Improved Polyhydroxyalkanoate Production from Selected Volatile Fatty Acids using Cupriavidus necator Pearl Passanha A submission in partial fulfilment of the requirements of the University of South Wales/Prifysgol De Cymru for the degree of Doctor of Philosophy October 2014 Graduate Research Office Swyddfa Ymchwil Graddedigion R10 Approval for Submission of a Thesis Student Name: Pearl Passanha Award: Doctor of Philosophy (PhD) This is to confirm that the thesis entitled: Improved Polyhydroxyalkanoate Production from Selected Volatile Fatty Acids using Cupriavidus necator has been approved for submission by the student's supervisory team and can therefore be forwarded to the examiners. Signed Dr. Sandra Esteves Director of Studies Date 16/10/2014 i Graduate Research Office Swyddfa Ymchwil Graddedigion R11 Certificate of Research This is to certify that, except where specific reference is made, the work described in this thesis is the result of the candidate’s research. Neither this thesis, nor any part of it, has been presented, or is currently submitted, in candidature for any degree at any other University. Signed Pearl Passanha Candidate Date 16/10/2014 Signed Dr. Sandra Esteves Director of Studies Date 16/10/2014 ii Contents Contents List of Figures ................................................................................................................. vi List of Tables .................................................................................................................. ix List of Abbreviations ...................................................................................................... x Abstract ........................................................................................................................... xi Acknowledgements ...................................................................................................... xiv 1. Introduction ................................................................................................................. 1 1.1 General Overview ................................................................................................... 2 1.2 Polyhydroxyalkanoates (PHAs) .............................................................................. 4 1.3 Aims and Objectives ............................................................................................... 7 1.4 Structure of the thesis ............................................................................................. 8 2. Literature Review ..................................................................................................... 10 2.1 Concepts Relevant to Biopolymers ....................................................................... 11 2.2 Need and importance of biodegradable polymers in society and the environment .................................................................................................................................... 12 2.3 Polyhydroxyalkanoate (PHA) related history ....................................................... 15 2.4 Biosynthesis of PHA ............................................................................................. 17 2.5 Structure, characteristics, types, composition and properties of PHA .................. 20 2.6 PHA producing microorganisms .......................................................................... 23 2.7 PHA production using different feedstocks and nutrients .................................... 29 2.8 PHA process control and optimization ................................................................. 38 2.9 Downstream Processing of PHA .......................................................................... 48 2.10 Biodegradation .................................................................................................... 54 2.11 Applications of PHA based polymers ................................................................. 55 iii Contents 3. Materials and Methods ............................................................................................. 59 3.1 Microorganism and maintenance .......................................................................... 60 3.2 Media .................................................................................................................... 60 3.3 Carbon sources ...................................................................................................... 63 3.4 Fed-batch fermentations and fermenter design and control .................................. 64 3.5 Bacterial cell harvesting ........................................................................................ 65 3.6 Soxhlet extraction of PHA .................................................................................... 66 3.7 Capacitance measurement ..................................................................................... 68 3.8 Measuring optical density of samples (OD) ......................................................... 69 3.9 Measuring cell dry weight (CDW) ....................................................................... 69 3.10 Measuring Volatile fatty Acid Content (VFAs) ................................................. 70 3.11 Ion analysis ......................................................................................................... 71 3.12 Inductively coupled plasma atomic emission spectroscopy (ICP-AES) ............ 72 3.13 Polyhydroxyalkanoate (PHA) quantification ...................................................... 72 3.14 Experimental Regimes Methodologies ............................................................... 75 4. Investigating the effects of environmental conditions and VFA feeding regimes for optimal PHA accumulation by Cupriavidus necator ............................................ 81 4.1 Shake flask study to determine the optimum basic medium for the growth of C. necator ........................................................................................................................ 82 4.2 Determining the optimum temperature for growth of C. necator ......................... 84 4.3 Investigating VFA feeding regimes for optimal PHA accumulation in C. necator .................................................................................................................................... 85 4.4 Conclusion .......................................................................................................... 107 5. Monitoring Real Time Process and Biopolymer Accumulation in Cupriavidus necator using Dielectric Spectroscopy for Optimal Polyhydroxyalkanoate Harvesting .................................................................................................................... 108 iv Contents 5.1 Monitoring of bacterial growth and PHA production in an aerobic fed batch reactor fed with acetic acid (Kindly refer to section 4.3.2. Some graphs have been re- presented with more relevant data to this section) .................................................... 109 5.2 Monitoring biomass growth and PHA production in an aerobic fed batch reactor supplied with butyric acid ......................................................................................... 121 5.3 Conclusions ......................................................................................................... 125 6. Evaluation of the effect of trace metals and sodium chloride on Cupriavidus necator during growth and PHA accumulation ....................................................... 126 6.1 Shake flask study to investigate and compare the effects of certain trace elements, copper and sodium chloride additions on bacterial cell growth in C. necator ......... 128 6.2 Effect of NaCl at different concentrations on the growth of C. necator ............ 131 6.3 Effect of NaCl salt at different concentrations on PHA accumulation in C. necator .................................................................................................................................. 135 6.4 Conclusions ......................................................................................................... 146 7. Increasing PHA yields from Cupriavidus necator by using Filtered Digestate Liquors ......................................................................................................................... 148 7.1 PHA production from nutrient media and digestates .......................................... 150 7.2 Individual assessment of the effects of nutrients and metals on PHA production .................................................................................................................................. 156 7.3 The influence of nutrients and metals on PHA production ................................ 174 8. Overall Results, Conclusions and Future Work .................................................. 179 8.1 Overall Results ............................................................................................... 180 8.2 Conclusions .................................................................................................... 192 8.3 Future Work ................................................................................................... 196 9. References ................................................................................................................ 202 Appendix ...................................................................................................................... 230 v List of Figures List of Figures Figure 1- Biosynthesis