University of Bath PHD The development of industrially-relevant lipids from Rhodotorula species as a feedstock for fuels and commodity products Sargeant, Lisa Award date: 2015 Awarding institution: University of Bath Link to publication Alternative formats If you require this document in an alternative format, please contact: [email protected] General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal ? Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Download date: 07. Oct. 2021 THE DEVELOPMENT OF INDUSTRIALLY-RELEVANT LIPIDS FROM RHODOTORULA SPECIES AS A FEEDSTOCK FOR FUELS AND COMMODITY PRODUCTS. LISA A. SARGEANT A thesis submitted for the degree of Doctor of Philosophy University of Bath Department of Chemical Engineering October 2014 COPYRIGHT Attention is drawn to the fact that copyright of this thesis rests with the author. A copy of this thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with the author and that they must not copy it or use material from it except as permitted by law or with the consent of the author. This thesis may be made available for consultation within the University Library and may be photocopied or lent to other libraries for the purpose of consultation with effect from ……………………………………………….. Signed on behalf of the Faculty of Engineering Acknowledgements I would especially like to thank my lead supervisor, Dr. Chris Chuck in the Dept. of Chemical engineering for your dedication, enthusiasm and patience throughout this project. I feel especially flattered to be one of your first graduating PhD students and I wish you every success in what I’m sure will be a very prosperous career. My thanks also go to my other supervisors: Prof. Rod Scott in the Dept. of Biology and Biochemistry, and Prof. Matthew Davison in the Dept. of Chemistry, for your guidance and expertise throughout my PhD. Thanks also to Dr. Chris Bannister in the Dept. of Mechanical Engineering for help with the Design of Experiments work. I am also particularly thankful to the EPSRC Centre for Sustainable Chemical Technologies for providing the funding for this PhD, as well as the behind-the- scenes support from Janet Scott, Sheila Apps and Jez Cope. Many thanks also to the industrial partners that have been involved throughout this PhD, especially Airbus Group for providing financial support. Firstly, those at Airbus Innovation Group: John Price, Sarah Nash, Solange Baena, Odile Pepillon, Isabelle Lombaert-Valot, Stephanie Bricout, Mohammed Yahyaoui and all of those who made my time living and working in Paris so enjoyable. Thanks also to the team at Almac: Iain Miskelly, Derek Quinn and Tom Moody for enabling me to experience fermentation technologies within an industrial lab. A huge thank you also goes to the technical team in the Dept. of Chemical Engineering, most notably Marianne Harkins and Daniel Lou-Hing for your hard work and perseverance to help us set-up the lab, and Fernando Acosta for your endless help and advice with the HPLC. Thanks especially to Kirsty Mokebo and Holly Smith-Baedorf for your expertise and support at the beginning of my PhD as well as to Rhodri Owen and Heather Parker for your help and guidance with the GC-MS. I am also grateful to the undergraduate students that have helped contribute to the work contained within this thesis, especially Rebecca Dean and Matthew Mardel, as well as to Ali Hussein for providing the Miscanthus hydrolysates, in what turned out to be a more involved task than was envisioned. i To Rhod, thank you for the endless coffee supplies, listening to my rants, providing healthy competition, and more than anything else, being the most amazing friend. Joe Donnelly, I thank you for always being relied on for a good political debate or knowledge of the UK housing market. And to Dave Miles; I dread to think how many crosswords would have been left unfinished without your brainpower! Thank you to such a fantastic group of friends who I haven’t mentioned by name both in Bath and further afield, for providing many nights of fun and laughter as well as your support on the rollercoaster that is a PhD. To Dave, I thank you for your kindness, encouragement and endless humour. You always manage to bring a smile to my face no matter how bad the day! Finally, thank you to my Mum and Dad for always believing that this was possible. ii Abstract Lipids are becoming an increasingly important chemical feedstock for the manufacture of biofuels, bioplastics, care-products and as a food source. Many of these consumer products are derived from petroleum resources, and therefore finding suitable replacements is a key engineering challenge. While first generation lipid feedstocks have shown potential to displace some fossil fuel use, lipids produced from current sources such as oil crops, cannot realistically meet the demand for these uses sustainably. One alternative is to produce microbial oils from oleaginous yeast. These have many advantages such as high growth rates, year-round productivity and high lipid yield. The fatty acid profile of the lipids is extremely important in determining their eventual use. Oils high in oleic acid such as rapeseed oil are the most suitable biodiesel feedstock and also offer the highest potential for further chemical upgrading to polymers, higher value chemicals or aviation fuels. Alternatively, to replace palm oils in the cosmetic or food industries, high levels of saturated lipids are necessary. Rhodotorula sp., can produce high yields of lipid and has a simple fatty acid profile, composed mainly of C16 and C18 fatty acids. Using Design of Experiments it was shown that the fatty acid profile of R. glutinis could be tailored towards a desired application. A high culture temperature and high nitrogen ratio yielded mainly unsaturated oil, whereas a low culture temperature and high glucose loadings gave a more saturated profile. On transesterification, the oil high in monounsaturated esters yielded biodiesel with fuel properties akin to rapeseed methyl ester (RME), whereas the oil high in saturates was found to be suitable as a substitute for palm oil. In contrast, the lipid profile for R. minuta showed no such fluctuation. One of the drawbacks to the commercialisation of this technology is the high production costs involved. Low energy ultrasound is known to have a positive effect on both biomass and ethanol production in S. cerevisiae. In an attempt to reduce processing costs, intermittent ultrasound with R. glutinis was undertaken to aim to improve glucose conversion efficiencies. Sonication was found to have no positive iii effect on the biomass or lipid accumulation when applied in the exponential phase of R. glutinis growth. However, on applying the sonication in the stationary phase, a beneficial impact was observed with the lipid coefficient being increased by 24%. While it is unlikely to be economic to produce lipids from refined sugars, inexpensive carbon sources such as lignocellulosic hydrolysates or waste streams offer a promising alternative. Microbial growth on these feedstocks can however be challenging, due to the large range of sugars present in the hydrolysates as well as toxic compounds formed during the sugar extraction process. The potential of two biomass hydrolysates: depolymerised Miscanthus and household food waste were investigated, alongside the effects of the model inhibitory compounds and sugar substrates on the growth of Rhodotorula sp. While the Miscanthus hydrolysate was deemed unsuitable as a feedstock for lipid production, acid-hydrolysed food waste produced a promising feedstock for Rhodotorula sp. Biomass yields of approximately 10 g/L were produced for R. minuta and R. glutinis, with the resulting lipid profile being approximately 65% oleic acid (18:1) for both species. One obstacle for lipid production from oleaginous microbes are the energy costs associated with the extraction and subsequent conversion into biodiesel (FAME). A one-step method to produce FAME by combining lipid extraction from R. glutinis using a microwave reactor with acid-catalysed transesterification was developed. Over 99% of the lipid was extracted using 25 wt.% H2SO4 over 20 min at 120 °C. At higher catalyst loadings, similar yields were achieved at a reaction time of 30 s. Equivalent yields of FAME were achieved compared to the traditional method of Soxhlet extraction, run with the same solvent system for 4 h. Under the best conditions, the energy required by the microwave was less than 20% of the energy content of the biodiesel produced. Finally, the energetics of the conversion of household food waste to oil (SCO process) using Rhodotorula sp. was compared to that of the anaerobic digestion (AD) of food waste. Oil production alone was deemed energetically unfeasible. However, a coupled SCO and AD plant may have economic viability as a waste-to-energy route, especially for the production of bulk commodities such as jet fuel, in which the energy generated from the methane can be used to power the SCO process. iv Contents Acknowledgements i Abstract iii Abbreviations and acronyms x List of figures xi List of tables xiv 1.