Exploiting yeast diversity to produce renewable chemicals from rice straw and husk Jia Wu 100030434 A thesis submitted for the degree of Doctor of Philosophy to The University of East Anglia 2018 Quadram Institute Bioscience Norwich Research Park, Norwich, NR4 7UA ©This copy of the thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with the author and that use of any information derived therefrom must be in accordance with Current UK Copyright Law. In addition, any quotation or extract must include full attribution. Declaration Exploiting yeast diversity to produce renewable chemicals from rice straw and husk I certify that the work contained in this thesis is entirely the result of my own work, except where due reference is made to other authors as part of a joint piece of work. It has not previously been submitted in any form to the University of East Anglia or any other University. Jia Wu Abstract Exploiting organic lignocellulosic wastes via bio-refining processes has been widely accepted as one of the renewable, environmentally friendly solutions to producing platform chemicals and liquid fuels. Pre-treatment serves as an initial step to improve the accessibility of lignocellulosic polysaccharides to enzymes, and fermentation is a core step to obtain a range of products from the sugars. However, inhibitors of enzymatic saccharification and fermentation are unavoidably generated during hydrothermal pre-treatment. Therefore, the aim of this study has been to assess the associations and possibly correlations between severities of pre-treatment, yield of fermentable sugars and formation of inhibitors, and to evaluate the potential of 11 yeast diverse yeast strains for the potential to produce not only ethanol but also some highly- sought-after platform chemicals. Air dried rice husk (RH) and rice straw (RS) from the same rice cultivar (Oryza sativa, cv. KhangDan18) were used as substrates. Carbohydrate compositions of each were similar whereas lignin contents differed significantly. Using complementary analytical approaches including a new, rapid NMR screening method, 40 compounds including carbohydrates, organic acids, phenolics and furans were identified from the solids and liquors of pre-treated RH and RS. However, the quantities of compounds differed between the two substrates. Fermentation inhibitors included 5-HMF, 2-FA and phenolic acids such as para-couamric acid (pCA) and trans-ferulic acid (tFA). Differences in lignin, tFA, diferulic acids (DiFA) and pCA between RS and RH reflect differences in cell wall physiology and are probably responsible for the higher recalcitrance of RH. After pre-treatment at a severity of 3.65, ethanol was produced from RS with a yield double that from RH. Above a severity of 5, fermentation was completely inhibited in both RH and RS. More careful control of pre-treatment may be sufficient to reduce the levels of fermentation inhibitors. Such inhibition was found to occur with a range of genetically diverse yeast strains which differed considerably in their metabolic capabilities and production of ethanol. A number could produce significant amounts of ethyl acetate, arabinitol, glycerol and acetate in addition to ethanol, including from hitherto unreported carbon sources. Moreover, a new catabolic property of Rhodotorula mucilaginosa (NCYC 65) was discovered in which sucrose is cleaved into glucose and fructose but they are not metabolised. Engineering some of properties discovered in this study and transferring such properties to conventional industrial yeast strains could greatly expand their biotechnological utility. Acknowledgments I would like to thank my supervisors Professor Keith Waldron and Dr Ian Roberts, supervisory team members Dr Jo Dicks and Dr Adam Elliston for their guidance, support and patience. I am grateful to our group members Mr Graham Moates, Dr Ian Wood and Mr Samuel Collins for their great help with the laboratory work and experimental design. Also thank Dr Gwenaelle Le Gall, Dr Ian Colquhoun, Dr Klaus Wellner and Dr Mary Parker for helping me with Nuclear Magnetic Resonance, Fourier Transform Infrared and Florescence Microscopy. Finally, special thanks to my family who have given me love, support and encouragement always. Especially to my wife who has travelled 10,000 kilometres to accompany with me every day and take care of my daily life. I would never reach this point without her and my parents’ support. List of contents Chapter 1 General Introduction ..................................................................................... 1 1.1 Opportunities and challenges of bio-refining ....................................................... 2 1.2 Chemical compositions of lignocellulosic plant cell wall .................................... 4 1.3 Processes of bio-refining .................................................................................... 10 1.3.1 Pre-treatments and pre-treatment related inhibitory compounds ................ 11 1.3.2 Physical pre-treatments ............................................................................... 14 1.3.3 Chemical pre-treatments .............................................................................. 14 1.3.4 Biological pre-treatments ............................................................................ 17 1.3.5 Physicochemical pre-treatments .................................................................. 18 1.3.6 Inhibitory components generated during pre-treatment .............................. 19 1.4 Enzymatic hydrolysis of lignocellulosic polysaccharides ................................. 21 1.4.1 Enzymatic hydrolysis of cellulose ............................................................... 22 1.4.2 Enzymatic hydrolysis of hemicellulose ....................................................... 22 1.4.3 Enzymatic hydrolysis of pectin ................................................................... 23 1.5 Fermentation ...................................................................................................... 24 1.6 Purification ......................................................................................................... 26 1.7 Overall aims and objectives ............................................................................... 27 Chapter 2 General Materials and Methodology ..................................................... 31 2.1 Raw lignocellulosic biomass.............................................................................. 32 2.2 Milling and freeze mill of air dried raw materials ............................................. 32 2.3 Sugar analysis by using Gas Chromatography (GC) ......................................... 32 2.4 Hydrothermal pre-treatment of RH and RS ....................................................... 34 2.5 Klason lignin analysis of raw and PT lignocellulosic biomass.......................... 36 2.6 Enzymatic hydrolysis of pre-treated RH and RS ............................................... 37 2.7 Glucose analysis of PT RH and RS by using GOPOD ...................................... 38 2.8 Quantification of reducing sugars by using DNS .............................................. 38 2.9 Selection of genetically diverse yeast strains ..................................................... 39 2.10 Growth and fermentation of 11 diverse yeast strains on 13 lab purified sugars .......................................................................................................................... 41 2.10.1 Aerobic growth .......................................................................................... 41 2.10.2 Anaerobic fermentation ............................................................................. 41 2.10.3 Partial anaerobic fermentation ................................................................... 42 2.10.4 Small-scale simultaneous saccharification and fermentation .................... 42 2.11 Chemical compounds and yeast metabolites analysis of liquid samples by using 1H NMR............................................................................................................ 43 2.12 Fluorescence Microscopy of raw and PT lignocellulosic materials ................ 44 2.13 FTIR-ATR of raw and PT lignocellulosic materials........................................ 45 2.14 Analysis of phenolic compounds .................................................................... 45 2.14.1 Phenolics analysis of raw and PT RH & RS solids (dry) .......................... 45 2.14.2 Phenolics analysis of liquors collected from PT RH and RS .................... 46 2.14.3 Phenolics analysis of liquors with additions of enzymes .......................... 47 2.15 Effects of enzymes on esterified phenolics in liquors from PT samples ......... 48 Chapter 3 Comparison of RH and RS as substrates form industrial bio-technology .................................................................................................................... 49 3.1 Introduction ........................................................................................................ 50 3.2 Methodology ...................................................................................................... 52 3.2.1 Small-scale SSF of pre-treated RH and RS ................................................. 52 3.2.2 Hydrothermal pre-treatment of RH and RS................................................. 52 3.2.3 PCA of chemical compounds from the liquors of PT RH and RS