement of contribution Laycock, Bronwyn Conceived the research (20%) Designed and performed experiments (40%) Analysed and interpreted data (20%) Wrote and edited paper (10%) Lu, Yang Analysed and interpreted data (20%) Arcos-Hernández, Monica Vanessa Analysed and interpreted data (20%) Werker, Alan Conceived the research (20%) Wrote and edited paper (10%) Pratt, Steven Conceived the research (20%) Analysed and interpreted data (20%) Wrote and edited
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