Demystifying Brettanomyces bruxellensis: Fermentation kinetics, flavour compound production, and nutrient requirements during wort fermentation by Caroline S Tyrawa A Thesis presented to The University of Guelph In partial fulfilment of requirements for the degree of Master of Science in Molecular and Cellular Biology Guelph, Ontario, Canada © Caroline S Tyrawa, January 2019 ABSTRACT DEMYSTIFYING BRETTANOMYCES BRUXELLENSIS: FERMENTATION KINETICS, FLAVOUR COMPOUND PRODUCTION, AND NUTRIENT REQUIREMENTS DURING WORT FERMENTATION Caroline S. Tyrawa Advisor: University of Guelph, 2018 Dr. G. van der Merwe Brettanomyces bruxellensis is beginning to gain popularity in the craft brewing industry; however, a lack of research on this yeast and lengthy fermentation times present challenges for brewers. Here eight novel Brettanomyces strains were characterized and two were chosen for secondary and co-pitch fermentations, along with the industry standard BSI Drei. The ester and phenol content of these beers was slightly lower than that found in primary Brettanomyces fermentations. Regardless, mixed fermentations proved to be a viable approach to developing beers with “Brett character” in a shortened timeframe. It was also observed that ester and phenol synthesis peaked around day 14 and near the end of the fermentation, respectively. Furthermore, supplementation of thiamine or various amino acids to the pre-growth or fermentation appeared to have a positive effect on fermentation rate in a strain- dependent manner. Overall, these findings will allow brewers to be better informed when developing products using Brettanomyces. Acknowledgments Despite what the subject matter of my thesis may suggest, a lot of hard work went into creating the data found within these pages and none of it could have happened without the help and support of various Van der Merwe lab members, both past and present. In particular, thank you to Jordan Willis, Richard Preiss, and Júlia Santos for their friendship and many lively discussions. Richard, thank you for introducing me to this crazy world in the first place. It’s been a blast. George, you have provided me with every opportunity and encouraged me every step of the way. Thank you for being an amazing advisor. I’m looking forward to seeing what the lab brews up next. To my advisory committee: Emma Allen-Vercoe. I want to thank you for all of the encouragement and help throughout my time here. I would also like to thank Escarpment Laboratories for their guidance on my project and for providing me with strains and wort. In addition, this thesis would not be possible without a number of people: Royal City, Dyanne Brewer and Armen Charchoglyan from the Mass Spec facility, and Paramjit Bajwa from the Lee lab. To all of my friends: thank you for listening to me talk about my research and complain when things weren’t working out, even if you didn’t understand any of it. You kept me sane all of these years. To my sister and brother-in-law: Anna and Stefan Brierley. Thank you for the years of pep talks and motivation. You always helped me to see the light when I was freaking out about having to become a “real adult”. I will be forever grateful. Last but definitely not least, my parents: Joseph and Justyna Tyrawa. Thank you for always providing me with every opportunity and believing in me even if I didn’t believe in myself. I wouldn’t be who I am without your love, support, and many sacrifices. It means more to me than you could ever know. I only hope that I can repay you one day. Kocham was. iii Statement of Work Thiamine supplementation fermentations were performed by Fariha Hossain. Caroline Tyrawa collected and analyzed data for this experiment. Richard Preiss helped with figure construction in Chapter 3, particularly Figures 7, 10, and 11. All other work was performed by Caroline Tyrawa. iv Table of Contents Abstract…………………………………………………………………………………………...ii Acknowledgements……………………………………………………………………………..iii Statement of Work……………………………………………………………………………...iv Table of Contents………………………………………………………………………………..v List of Tables……………………………………………………………………………………vii List of Abbreviations…………………………………………………………………………..viii List of Figures……………………………………………………………………………………x Chapter 1 – Introduction ……………………………………………………………………..1 1.1: Brettanomyces evolution anD genomic characteristics…………………………..1 1.1.1: Evolution of Brettanomyces bruxellensis’ fermentation capabilities………..2 1.1.2: Ploidy and karyotype…………………………………………………………….6 1.1.3: Brettanomyces reproduction and mating……………………………………...7 1.1.4: Domestication markers………………………………………………………….8 1.1.5: Carbon source utilization………………………………………………………..9 1.1.6: Nitrogen metabolism…………………………………………………………...11 1.1.7: Flavour development in B. bruxellensis beers………………………………12 1.2: Brettanomyces in InDustry……………………………………………………………15 1.2.1: Brettanomyces as a spoilage organism……………………………………...16 1.2.2: The Canadian beer industry and its economic impact……………………..17 1.2.3: Primary, secondary, and co-pitch/mixed fermentations……………………18 1.2.4: Brettanomyces presents some challenges………………………………….20 1.3: Hypothesis anD Objectives……………………………………………………………25 Chapter 2 - Materials and methods……………………………………………………….26 2.1: Strains and growth media & conditions……………………………………….……26 2.2: Genetic identification of yeasts………………………………………………………27 2.3: Fermentation trials……………………………………………………………………...29 2.3.1: Pre-growth conditions………………………………………………………….29 2.3.2: Primary fermentations………………………………………………………….30 2.3.3: Fermentations with nutrient additions………………………………………..30 2.3.4: Primary, secondary, and co-pitch fermentation……………………………..32 2.3.5: Timecourse fermentations…………………………………………………….33 2.4: Metabolite analysis……………………………………………………………………..34 2.4.1: HS-SPME-GC-MS……………………………………………………………...34 2.4.2: HPLC…………………………………………………………………………….34 2.5: Carbon source growth assay…………………………………………………………34 v Chapter 3 - Results: Screening novel Brettanomyces strains……………………….36 3.1: Phylogenetic analysis of eight Brettanomyces strains reveals distinct groupings...36 3.2: Fermentation temperature impacts attenuation efficiency of Brettanomyces strains in primary wort fermentation………………………………………………………………….37 3.3: Fermentation temperature impacts production of several, but not all, Brettanomyces-produced volatile flavour compounds……………………………………..42 3.4: Brettanomyces isolates display variation in carbon utilization capabilities under aerobic conditions……………………………………………………………………………..46 Chapter 4 - Results: Primary, secondary, and co-pitch fermentations…………….49 4.1: Brettanomyces strain selection impacts secondary fermentation profiles more than co-pitch………………………………………………………………………………………….49 4.2: Secondary and co-pitch fermentations produce higher ethanol concentrations compared to primary…………………………………………………………………………..53 4.3: Secondary and co-pitch ferments produce decreased ester and phenolic compounds compared to primary……………………………………………………………55 Chapter 5 - Results: Timecourse fermentations………………………………………..61 5.1: Strains show different rates of sugar consumption and ethanol production……….61 5.2: Brettanomyces’ production of esters peaks around day 14 of the fermentation…..65 5.3: Phenolic compound production begins at the start of fermentation………………...69 Chapter 6 - Results: Nutrient supplementation…………………………………………72 6.1: Impact of various nutrient additions is strain-dependent……………………………..72 Chapter 7 – Discussion……………………………………………………………………..86 Selecting strong candidates for further experiments………………………………86 Benefits to the industry………………………………………………………………..90 Media customization is necessary for optimal Brettanomyces fermentations…..92 This is not the end of the road………………………………………………………..95 References…………………………………………………………………………………….98 vi List of Tables Table 1. Compounds that were found to abolish the Custer’s effect in B. bruxellensis..23 Table 2. Strains used in this study…………………………………………………………..27 Table 3. Nutrient supplementations in yeast pre-growth cultures………………………..30 Table 4. Strains used in the primary, secondary, and co-pitch fermentation experiment …………………………………………………………………………………………………..32 Table 5. Pitch day and rate for primary, secondary, and co-pitch fermentations………33 Table 6. Initial sugar concentrations for wort used in timecourse experiment………….61 vii List of Abbreviations Abbreviation Meaning Mya Million years ago WGD Whole genome duplication MRP Mitochondrial ribosomal proteins DHODase Dihydroorotate dehydrogenase PDC Pyruvate decarboxylase ADH Alcohol dehydrogenase DNA Deoxyribonucleic acid PCR Polymerase chain reaction SNP Single nucleotide polymorphism PFGE Pulsed-field gel electrophoresis NMR Nitrogen metabolite repression IPA India pale ale CD p-coumarate decarboxylase VR Vinylphenol reductase POF Phenolic off-flavour 4-VG 4-vinylguaiacol 4-EG 4-ethylguaiacol viii 4-EP 4-ethylphenol GDP Gross national product NAD+ Nicotinamide adenine dinucleotide (oxidized) NADH Nicotinamide adenine dinucleotide hydride (reduced) HPLC High performance liquid chromatography HS Head space SPME Solid phase microextraction GC Gas chromatography MS Mass spectrometry WLN Wallerstein nutrient YPD Yeast extract peptone dextrose dNTP Deoxynucleotide triphosphate RAPD Random amplification of polymorphic DNA ITS Internally transcribed spacer region DVB/CAR/PDMS Divinylbenzene carboxen polydimethylsiloxane RI Refractive index OD Optical density SEM Standard error of the mean PCA Principle component analysis ix List of Figures Figure 1. Microscopic comparison of S. cerevisiae (left) and B. bruxellensis (right) yeast cells.