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. ……………………………………………………………………………………………………………...1 Figure 2. Aroma wheel displaying various Brettanomyces descriptors as determined in synthetic wine by a trained panel of judges. The wheel has twelve general categories that are then further broken down to more specific terms [81]……………………………………………………………..13 Figure 3. Summary of primary, secondary, and co-pitch fermentation showing the timing of Saccharomyces and Brettanomyces addition to the medium………………………………………20 Figure 4. The Custer’s effect experienced by Brettanomyces. The Custer’s effect or lag phase experienced by B. bruxellensis upon shifting from an aerobic to an anaerobic environment (A) is due to a redox imbalance resulting from a buildup of NADH. S. cerevisiae overcomes this imbalance by producing glycerol, which restores NAD+; however, due to B. bruxellensis’ inability to produce glycerol, nitrate assimilation must occur in order to restore the NAD+ levels (B) [17]. …………………………………………………………………………………………………………….22 Figure 5. Spice jars fitted with fermentation locks that were used for all fermentation experiments in this thesis………………………………………………………………………………29 Figure 6. Schematic of fermentations with nutrient additions. Cultures are grown in YPD and then transferred to wort or wort containing the nutrient addition for pre-growth. Following that the cultures are pitched into spice jars for fermentations that either contain nutrient supplementation or do not receive supplementation. This produces four possible combinations: nutrient addition in pre-growth and in fermentation (w/ w/); nutrient addition in pre-growth but not in fermentation (w/ w/o); no nutrient addition in pre-growth and addition in fermentation (w/o w/); and no nutrient addition at either step (w/o w/o)……………………………………………………………………….31 Figure 7. Dendrogram of studied strains generated via OPC20 RAPD PCR analysis. RAPD PCR was performed, and the banding patterns imaged using capillary electrophoresis (Agilent Bioanalyzer). The resultant data were clustered by GelJ software using Pearson correlation and UPGMA…………………………………………………………………………………………………..37 Figure 8. Fermentation profiles at 15°C (A) and 22.5°C (B) for 7 novel B. bruxellensis strains, and the B. bruxellensis BSI Drei and S. cerevisiae Cali controls. Strains were pre-cultured and wort inoculated as described (see Methods). The fermentation profiles were obtained by weighing the spice jars to determine weight loss. Data points represent the mean of biological replicates (n=3). Data was normalized to account for water loss from the fermentation locks. Final ethanol concentrations (C) of fermentations performed at 15°C (filled bars) and 22.5°C (hashed bars) were measured using HPLC as described (see Methods). Statistical significance was calculated using an unpaired t-test. * p<0.05 in comparison to the same strain at 15°C; † p<0.05 in comparison to the BSI Drei control at 15°C………………………………………………41 Figure 9. Volatile flavour metabolite analysis following a 28-day fermentation. Following the fermentation, samples were filtered and analyzed by HS-SPME-GC-MS (see Methods). Four volatile flavour compounds are shown and the thresholds of detection are indicated. Error bars represent SEM (n=3). Statistical significance was calculated using an unpaired t-test. * p<0.05 in comparison to control (BSI Drei) at the same temperature; † p<0.05 among samples (same strain) in comparison to lower temperature (15°C)………………………………………………….44 Figure 10. Heat map and cluster analysis of volatiles produced at two different temperatures. Analysis was performed using ClustVis software [134]. Columns are centered; unit variance
x scaling is applied to columns. Both rows and columns are clustered using correlation distance and average linkage…………………………………………………………………………………….45 Figure 11. PCA plot of HS-SPME-GC-MS metabolite profiles of Cali (Saccharomyces control; squares), Brettanomyces beer strains (circles) and Brettanomyces wine strains (triangles) at 15°C (red) and 22.5°C (blue). Plot was generated using ClustVis software [134]. Unit variance scaling is applied to rows; SVD with imputation is used to calculate principal components. X and Y axis show principal component 1 and principal component 2 that explain 48.2% and 27.8% of the total variance, respectively………………………………………………………………………...46
Figure 12. Number of days required for each of the strains to reach an OD595 >0.5 at (A) 15°C and (B) 22.5°C. Growth of the strains was tested on 7 different carbon sources: Glucose, fructose, maltose, sucrose, maltotriose, cellobiose, and maltodextrin. OD595 readings were taken at various time-points over the course of 5 days to monitor growth progress (see Methods). Each of the numbers represents the mean of biological replicates (n=3). Strains that failed to reach this OD are marked with NR (Not Reached). Shading increases with prolonged incubation………………………………………………………………………………………………...48 Figure 13. Fermentation profiles for primary, secondary, and co-pitch fermentation models. Timepoints represent the mean of biological replicates (n=3)……………………………………...52 Figure 14. Final ethanol concentrations for primary, secondary, and co-pitch fermentations. The bars represent the mean of biological replicates (n=3)……………………………………………..54 Figure 15. Heat map and cluster analysis of different strains in primary, secondary (S), and co-pitch (Co) fermentation. Analysis was performed using the Heatmap3 package in R. …………………………………………………………………………………………………………….57 Figure 16. Volatile flavour metabolite analysis following a 21-day primary (P), secondary (S), or co-pitch (C) fermentation. Following the fermentation, samples were filtered and analyzed by HS-SPME-GC-MS. Error bars represent SEM (n=3)………………………………………………..58 Figure 17. Sugar utilization and ethanol production profiles for Cali, BSI Drei, BBY011, Cali x BSI Drei, and Cali x BBY011 during a 21-day fermentation at 22°C. Samples were collected on the indicated days. Each bar represents the mean of biological replicates (n=3)………………..64 Figure 18. Volatile esters at each timepoint during the timecourse fermentations. Samples were collected and filtered at each timepoint and frozen until the end of the experiment. Upon completion of the experiment, the metabolites were measured using HS-SPME-GC-MS. Error bars represent the SEM (n=3)…………………………………………………………………………66 Figure 19. Volatile phenols at each timepoint during the timecourse fermentations. Samples were collected and filtered at each timepoint and frozen until the end of the experiment. Upon completion of the experiment, the metabolites were measured using HS-SPME-GC-MS. Error bars represent the SEM (n=3)…………………………………………………………………………70 Figure 20. Fermentation curves for BSI Drei, BBY011, and PEST IV with thiamine supplementation in pre-growth and fermentation (w/ w/); with thiamine supplementation in pre- growth only (w/ w/o); with supplementation in fermentation only (w/o w/); and a control containing no supplementation (w/o w/o). The data points represent the SEM (n=3)…………..74 Figure 21. Final ethanol, maltose, and maltotriose concentrations in thiamine supplementation fermentations. Concentrations were determined through HPLC analysis and error bars represent the SEM (n=3)……………………………………………………………………………….75
xi Figure 22. Volatile flavour metabolite analysis following a 28-day thiamine supplementation fermentation. Error bars represent SEM (n=3). Statistical significance was calculated using an unpaired t-test (* represents p<0.05)………………………………………………………………….76 Figure 23. Fermentation curves for BSI Drei, BBY011, and PEST IV with amino acid supplementation in pre-growth & fermentation (w/ w/); with amino acid supplementation in pre- growth only (w/ w/o); with supplementation in fermentation only (w/o w/); and a control containing no supplementation (w/o w/o). The data points represent the SEM (n=3)…………..80 Figure 24. Final ethanol concentrations for BSI Drei, BBY011, and PEST IV with amino acid supplementation of aspartate (A), glutamate (G), or glutamine (GL) in pre-growth and fermentation (w/ w/); with supplementation in pre-growth only (w/ w/o); with supplementation in fermentation only (w/o w/); and a control containing no supplementation (w/o w/o). Error bars represent the SEM (n=3)……………………………………………………………………………….81 Figure 25. Final concentrations of volatile flavour metabolites following a 28-day fermentation with aspartate (A), glutamate (G), or glutamine (GL) supplementation in pre-growth and fermentation (w/ w/); pre-growth only (w/ w/o); fermentation only (w/o w/); compared to a control that received no supplementation (w/o w/o)………………………………………………………….82
xii Chapter 1 - Introduction
1.1: Brettanomyces evolution and genomic characteristics
The Brettanomyces genus is comprised of five species: B. bruxellensis, B. anomala, B. naardenensis, B. nanus, and B. custersianus [1]. Of these, B. bruxellensis is the most well-known and studied, particularly in relation to industrial practices [2]. It can be distinguished microscopically from Saccharomyces cerevisiae on the basis of size and morphology [3, 4]. S. cerevisiae has round cells that display multilateral budding, while B. bruxellensis undergoes apiculate budding producing ovoid cells that are often described as “lemon-shaped” as a result of the bud scars that are found on either end of the cell (Figure 1) [3, 4]. Currently, there is a teleomorphic designation for two of the species: Dekkera bruxellensis and Dekkera anomala, for B. bruxellensis and
B. anomala, respectively [5, 6]. However, Brettanomyces is more commonly used in the brewing industry and as such will be used in this thesis. The following sections will discuss B. bruxellensis’ evolution and the adaptations that have allowed it to become successful in a fermentation.
Figure 1. Microscopic comparison of S. cerevisiae (left) and B. bruxellensis (right) yeast cells.
1 1.1.1: Evolution of Brettanomyces bruxellensis’ fermentative capabilities B. bruxellensis shares a remarkably similar niche and function to S. cerevisiae, regardless of the fact that divergence of the two yeasts occurred nearly 200 million years ago (mya) [2, 7]. Although B. bruxellensis and S. cerevisiae once shared a common ancestor, B. bruxellensis now belongs to an ‘intermediate’ evolutionary group that separated from the progenitor of the CTG-clade [2]. This group also consists of
Komagetalla pastoris, Kuraishia capsulata, and Ogataea polymorpha, with B. bruxellensis and O. polymorpha likely sharing the most recent common ancestor due to the greater presence of closely related orthologous sequences and conservation of synteny amongst this group [8, 9, 10, 11]. B. bruxellensis is the only organism shown to have fermentative capabilities within this group [2].
While alcoholic fermentation proves stressful due to a number of factors, B. bruxellensis has evolved a number of traits that make it particularly well-suited for this environment [12]. Much like S. cerevisiae, it exhibits a high resistance to the ethanol and osmotic stress it encounters and can grow in oxygen-limited environments, as well as at a low pH [7, 13, 14, 15, 16]. These are all traits that are important not only for its survival but also its ability to ferment beer [17]. Despite sharing these traits, B. bruxellensis and S. cerevisiae likely developed them independently, as a result of pressures found in a shared environment [7]. While the molecular events that resulted in these adaptations are well studied and understood in S. cerevisiae, the same is not true of B. bruxellensis [7, 8, 18, 19].
Much like S. cerevisiae, B. bruxellensis employs a ‘make-accumulate-consume’ strategy for ethanol production [20]. This trait evolved seemingly independently in the two yeasts and is now referred to as the ‘Crabtree effect’ [21, 22]. It results in the
2 production of large quantities of ethanol from sugars present in the medium under aerobic conditions, where respiration would normally be favoured [22]. Once the glucose has been depleted, B. bruxellensis consumes the ethanol that has accumulated. This mechanism is thought to have evolved as a strategy to outcompete other organisms that do not possess the high alcohol tolerance that B. bruxellensis has
[7, 21].
S. cerevisiae evolved this ability through whole genome duplication (WGD), promoter rewiring, and loss of a cis-regulatory motif, AATTTT, in genes involved in respiration [23, 24]. These events occurred approximately 100 mya, long after the separation of the Saccharomyces and Brettanomyces lineages. B. bruxellensis does not appear to have a large number of duplicated genes suggesting that it did not undergo
WGD at some point in its evolution as a means of developing traits that would make it suitable for a fermentative environment [7]. However, numerous studies have found that global promoter rewiring in genes associated with respiration was responsible for the development of the ‘make-accumulate-consume’ strategy in B. bruxellensis [7, 25]. The
AATTTT motif is found in a fixed position in rapid growth associated genes in both S. cerevisiae and B. bruxellensis; however, both yeasts lack this motif in the fixed position of genes associated with respiration [7]. The MRP set of genes in B. bruxellensis that have the AATTTT motif at the fixed position was approximately half of that found in other closely related non-WGD yeasts, such as Kluveromyces lactis and Lachancea waltii [25]. Another yeast, Yarrowia lipolytica, has the AATTTT motif in the majority of both its respiration and rapid growth genes [25]. This yeast separated from the
Saccharomyces lineage prior to Brettanomyces, thereby suggesting that the common
3 yeast progenitor of these three yeasts also had the motif present in both sets of genes and that S. cerevisiae and B. bruxellensis independently developed the ‘make- accumulate-consume’ strategy through the use of global promoter rewiring [7, 26].
A study conducted by Woolfit et al. found that B. bruxellensis has the complete
URA9 gene, which S. cerevisiae has lost at some point, coding for a mitochondrial dihydroorotate dehydrogenase (DHODase) [19, 27]. This enzyme is involved in the de novo synthesis of pyrimidines and its activity is coupled to the mitochondrial respiratory chain [28, 29]. Instead of URA9, S. cerevisiae contains a URA1 gene, which codes for a cytoplasmic DHODase that catalyzes a step involved in pyrimidine biosynthesis in the absence of oxygen [30]. Horizontal gene transfer of the URA1 gene was thought to have occurred between 100-150 mya in S. cerevisiae, giving it the ability to grow anaerobically [31]. Further research on this subject led to the reconstruction of a
URA1/URA9 homolog gene tree, that determined that while B. bruxellensis has a URA9 gene, it does not have a URA1 gene [8]. This provided further proof that the horizontal gene transfer of the URA1 gene occurred following the S. cerevisiae and B. bruxellensis separation and that B. bruxellensis did not acquire this gene through a parallel evolutionary mechanism [8]. Despite only having the URA9 gene, B. bruxellensis is capable of growing anaerobically [8, 19].
Many genes involved in the glycolysis pathway, which converts glucose to pyruvate that can then be further metabolized into ethanol during fermentation, show altered evolution in B. bruxellensis [25]. HXK2, LAT1, ACS2, and PDB1 all show significantly slower evolution, while ALD2, PYK1, PRM15, PGK1, and YOR283W all show significantly faster evolution when compared with other non-WGD yeasts, such as
4 Y. lipolytica, K. lactis, L. waltii, Lachancea kluyveri, and Eremothecium gossypii [25]. In addition, the PDC gene in B. bruxellensis more closely resembles the PDC6 gene in S. cerevisiae than the PDC1 gene in other non-WGD yeasts [25]. The PDC genes encode enzymes that decarboxylate pyruvate into acetaldehyde, with PDC1 activity being high when yeast is grown in the presence of glucose, while PDC6 activity appears highest when yeasts are grown with a non-fermentable carbon source, such as ethanol [32]. B. bruxellensis shows high expression of its PDC6-like gene when it is in an environment with low oxygen and sugar levels, which may represent an evolutionary strategy for surviving in environments where S. cerevisiae has converted the majority of sugars to ethanol [25].
S. cerevisiae has seven ADH genes, with five of these encoding alcohol dehydrogenases responsible for converting aldehyde to ethanol in a reversible reaction
[33]. Of these five genes, ADH1, ADH3, ADH4, and ADH5 encode enzymes that reduce acetaldehyde to ethanol, while the enzyme encoded for by the fifth gene, ADH2, oxidizes ethanol to acetaldehyde [34, 35]. The ADH1 and ADH2 genes were recently duplicated in the Saccharomyces lineage [36]. Upon analyzing the B. bruxellensis genome it was found that this yeast has a lineage-specific duplication in three of its genes that have homologs in the ADH1, 3, 4, 5 group of S. cerevisiae, which may serve a similar function to the ADH1 and ADH2 genes that have been duplicated in S. cerevisiae, as well as allowing for more efficient ethanol production [8]. The remaining two ADH genes, ADH6 and ADH7, are involved in the synthesis of higher alcohols that serve as aromatic compounds in beer and precursors for aromatic esters through the conversion of longer chain aldehydes and alcohols [8]. Reports of strong aromatic
5 profiles in B. bruxellensis may be explained by a duplication found in homologs of ADH6 and ADH7 genes [8].
1.1.2: Ploidy and karyotype
Previous studies, relying on DNA fingerprinting techniques, have shown that there is a considerable amount of genetic variance between different strains of B. bruxellensis [37, 38, 39, 40, 41]. There has been some recent evidence linking the genotype of the strain to its isolation source, suggesting that the yeast is capable of niche adaptation [42]. A screening using PCR fingerprinting of 50 B. bruxellensis strains isolated from soft drinks, beer, and wine found that there was a link between isolation source and strains that grouped together, with all of the spoilage isolates clustering apart from the wine and beer isolates [37]. Through cluster analysis it was determined that the isolation source was more important than the geographic origin or year of isolation in regards to the genetic profiles observed amongst the isolates [37].
B. bruxellensis strains have been identified as being either diploid or triploid, with triploidy most commonly seen in wine isolates [43, 44]. These strains have a genomic complement in addition to a core diploid genome, which may confer a selective advantage in a wine environment [44]. A comparison of two wine and one beer isolate found that the beer strain greatly differed from the wine strains on the basis of single nucleotide polymorphisms (SNPs) [37]. In addition, the wine strains contained 20 genes that were missing in the beer strain [37]. A similar study found that the third set of chromosomes present in the triploid strains were not closely related to each other [44].
This indicates that they were likely acquired through independent hybridization events involving a distantly related Brettanomyces strain or a non-Brettanomyces species [44].
6 These genetic variations may account for differences in flavour profiles of beers fermented with beer isolates of B. bruxellensis, as opposed to wine isolates; however, further research must be conducted in this area.
B. bruxellensis karyotypes also show considerable variability, with strains containing between 4 and 9 chromosomes [45]. Hellborg and Piskur (2009) isolated
DNA from various strains of B. bruxellensis, along with controls Schizosaccharomyces pombe and Hansenula wingei, and used PFGE to compare chromosome size [45]. The chromosomes varied in size from less than 1 Mbp to more than 6 Mbp, with some bands having a higher intensity indicating that some chromosomes in certain strains may overlap in size [45]. These results seem to suggest that B. bruxellensis evolution was a quick process following lineage separation resulting in rapid genome rearrangements and karyotype variability [45].
1.1.3: Brettanomyces reproduction and mating
Decades after the isolation of the first non-sporulating Brettanomyces cells, Van der Walt and Van Kerken observed spore formation in some cells, albeit at a low frequency [46, 47]. They claimed that B. bruxellensis formed 1-4 hat-shaped ascospores and renamed the ascosporogenous form of this yeast Dekkera [47, 48].
However, since then most follow-up experiments have failed to produce or observe spores in Brettanomyces cells and there has been a lack of evidence for the presence of mating types, mating events, or crosses of two haploid strains in this species [49].
Based on this evidence it is likely that B. bruxellensis is asexual [49]. Indeed mating of two strains with different karyotypes, such as that seen in B. bruxellensis strains, presents a challenge in terms of chromosome segregation during meiosis [45]. Zygotes
7 produced through mating would likely not be viable due to the difficulty in segregating chromosomes during this process [45].
1.1.4: Domestication markers
Domestication is a process through which wild yeast evolve traits that allow them to survive in man-made environments, at the expense of thriving in nature, due to selection and breeding by humans [50]. This process has been observed in S. cerevisiae and is thought to have begun hundreds of years ago amongst the beer strains [50]. Hallmarks of domestication include: the ability to utilize maltose and maltotriose efficiently as a result of MAL gene duplications; loss of phenolic off-flavour production through nonsense mutations in the PAD1 and FDC1 genes; and the ability to flocculate efficiently [50, 51, 52, 53, 54, 55]. In addition, the majority of S. cerevisiae beer isolates lack a sexual reproductive cycle, as this trait likely wasn’t advantageous in a man-made brewing environment where conditions did not change frequently and adaptation was not as necessary for survival [51].
B. bruxellensis is not considered a domesticated yeast species as it lacks some of the markers of this process. Perhaps most obvious, are its functional PAD1 and
FDC1 genes that facilitate the production of phenolic compounds in beer producing characteristic smoky and clovey aromas [56]. B. bruxellensis’ inability to flocculate also differentiates it from S. cerevisiae and makes it more challenging for brewers to process the finished beer product [57]. S. cerevisiae strains that were flocculant were likely chosen by brewers, aiding in domestication, as they made filtering finished beer simpler and less costly [58, 59]. The ability of B. bruxellensis to utilize maltose and maltotriose will be discussed in the next section; however, it is important to note that a duplication of
8 MAL genes has not been reported to date. In addition, as mentioned above, sexual reproduction in Brettanomyces has never been reported [49].
Since B. bruxellensis has not been as popular in the brewing industry as S. cerevisiae and in fact has only recently been intentionally used for brewing, it is unlikely that it was ever placed under the same selective pressure that resulted in S. cerevisiae domestication. Brewing practices have changed over time and it is no longer common practice to reuse yeast cultures indefinitely [50, 51]. Often brewers will reuse yeast for a period of time and then reorder or regrow the yeast from the original stock in order to maintain consistency in their products [51]. This has caused further domestication to halt within beer yeast and as a result it is uncertain whether B. bruxellensis will ever undergo domestication to the same extent that S. cerevisiae has [51].
1.1.5: Carbon Source Utilization
B. bruxellensis is capable of fermenting a variety of carbon sources albeit at different rates [60]. B. bruxellensis has been shown to ferment monosaccharides, such as glucose, fructose, and galactose; disaccharides, such as sucrose, maltose, and cellobiose; and trisaccharides, such as maltotriose [61]. Of these sugars, the mono- and disaccharides are more commonly utilized [62]. Glucose is the preferential carbon source and is fermented more quickly than fructose, maltose, and sucrose [17, 63]. This could suggest that catabolic repression is occurring as a result of glucose being present in the medium, a phenomenon that occurs in S. cerevisiae [64].
B. bruxellensis’ ability to survive in environments where other organisms may perish as a result of limited energy sources or nutrients has led to it being branded as a
‘survivalist’ and ‘scavenger’ [12]. Indeed, its ability to consume a broader range of
9 sugars when compared with S. cerevisiae is thought to be one of the factors that allow
B. bruxellensis to outcompete S. cerevisiae in a fermentation setting, particularly if resources are scarce [65, 66]. B. bruxellensis, unlike its counterpart, is capable of fermenting complex sugars, such as cellobiose and dextrins [17, 64, 67]. The ability to degrade these sugars is a result of α-glucosidase and β-glucosidase enzymes, respectively [17, 68]. The fermentation of these additional sugars produces a beer that is ‘superattenuated’, or contains a slightly higher alcohol concentration, and lower residual sugar content [17, 67].
Dextrin sugars are often found in beer even after the fermentation has occurred, making them an ideal resource for B. bruxellensis in a secondary or co-pitch fermentation where S. cerevisiae will have consumed the majority of the sugars by the time B. bruxellensis begins to grow [17,67]. The α-glucosidase enzymes produced by B. bruxellensis degrade the dextrin sugars into glucose units, allowing them to be further fermented [67]. These enzymes are found intracellularly, associated with the cell wall, and extracellularly following secretion by the yeast, and are most effective in degrading dextrins with low degrees of polymerization [67].
Cellobiose is found in wood, such as the barrels that are often used in the ageing process for both wine and beer [17]. Again, much like with the dextrins, the ability to assimilate cellobiose allows B. bruxellensis to flourish despite low sugar availability in these environments following S. cerevisiae fermentation [69, 70]. Cellobiose is not hydrolyzed by S. cerevisiae as it is lacking the necessary β-glucosidase enzyme [65].
The ability to degrade this sugar is a trait often found in wine isolates of this yeast, suggesting niche adaptation [62].
10 1.1.6: Nitrogen Metabolism
Yeast are known to use inorganic nitrogen sources, such as ammonium and various amino acids, preferentially [71]. Indeed this has been shown in S. cerevisiae and, more recently, in B. bruxellensis [72, 73, 74]. In B. bruxellensis, glutamine has been shown to be the most readily assimilated amino acid in both aerobic and anaerobic conditions [75]. In addition to glutamine, glutamic and aspartic acid are preferentially used in anaerobic conditions [75]. A number of other amino acids, such as arginine, lysine, and methionine, can also be assimilated to varying degrees [73].
Interestingly, for many of these amino acids only the L-isomer can be readily used, with
D-alanine being the sole exception [62]. The ability to assimilate a number of different amino acids likely represents a need for organic compounds as opposed to a specific auxotrophy [73].
Nitrogen metabolite repression (NMR) occurs when preferentially used nitrogen sources are present in the medium, causing down-regulation of genes involved in uptake and utilization of secondary nitrogen sources [76, 77, 78]. However, if no preferential nitrogen sources are present or levels of these sources are low, this repression is lifted to allow for assimilation of secondary nitrogen sources [78, 79]. In B. bruxellensis, presence of nitrate and nitrite can alleviate NMR [71]. The ability to use nitrate as a nitrogen source confers a selective advantage to this yeast, as S. cerevisiae is unable to utilize this substrate [76]. Genes encoding necessary enzymes for the nitrate assimilation pathway can be found in the Brettanomyces genome [19]. Among these genes is a nitrate transporter, a nitrate reductase, and nitrite reductase [76].
Nitrate is transported into the cell by the nitrate transporter and then subsequently
11 reduced to ammonium in two reactions catalyzed by the nitrate and nitrite reductases
[79]. This process requires eight electrons, making it an energetically unfavourable reaction [79]. For this reason, slower growth and ethanol production is observed when
B. bruxellensis is grown on this nitrogen source [74]. Despite this, by being able to assimilate nitrate B. bruxellensis is able to grow in environments where the ammonium or amino acid content has been greatly depleted by S. cerevisiae [76]. Numerous studies have shown that although its growth is slower on this substrate, B. bruxellensis is eventually able to overtake S. cerevisiae populations and become the dominant species in fermentations [76, 80].
1.1.7: Flavour development in B. bruxellensis beers
The aromas associated with B. bruxellensis in wine have been well characterized and a flavour wheel depicting the various aromas has been developed (Figure 2) [81].
While low concentrations of some of these compounds can be considered a positive attribute in certain styles of red wine, most often the presence of these metabolites is considered a sign of wine spoilage, with aromas such as ‘wet dog’, ‘leather’, and ‘band- aid’ being described [82, 83, 84, 85]. Despite this negative connotation frequently being associated with “Brett character” in the wine industry, the aromas and compounds produced by B. bruxellensis are considered a positive contribution to the flavour profile of certain styles of beer, yielding a “funky” taste and aroma [86, 87, 88, 89]. Indeed, in certain styles of beer, such as Belgian lambics or guezes, the “Brett character” is an essential component of the flavour profile [9]. In recent years brewers in North America have also started to experiment with this yeast in other styles of beer, such as IPAs or pilsners [86].
12
Figure 2. Aroma wheel displaying various Brettanomyces descriptors as determined in synthetic wine by a trained panel of judges. The wheel has twelve general categories that are then further broken down to more specific terms [81].
B. bruxellensis is well known for its phenolic aromas, which are often described as “barnyard” or “clove”; however, certain strains also produce pleasant fruity aromas
[86, 90]. Volatile phenols, 4-ethylphenol and 4-ethylguaiacol, are responsible for the barnyard and clove aromas, respectively [91]. They are synthesized from p-coumaric
13 acid and ferulic acid through a decarboxylation reaction [92]. The abundance of these volatile phenols is dependent on the presence of precursors, which can differ depending on the wort used, resulting in high variability of production [93]. The fruity aromas result from the production of esters, such as ethyl caproate and ethyl caprylate, from higher fatty acid precursors [91]. These metabolites are often described as imparting an apple, pear, or anise aroma to the beer [91]. In addition, the presence of esterases in B. bruxellensis allows for the production of ethyl acetate from higher fatty acids [91]. Ethyl acetate is produced during all beer fermentations; however, high levels of it are considered an off-flavour, as it gives the beer an acetone or nail varnish aroma and flavour [91].
Volatile phenols produced by B. bruxellensis are synthesized from hydroxycinnamic acids in a sequential reaction carried out by p-coumarate decarboxylase (CD) and vinylphenol reductase (VR) enzymes [94]. The first step in this reaction is the conversion of hydroxycinnamic acids to their vinyl derivatives by CD enzymes encoded by PAD1 and FDC1 genes [95, 96]. Studies have shown that the presence of either p-coumaric acid or 4-vinylphenol (4-VP) in the culture media stimulates the activity of this enzyme [97]. Following this step, VR converts the vinylphenol to its ethyl derivative, 4-ethylphenol (4-EP) or 4-ethylguaiacol (4-EG) [98].
The presence of 4-vinylphenol in the culture media was shown to induce activity of the
VR enzyme [97]. While certain S. cerevisiae strains have the CD enzyme (making them
POF +), they do not possess the VR enzyme and are therefore unable to convert vinyl forms of these volatile phenols to their ethyl derivatives. 4-EG and 4-EP are therefore characteristic Brettanomyces aroma compounds [97].
14 While the enzymes responsible for the synthesis of phenolic compounds have been identified and characterized, the same is not true for ester-synthesizing enzymes.
Some of these enzymes have been identified in S. cerevisiae, which produces a number of the same flavour compounds, thereby providing possible clues into what enzymes may be responsible for the same function in B. bruxellensis.
Acetate and ethyl esters are the two main groups of esters produced during the fermentation process [99]. To date, three enzymes, Atf1, Lg-Atf1, and Atf2, have been implicated in the synthesis of acetate esters in Saccharomyces, with Atf1 being the most important [100]. These enzymes are alcohol O-acetyltransferases that catalyze a reaction between acetyl-CoA and ethanol or a higher alcohol to produce acetate esters, such as ethyl acetate or isoamylacetate [99, 100]. Ethyl esters, such as ethyl caproate and ethyl caprylate, are synthesized from the transfer of a fatty acid group to an alcohol by acyl coenzyme A: ethanol O-acyltransferase enzymes [100]. Two such enzymes,
Eht1 and Eeb1, have been discovered in S. cerevisiae [100]. The production of esters has been shown to be highly strain-dependent, producing unique flavour profiles associated with specific strains [99]. In addition, ester production increases with an increase in fermentation temperature, while aeration of the medium causes a decrease in the concentration of both acetate and ethyl esters [99].
1.2: Brettanomyces in Industry
The fermentation of beer through the conversion of sugars to ethanol and carbon dioxide dates back to Neolithic times [101]. This process is most often associated with
Saccharomyces cerevisiae, or “brewer’s yeast” as it is also known; however, limited diversity amongst fermentative strains is causing brewers to turn to non-conventional
15 yeasts to extend the range of products that they can produce [54]. Once solely considered an agent of wine spoilage, B. bruxellensis is getting a new reputation as a positive contributor to beer fermentations with the production of unique flavours and characteristics [102]. The following sections will discuss its role in the wine and beer industry, as well as where Canada’s beer industry stands today.
1.2.1: Brettanomyces as a spoilage organism
B. bruxellensis is most widely known as a wine spoilage organism that imparts negative flavours and aromas to wines [103, 104]. Wine contains hydroxycinnamic acids that B. bruxellensis is able to convert into ethylphenols, which impart ‘phenolic’, ‘animal’, and ‘stable’ odours [105]. These are collectively referred to as “Brett character” and in high concentrations they are considered undesirable and off-putting [106]. It is only in recent years that this yeast has been considered a positive and desirable addition to industrial fermentations, most notably in beer [17, 90]. This is likely due to the fact that wine generally contains higher levels of 4-EP (barnyard, stable), while 4-EG (smoky, clove) is present in higher amounts in beer [9]. Prior to this shift, most research that was done on Brettanomyces yeasts focused on its detection, growth inhibition, and minimizing its presence and off-flavour production in wine and only recently has the research and focus shifted towards its other potential applications [107, 108, 109].
Unfortunately, its long-standing status as a spoilage organism has resulted in minimal understanding of its biology and fermentation capabilities [2]. Recent research has begun to shift its focus from identifying mechanisms to inhibit its presence in wineries to exploiting its positive attributes during beer fermentations.
16 1.2.2: The Canadian beer industry and its economic impact
Canada’s craft brewing industry has experienced a significant boom in recent years, with an 108% increase in the number of licensed breweries open nationwide between 2011 and 2016 [110]. Canada had a record number of 817 brewing facilities in
2017, with more than half of these located in either Ontario or Quebec [110]. However, the growth seen in the craft brewery sector has not translated to an increase in national production or national volume sales, which have stayed relatively flat with only minor increases or decreases seen from year to year [110]. This may be due to the fact that craft beer is generally more expensive per bottle or can than beer brewed by large breweries such as Molson Canadian or Budweiser causing consumers to purchase smaller quantities of the higher cost beer or reducing their overall alcohol purchases
[111]. Despite this, the consumer demand for beer has remained high, with the beer industry having three times the economic impact of other alcoholic beverages, such as wine or spirits, and contributing $13.6 billion towards Canada’s GDP in 2016 alone [110,
111].
With all of the local competition, particularly within Ontario and Quebec, it is important for craft brewers to produce high-quality beers with unique characteristics to continue to establish themselves within the brewing industry and to continue to draw consumers to purchase their product. Many breweries are beginning to turn to alternative brewing yeasts, such as Brettanomyces bruxellensis, to achieve unique flavours and aromas within their beers [90]. This yeast is often also used in barrel fermentations, with numerous breweries starting an in-house barrel program [112].
These beers generally have lengthy aging periods, ranging from a couple of months to
17 years thereby increasing production costs but also allowing breweries to charge more per bottle [113]. For example, Bellwoods Brewery, located in Toronto, Ontario, produces a barrel-aged, mixed fermentation beer called Barn Owl that sells for $14 a bottle and often has a limit on how many bottles each consumer can purchase [114]. When investing the time and money into producing a beer such as this it is important for the brewers to know the organisms they are working with in order to avoid unexpected flavours or aromas in their final beer product. Despite this, not much is currently known about the newly popular Brettanomyces bruxellensis yeast in the context of beer fermentation.
1.2.3: Primary, secondary, and co-pitch fermentations
In the beer world, B. bruxellensis is most well-known for its contributions to various spontaneously fermented styles of beer, such as Belgian Lambics, Guezes, or
Krieks [113, 115]. In this context, B. bruxellensis is often found in barrels used in the maturation process, where it is responsible for carrying out a secondary fermentation following the initial primary alcohol fermentation that is completed by S. cerevisiae [12,
113]. This secondary fermentation adds a depth of flavour to the beer’s profile with the addition of various esters and particularly phenols that are characteristic of these beer styles [90]. It is important to note that spontaneous fermentations utilize organisms found naturally in the brewing environment, as opposed to fermentations that are intentionally inoculated with a yeast culture.
Despite historically being associated with spontaneous secondary fermentations, recent years have seen craft breweries beginning to experiment with primary
Brettanomyces fermentations and co-pitch fermentations consisting of S. cerevisiae and
18 B. bruxellensis [54]. The difference between a secondary and a co-pitch fermentation is dependent upon the timing of the B. bruxellensis inoculation, with the yeast being inoculated following completion of the alcoholic fermentation by S. cerevisiae in a secondary fermentation versus at the start of the fermentation along with S. cerevisiae in a co-pitch fermentation [2, 116]. By incorporating Brettanomyces into beers fermented with S. cerevisiae, brewers are able to alter the flavour profile of the beer, resulting in a more unique final product [2]. Alternatively, a primary fermentation is inoculated with B. bruxellensis in the absence of S. cerevisiae, allowing it to perform the alcoholic fermentation, in addition to developing the flavour profile of the beer [91].
Figure 3 summarizes the three types of fermentation.
Figure 3. Summary of primary, secondary, and co-pitch fermentation showing the timing of Saccharomyces and Brettanomyces addition to the medium.
19 Despite an increase in the use of Brettanomyces, the effect that it has on the different types of fermentation is not well characterized. The majority of the evidence is largely anecdotal and has not been confirmed through scientific research. This provides an interesting area of study that has the potential to answer numerous questions, such as the effect and extent of flavour compound production in different fermentations, whether the Saccharomyces strain selection has an impact on how Brettanomyces fermentation proceeds, or whether there is a difference between secondary and co-pitch fermentations in terms of kinetics and flavour compound production.
1.2.4: Brettanomyces presents some challenges
B. bruxellensis has been shown to have decreased fermentation capabilities under conditions of complete anaerobiosis, a phenomenon referred to as the ‘Custer’s effect’ or colloquially as the ‘negative Pasteur effect’ [117]. Unlike the Pasteur effect, whereby yeasts favour respiration over fermentation in the presence of oxygen, B. bruxellensis has higher fermentative capabilities when oxygen is present [117]. This effect has been attributed to a redox imbalance that occurs when B. bruxellensis is transferred to anaerobic conditions [17, 71]. When B. bruxellensis is grown in aerobic conditions (as is often the case during propagation of this yeast) it converts acetaldehyde to acetic acid via NAD+-aldehyde dehydrogenase [17]. This process produces NADH through an irreversible oxidative conversion [17]. This NADH can be converted back to NAD+ in the presence of oxygen or an alternative electron acceptor
[17]. However, once the yeast is switched to an anaerobic environment this process results in a buildup of NADH and a shortage of NAD+, causing glycolysis to halt [17,
118]. This results in a considerable lag phase when B. bruxellensis is transferred to an
20 anaerobic environment following aerobic growth (Figure 4A) [118]. In addition to the challenges exhibited during anaerobic growth, Brettanomyces displays slow growth during aerobic growth compared to Saccharomyces. This is thought to be due to acetic acid buildup, as the yeast is known to produce this in the presence of oxygen [17]. This could cause the vitality of the cells to be diminished upon transfer to an anaerobic setting, thereby further exacerbating the lag phase.
Other yeasts, such as S. cerevisiae, can overcome the buildup of NADH by restoring their redox balance through the production of glycerol as a secondary metabolite, which aids in the reoxidation of NADH in anaerobic conditions [119,120]. B. bruxellensis is unable to do this as a result of a lacking glycerol 3-phosphate phosphatase enzyme [65]. The Custer’s effect can be overcome in B. bruxellensis following nitrate assimilation (Figure 4B) [71]. This allows for the reduction of nitrate to ammonium, thereby restoring NAD+ levels and allowing for fermentation to occur [17,
71]. The lag phase observed in B. bruxellensis following transfer to an anaerobic environment is a result of this being a slower route to restoring NAD+ levels than the production of glycerol [17].
Figure 4. The Custer’s effect experienced by Brettanomyces. The Custer’s effect or lag phase experienced by B. bruxellensis upon shifting from an aerobic to an anaerobic environment (A) is
21 due to a redox imbalance resulting from a buildup of NADH. S. cerevisiae overcomes this imbalance by producing glycerol, which restores NAD+; however, due to B. bruxellensis’ inability to produce glycerol nitrate assimilation must occur in order to restore the NAD+ levels (B) [17].
Studies in the 1960s aimed to circumvent this lag phase through various means
(Table 1) [121, 122]. Experiments during this time revealed that compounds such as acetaldehyde, acetone, formaldehyde, or acetoin all have the ability to speed up anaerobic fermentation by decreasing the lag phase [121]. Despite theoretically solving this problem, none of these compounds provide a reasonable solution for the brewing industry due to them being either dangerous or contributing a negative or undesirable flavour or aroma to the beer product. The addition of oxygen has also been shown to increase the fermentation rate; however, this creates an environment that isn’t entirely anaerobic [122]. In addition, an aerobic environment causes B. bruxellensis to produce acetic acid, which is perceived as an off-flavour in beer [123, 124].
Along with the challenges exhibited during anaerobic growth, Brettanomyces displays slower growth during aerobiosis compared to Saccharomyces, resulting in longer propagation times. This is thought to be due to the eventual buildup of acetic acid, which lowers the pH of the medium [17]. In addition to having to grow the culture for longer periods of time prior to pitching into a fermentation the vitality of the cells may be diminished following propagation due to the lowered pH. Pitching cells that are not healthy may further exacerbate the lag phase. Determining a method or strategy for decreasing this lag phase will provide beneficial information for brewers and the brewing industry, as it will allow them to grow B. bruxellensis and ferment beer with it on a shorter timeline, thereby increasing production while decreasing costs.
22 Table 1. Compounds that were found to abolish the Custer’s effect in B. bruxellensis Compound that was added Impact on beer/consumer Reference
Acetaldehyde Carcinogen 121
Formaldehyde Toxic 121
Acetone Solvent aroma 121
Acetoin Buttery aroma; Carcinogen 121; 122
Eliminates the anaerobic Oxygen environment; causes an increase in 121; 122 acetic acid production
A possible approach to decreasing the lag time is the addition of various nutrients, such as thiamine, to the growth or fermentation medium. Thiamine is an important cofactor for many enzymes involved in carbohydrate breakdown and metabolism [125]. It has been shown that yeasts, such as S. cerevisiae, are able to synthesize thiamine de novo through a series of reactions [125]. One of the precursors for thiamine, 5-(2-hydroxyethyl)-4-methylthiazole phosphate (HET-P), is synthesized through a reaction that requires NAD+ as a cofactor [125]. The redox imbalance experienced by B. bruxellensis upon transfer to an anaerobic environment might hinder
HET-P synthesis, thereby impeding thiamine synthesis until the redox balance can be restored. Carbohydrate-metabolizing enzymes are unable to function properly without thiamine, providing a possible explanation for the lag phase experienced at the beginning of anaerobic fermentation [17, 125].
23 1.3: Hypothesis and Objectives
Due to the gaps in knowledge pertaining to Brettanomyces bruxellensis and its role in beer fermentation, the nature of this thesis is more exploratory and information- driven as opposed to hypothesis-driven. Differences are expected between different strains and particularly between Brettanomyces and Saccharomyces strains in regard to fermentation efficiency, flavour compound production, and nutrient requirements; however, it is difficult to speculate beyond that. In order to gather more information and gain a better understanding of how Brettanomyces behaves and contributes to beer fermentations three objectives have been proposed:
1. Determine fermentation kinetics, flavour profiles, and sugar utilization of eight novel
B. bruxellensis strains to identify candidates for further experiments.
This initial experiment will characterize eight novel B. bruxellensis isolates to
better understand their performance in a fermentation setting. Fermentation proves
to be a stressful environment for many reasons, making some isolates unsuitable
candidates for this process, particularly when the burden of both alcoholic
fermentation and flavour production are tasked to B. bruxellensis in a primary
fermentation setting. It is important to identify strong candidates that can both
ferment well and produce a pleasant flavour profile in the finished beer product. To
this end, the novel isolates will be tested in a lab-scale wort fermentation at two
temperatures that represent industry temperatures for various beer styles. The
fermentations will be carried out for 28 days and monitored for weight loss as a
means of tracking fermentation rate. Following fermentation, the ethanol content will
be measured using HPLC and the volatile flavour profile will be determined using
24 HS-SPME-GC-MS. The data generated in this objective will help in the selection
process of strong candidates for experiments carried out in objective 2.
2. Investigate the effect of B. bruxellensis on fermentation rate, flavour profile, sugar
consumption and ethanol production in a primary fermentation and in combination
with various strains of S. cerevisiae in a secondary or co-pitch fermentation.
Experiments will be conducted with the best B. bruxellensis isolates identified in
the previous objective. These isolates will be tested in combination with strains of S.
cerevisiae chosen to represent a neutral, fruity, or phenolic flavour profile in lab-
scale fermentations. These experiments will provide a better understanding of how
Brettanomyces impacts a fermentation and the differences in volatile metabolite
perception when used individually or in combination with a Saccharomyces strain.
Analysis will be performed using the same methods employed in objective 1.
3. Test whether various nutrient additions will impact B. bruxellensis during pre-growth
and fermentation.
The impact of various nutrient supplementations will be tested in both pre-growth
and fermentation and combinations of the two to determine if there is an impact at
any point during the growth or fermentation process. This experiment will be carried
out using lab-scale fermentations and the end products of the fermentation will be
subjected to HPLC and HS-SPME-GC-MS to determine if the supplementation had
any effect on sugar consumption or ethanol and volatile metabolite production.
25 Chapter 2 - Materials and Methods
2.1: Strains and growth media & conditions
The yeast strains used in this study and their origins are listed in Table 2. The S. cerevisiae Cali ale yeast (Fermentis) and B. bruxellensis BSI Drei (Brewing Science
Institute) yeast strains were used as known commercially used controls. A sampling of eight novel B. bruxellensis yeast strains were characterized. Following this, a subset of
B. bruxellensis strains (BSI Drei, BBY011, and PEST IV) were chosen for further characterization, both alone and in combination with commercially available S. cerevisiae strains listed in Table 2.
Yeasts were streaked onto differential WLN media, containing bromocresol green as an indicator. This is a rich medium that supports the growth of both yeast and bacteria, while allowing for differentiation between different organisms and strains.
Bromocresol green is taken up at different rates by different yeast strains allowing for identification of distinct strains in a mixed culture or ensuring purity of a culture. Isolated colonies were cultured in standard YPD broth (1% (w/v) yeast extract, 2% (w/v) peptone, and 2% (w/v) dextrose) at 30°C with shaking at 170 rpm for 3 days for B. bruxellensis strains and 24 hours for S. cerevisiae strains.
26 Table 2. Strains used in this study
Strain Species Origin
Cali Saccharomyces cerevisiae Fermentis
BSI Drei Brettanomyces bruxellensis Brewing Science Institute
BBY011 Brettanomyces bruxellensis Bottled beer (Canada)
BBY017 Brettanomyces bruxellensis Bottled beer (USA)
BBY024 Brettanomyces bruxellensis Bottled beer (USA)
BBY026 Brettanomyces bruxellensis Bottled beer (USA)
BBY028 Brettanomyces bruxellensis Bottled beer (USA)
EBY010 Brettanomyces bruxellensis Lambic
PEST III Brettanomyces bruxellensis Wine (Canada)
PEST IV Brettanomyces bruxellensis Wine (Canada)
Vermont Saccharomyces cerevisiae Bottled beer (USA)
St. Remy Saccharomyces cerevisiae Bottled beer (Belgium)
Dupont Saccharomyces cerevisiae Bottled beer (Belgium)
2.2: Genetic identification of yeasts
The internal transcribed spacer (ITS) regions that separate conserved ribosomal
RNA genes can be used to identify species of the Brettanomyces/Dekkera genus [126].
Genomic DNA was isolated from YPD-grown yeast cultures using standard procedures
[127]. The ITS regions of the Brettanomyces strains were amplified using ITS1 (5’TCC
27 GTA GGT GAA CCT TGC GG 3’) and ITS4 (5’TCC TCC GCT TAT TGA TAT GC 3’) as primers [128]. PCR reactions contained 1 µL of genomic DNA, 2.5 µM of each primer,
0.4 mM dNTPs, 2.5 U of Taq DNA polymerase, and 1X Taq reaction buffer. The amplification reactions were carried out in a BioRad T100 Thermal cycler under conditions adapted from Pham et al. (2011). The DNA products were purified using a
QIAquick PCR purification kit and submitted for sequencing using an Applied
Biosystems 3730 DNA analyzer. The resulting sequences were identified using NCBI
BLAST.
PCR fingerprinting of the Brettanomyces strains was performed using the RAPD primer OPC20 (5’ACT TCG CCA C 3’), which we found to be the most reliable and discriminatory of the available RAPD primers for Brettanomyces [37]. PCR reactions contained 1µL of genomic DNA, 1.5 mM Mg2Cl, 2.5 µM primer, 0.4 mM dNTPs, 2.5 U of
Taq DNA polymerase, and 1X Taq reaction buffer. The amplification reactions were carried out in a BioRad T100 Thermal cycler under RAPD-PCR conditions described by
Crauwels et al. (2014).
Reaction products were confirmed through electrophoresis on a 1% agarose gel in 1X TAE buffer. PCR samples were then purified using a QIAquick PCR purification kit and submitted for analysis on an Agilent 2100 Bioanalyzer using the Agilent DNA 7500 chip. Banding patterns obtained using Bioanalyzer were analyzed using GelJ software and a dendrogram was built using the software, using Pearson correlation and the unweighted pair group method with arithmetic mean (UPGMA) cluster algorithm [129].
28 2.3: Fermentation trials
Lab-scale fermentation trials were carried out in 70 mL of wort in ‘spice jars’ fitted with fermentation locks (Figure 5). Wort for these experiments was obtained from either
Royal City Brewing or Escarpment Labs in Guelph, Ontario. The wort was autoclaved prior to use at 121ºC for 20 minutes and cooled to the desired fermentation or propagation temperature overnight. Fermentation profiles were obtained by weighing the spice jars to measure the weight loss (CO2 evolution) over the course of the fermentation. In addition, empty spice jars fitted with fermentation locks were weighed to determine the degree of water loss over the course of the fermentation, allowing data to be normalized for water loss from the fermentation locks.
Figure 5. Spice jars fitted with fermentation locks that were used for all fermentation experiments in this thesis.
2.3.1: Pre-growth conditions
Prior to fermentation, each strain was propagated in YPD at 30°C with shaking at
170 rpm. The YPD culture was then used to inoculate wort at a rate of 50x106 cells/mL.
The wort culture was also grown at 30°C with shaking at 170 rpm. B. bruxellensis cultures were grown for 3 days in both YPD and wort, while S. cerevisiae cultures were grown for 24 hours in each media. Nutrient additions used in nutrient fermentation
29 studies are summarized in Table 3. All nutrients were added to the wort at the start of propagation. Nutrient supplementation did not occur in the YPD growths. For all nutrient studies controls that received no supplementation were also grown.
Table 3. Nutrient supplementations in yeast pre-growth cultures
Experiment Nutrient Final Concentration
Thiamine Thiamine 0.48 mg/L
Aspartate 1.05 mg/mL
Individual Amino Acids Glutamate 1.05 mg/mL
Glutamine 1.05 mg/mL
2.3.2: Primary Fermentations
The eight novel B. bruxellensis strains were tested alongside the S. cerevisiae
(Cali; Fermentis) and B. bruxellensis (BSI Drei; Brewing Science Institute) standard controls in a 28-day fermentation at 15°C and 22.5°C. A standard pitching rate of
1.2x107 cells/mL was used to inoculate 70 mL of wort in triplicate for each strain, producing biological replicates.
2.3.3: Fermentations with nutrient additions
For this experiment BSI Drei, BBY011, and PEST IV were used to determine the effect of nutrient supplementation in three different combinations. These combinations were: supplementation in wort pre-growth and supplementation in fermentation (w/ w/), supplementation in wort pre-growth and no supplementation in fermentation (w/ w/o), and no supplementation in pre-growth and supplementation in fermentation (w/o w/)
30 (Figure 6). In addition, a control with no nutrient supplementation in pre-growth or fermentation (w/o w/o) was tested (Figure 6). These fermentations were set up in triplicate for each strain and condition. The experiment was conducted at 22°C for 28 days, following which the samples were filtered and analyzed.
The following nutrient additions were tested: thiamine, aspartate, glutamate and glutamine. Please refer to table 3 for final concentrations of each nutrient in the fermentation.
Figure 6. Schematic of fermentations with nutrient additions. Cultures are grown in YPD and then transferred to wort or wort containing the nutrient addition for pre-growth. Following that the cultures are pitched into spice jars for fermentations that either contain nutrient supplementation or are left unsupplemented. This produces four possible combinations: nutrient addition in pre-growth and in fermentation (w/ w/); nutrient addition in pre-growth but not in fermentation (w/ w/o); no nutrient addition in pre-growth and addition in fermentation (w/o w/); and no nutrient addition at either step (w/o w/o).
31 2.3.4: Primary, secondary, and co-pitch fermentations
Primary, secondary, and co-pitch fermentations were conducted at 22°C for 21 days. Each yeast strain was tested individually in a primary fermentation and a combination of each S. cerevisiae strain with each B. bruxellensis strain was tested in a secondary and co-pitch fermentation. Table 4 summarizes the yeast strains used in this experiment and their characteristics. Table 5 summarizes the pitch day and pitch rate for each type of fermentation.
Table 4. Strains used in the primary, secondary, and co-pitch/mixed fermentation experiment Yeast Species Strain Name Strain Characteristics
B. bruxellensis BSI Drei Beer isolate
B. bruxellensis BBY011 Beer isolate
B. bruxellensis PEST IV Wine isolate
S. cerevisiae Cali, American ale Neutral; POF -
S. cerevisiae Vermont, English ale Fruity; POF -
S. cerevisiae St. Remy, Belgian ale Phenolic; POF +
32 Table 5. Pitch day and rate for primary, secondary, and co-pitch fermentations.
Yeast Species Fermentation Type Pitch Day Pitch Rate
B. bruxellensis Primary 0 1.2x107
B. bruxellensis Secondary 7 1.0x106
B. bruxellensis Co-pitch 0 1.0x106
S. cerevisiae Primary 0 1.2x107
S. cerevisiae Secondary 0 1.2x107
S. cerevisiae Co-pitch 0 1.2x107
2.3.5: Timecourse fermentations
These fermentations tested BSI Drei and BBY011 individually in a primary fermentation and as a combination of Cali with each B. bruxellensis strain in a co- pitch/mixed fermentation. The experiment was carried out in two batches and using two different batches of wort. Batch 1 consisted of Cali, BSI Drei, and Cali x BSI Drei and batch 2 consisted of BBY011 and Cali x BBY011. Fermentations were performed in triplicate for each primary and co-pitch fermentation and for each of the timepoints (Day
1, 2, 3, 4, 5, 7, 10, 14, 17, & 21). These fermentations were discarded following sampling. A pitching rate of 1.2x107 cells/mL was used for each strain in the primary fermentation and Cali in the co-pitch fermentation, while a pitching rate of 1.0x106 cells/mL was used for the B. bruxellensis strains in the co-pitched fermentations. At each timepoint samples were filtered and then frozen to allow for simultaneous analysis of all samples at the end of the fermentation.
33 2.4: Metabolite Analysis
2.4.1: HS-SPME-GC-MS
Immediately following the fermentations, samples were filtered using a 0.45μm syringe filter. The samples were subjected to HS-SPME-GC-MS analysis as previously described [130]. 3-octanol and 3,4-dimethylphenol were used as internal standards and a DVB/CAR/PDMS fiber was used to capture the volatilized compounds. The resulting data was analyzed using R Studio and the data was visualized in bar plots using the ggplot2 package [131] and heatmaps using the heatmap3 package [132] to identify differences in fermentation profiles between different strains and conditions.
2.4.2: HPLC
The ethanol and sugar concentrations of each fermentation sample were measured using HPLC and a RI detector as previously described [133]. Samples contained 400μL of filtered sample and 50μL of 6% (w/v) arabinose as the run standard.
The samples were analyzed with a BioRad Aminex HPX-87H column, using 5 mM sulfuric acid as the mobile phase at a flow rate of 0.6 mL/min under ~600 psi at 60°C.
Ethanol and sugar concentrations were determined by comparison of peak heights with standard curves for each compound.
2.5: Carbon source growth assay
Following growth using standard conditions, outlined above, cells were inoculated into 250 μL of the test media (adapted from [61]) containing 2% of the carbon source of interest to a starting OD595 of 0.05. The experiment was done in a 96 well plate and in triplicate for each strain at 15°C and 22.5°C, with shaking at 130 rpm. The following carbon sources were tested: glucose, fructose, maltose, sucrose, maltotriose,
34 cellobiose, and maltodextrin. OD595 measurements were taken at several time points
(T=0, 2, 4, 8, 16, 24, 36, 48, 72, 96, & 120 hours) over a five-day period. The number of days required to reach an OD595 greater than 0.5 was calculated.
35 Chapter 3 - Results: Screening novel Brettanomyces strains
3.1: Phylogenetic analysis of eight Brettanomyces strains reveals distinct groupings
ITS PCR and sequencing (University of Guelph Genomics Facility) was used to confirm the identity of the strains used in this study. As expected, the B. bruxellensis and S. cerevisiae strains supported the production of PCR products of approximately
400 bp and 860 bp, respectively, and sequencing of these products confirmed the eight novel strains were indeed B. bruxellensis and that the species identities of the two controls were accurate (Table 2).
Using the above data, a simple phylogenetic analysis of these strains was performed by clustering the fingerprint data produced by the RAPD PCR primer OPC20
(37). The American ale yeast Cali (Fermentis) was clearly distinct from all the
Brettanomyces strains. Within the Brettanomyces grouping, two clusters were identified; the first contained all the beer isolates and the second contained the two wine isolates
(Figure 7). Thus, the wine isolates (PEST III and PEST IV) are likely genetically distinct from any of the beer isolates, regardless of the origin of the latter.
Two sub-clusters formed within the beer isolate cluster. Here, EBY010 and
BBY011 separated from the other Brettanomyces strains by a distinct margin suggesting that BBY011 (Canadian isolated) and EBY010 (Lambic isolated) are closely related (Figure 7). Next, a group of five strains form the second sub-cluster. BBY024,
BBY026 and BSI Drei form a distinct grouping within which BBY026 and BSI Drei are more closely related. Lastly, BBY017 and BBY028 are closely related, but clearly
36 separate from the other three strains within this sub-cluster. Interestingly, all the strains within this second cluster are strains isolated from North American beer.
Figure 7. Dendrogram of studied strains generated via OPC20 RAPD PCR analysis. RAPD PCR was performed, and the banding patterns imaged using capillary electrophoresis (Agilent Bioanalyzer). The resultant data were clustered by GelJ software using Pearson correlation and UPGMA.
3.2: Fermentation temperature impacts attenuation efficiency of
Brettanomyces strains in primary wort fermentation
The fermentation efficiencies of the individual Brettanomyces strains were analyzed in primary wort fermentations at 15°C and 22.5°C. Fermentation kinetics were monitored as a function of CO2 evolution measured as weight loss over a period of 28
37 days (Figure 8A and 8B). The initial gravity of the wort was 12.5°Plato. At 15°C, the
Saccharomyces beer yeast control Cali reached 50% attenuation at 7 days and its maximum attenuation (4.05 g weight loss) after 24 days of fermentation. As expected, this rate is slower than the corresponding attenuation rate at 22.5°C (50% attenuation after 5 days and maximum attenuation of 5.44 g weight loss after 12 days of fermentation). Similarly, the lower temperature also decreased the attenuation rate of the Brettanomyces control BSI Drei (3.1 g weight loss after 28 days at 15°C vs. 4.9 g weight loss after 17 days at 22.5°C). Unexpectedly, within the first five days of fermentation Cali and BSI Drei showed very similar attenuation profiles at 22.5°C while
BSI Drei attenuated slightly faster than Cali at 15°C. There was no prolonged lag phase for BSI Drei at either temperature. However, around day 5 at either temperature, the attenuation rate of BSI Drei started to decrease (more pronounced at 15°C) leading to prolonged attenuation times for this yeast.
The eight isolates tested showed significant diversity in fermentation profiles.
Only BBY024 showed fast attenuation, almost identical to BSI Drei, at both temperatures. The remaining 7 strains showed prolonged lag phases of 8-10 days at
15°C and shorter adaptations of 2-4 days at 22.5°C. At 15°C, 5 of the 7 strains accelerated attenuation following the prolonged lag phase, of which EBY010, BBY011 and PEST IV reached similar attenuation levels as BSI Drei (within the 2.35 to 3.1 g weight loss range; Figure 8A). BBY017 and BBY028 showed continued slow attenuation. Two strains, BBY026 and PEST III, did not support any significant attenuation over the 28-day period.
38 Also, all 7 strains exhibited accelerated attenuation at the warmer temperature following the initial adaptation (Figure 8B). Four strains, PEST III, PEST IV, BBY017 and BBY026, clustered together with the weakest attenuation after 28 days. By comparison, EBY010, BBY011 and BBY028 showed faster attenuation, resulting in weight loss within the same range as BSI Drei at the final time point. Collectively, these data show significant variation in wort attenuation capacities for different B. bruxellensis strains at different temperatures.
Measurements of the ethanol concentrations in the beer after 28 days of fermentation showed attenuation by several of the Brettanomyces strains was impacted by the fermentation temperature. The Cali control achieved similar ethanol concentrations at both fermentation temperatures (Figure 8C). By contrast, six
Brettanomyces strains produced significantly different concentrations of ethanol at the warmer temperature with five isolates showing increases and BSI Drei showing a small, but reproducible decrease in ethanol concentration (Figure 8C; * p<0.05). Interestingly, at 22.5°C almost all the Brettanomyces strains produced ethanol concentrations similar to that of Cali (Figure 8C; p>0.05). Collectively, these data suggest warmer fermentation temperatures supports better attenuation by Brettanomyces strains. By contrast, the colder fermentation temperatures clearly inhibited ethanol production by five of the
Brettanomyces strains (Figure 8C; † p<0.05). BBY024, EBY010 and PEST IV were the only isolates that produced ethanol at levels comparable to Cali and BSI Drei (Figure
8C; p>0.05). These yeasts were also the strains that showed the more efficient fermentation phenotypes at the colder temperature (Figure 8C). Therefore, some beer isolates were clearly capable of efficient attenuation at colder temperatures, while
39 others were not. Interestingly, PEST IV, a wine isolate, was capable of efficient attenuation of wort independent of temperature.
40
Figure 8. Fermentation profiles at 15°C (A) and 22.5°C (B) for 7 novel B. bruxellensis strains, and the B. bruxellensis BSI Drei and S. cerevisiae Cali controls. Strains were pre-cultured and wort inoculated as described (see Methods). The fermentation profiles were obtained by weighing the spice jars to determine weight loss. Data points represent the mean of biological replicates (n=3). Data was normalized to account for water loss from the fermentation locks. Final ethanol concentrations (C) of fermentations performed at 15°C (filled bars) and 22.5°C (hashed bars) were measured using HPLC as described (see Methods). Statistical significance was calculated using an unpaired t-test. * p<0.05 in comparison to the same strain at 15°C; † p<0.05 in comparison to the BSI Drei control at 15°C.
41 3.3: Fermentation temperature impacts production of several, but not all
Brettanomyces-produced volatile flavour compounds
The impact of fermentation temperature on the flavour profiles of each of the isolates was also analyzed by measuring selected volatile flavour compounds in the respective beers. The compounds were chosen based on those that have been identified as compounds produced by or characteristic of Brettanomyces and those that could be analyzed using HS-SPME-GC-MS. The final concentrations of volatile phenols, esters, higher alcohols, and medium chain fatty acids of samples collected on day 28 of the fermentations performed in Figure 8 were determined. Examples of the impact of temperature on the concentrations of individual volatile compound concentrations are presented in Figure 9, while all the data collected are summarized in a heat map (Figure
10). As expected, B. bruxellensis strains displayed significantly different levels of each of these metabolites when compared to Cali. While temperature impacted the production of 4-vinylguaiacol (4-VG) by Cali, albeit below the threshold of detection
(Figure 10), this yeast produced negligible amounts of 4-ethylguaiacol (4-EG) (Figure 9
& 10) or 4-ethylphenol (4-EP) (Figure 10). By contrast, while these volatile phenols were all produced by the Brettanomyces isolates tested, only 4-EG was produced at above threshold of detection concentrations by all B. bruxellensis strains (Figure 9). The 4-EG levels stayed relatively consistent between the BSI Drei control and Brettanomyces isolates at low fermentation temperatures. However, at the higher fermentation temperatures slightly higher levels were detected for some Brettanomyces strains, notably BBY011, BBY017, BBY026, BBY028, EBY010 and PEST IV (Figure 9).
42 Cali expectedly produced comparatively high levels of some esters such as isoamyl acetate and phenethyl acetate at both fermentation temperatures (Figure 10).
Similarly, it showed increased production of higher alcohols (phenethyl alcohol and isoamyl alcohol) at 22.5°C. By comparison, the Brettanomyces isolates produced several of these volatile compounds (isoamyl acetate, phenethyl acetate, isoamyl alcohol and phenethyl alcohol) at low levels at both fermentation temperatures (Figure
10). Interestingly, the Brettanomyces isolates produced ethyl acetate at slightly elevated levels at 22.5°C, with BBY026 producing the highest concentration (Figure 10).
Also, Cali produced low concentrations of medium chain fatty acids (hexanoic, octanoic and decanoic acids) and fatty acid esters (ethyl caproate, ethyl caprylate, ethyl decanoate and ethyl nonanoate) below the threshold levels of detection at either fermentation temperature tested (Figure 9 & 10). By contrast, several ethyl esters (ethyl caproate, ethyl caprylate, ethyl decanoate) were produced above the threshold of detection by most of the Brettanomyces strains analyzed (Figure 9). The wine isolates
(PEST III and PEST IV) generally produced lower concentrations of medium chain fatty acids and fatty acid esters than the beer isolates; often below the threshold of detection.
Also, several of the beer isolates (notably BBY011, BBY017, BBY024 and EBY010) had the tendency to produce more of these fatty acid esters at the higher fermentation temperature and at levels appreciably higher than the BSI Drei control strain (Figure 9).
43
Figure 9. Volatile flavour metabolite analysis following a 28-day fermentation. Following the fermentation, samples were filtered and analyzed by HS-SPME-GC-MS (see Methods). Four volatile flavour compounds are shown and the thresholds of detection are indicated. Error bars represent SEM (n=3). Statistical significance was calculated using an unpaired t-test. * p<0.05 in comparison to control (BSI Drei) at the same temperature; † p<0.05 among samples (same strain) in comparison to lower temperature (15°C).
44
Figure 10. Heat map and cluster analysis of volatiles produced at two different temperatures. Analysis was performed using ClustVis software [134]. Columns are centered; unit variance scaling is applied to columns. Both rows and columns are clustered using correlation distance and average linkage.
When performing Principle Component Analysis (PCA) with the flavour profile data produced at the two different fermentation temperatures, it was clear that the
Saccharomyces Cali strain separated significantly from the Brettanomyces strains as expected (Figure 11). Interestingly, when considering the Brettanomyces strains, temperature clearly has a significant impact on the flavour profiles produced as indicated by the pronounced shift of the beer Brettanomyces strains in particular. Also, some separation was observed between the BSI Drei control and Brettanomyces isolates at the higher temperature. Lastly, the flavour profiles of the beer Brettanomyces strains seemed to be affected more dramatically by temperature than those of the wine strains.
45
Figure 11. PCA plot of HS-SPME-GC-MS metabolite profiles of Cali (Saccharomyces control; squares), Brettanomyces beer strains (circles) and Brettanomyces wine strains (triangles) at 15°C (red) and 22.5°C (blue). Plot was generated using ClustVis software (27). Unit variance scaling is applied to rows; SVD with imputation is used to calculate principal components. X and Y axis show principal component 1 and principal component 2 that explain 48.2% and 27.8% of the total variance, respectively.
3.4: Brettanomyces isolates display variation in carbon utilization capabilities under aerobic conditions.
To gain some insight into the different fermentation profiles observed during small scale fermentation trials, the abilities of the isolates to utilize selected simple or complex sugars found in wort, including glucose, fructose, sucrose, maltose, maltotriose, cellobiose and maltodextrin, as sole carbon sources was tested. Carbon source utilization profiles differed among the strains and between the two temperatures with growth occurring more readily at the warmer temperature (Figure 12). Here, all strains, including Cali, experienced similar growth regardless of the carbon source with the exception of maltodextrin and cellobiose (Figure 12B). While Cali and two
46 Brettanomyces isolates (BBY026 and EBY010) did not reach the minimum growth on cellobiose, suggesting weak cellobiose metabolism, the remaining Brettanomyces isolates could metabolize this complex sugar with varying efficiencies. For example, the wine isolates (PEST III and IV) grew twice as fast as the beer isolate BBY017 (Figure
12B).
When considering growth at 15°C, more variation in the ability to metabolize the different carbon sources were observed among strains. Only glucose and fructose supported consistent growth by the strains analyzed. Notably, most beer isolates struggled to metabolize cellobiose at both temperatures tested. Only the control BSI
Drei and the wine isolates displayed consist growth with cellobiose as the sole carbon source. The data suggest that cellobiose utilization is more variable among
Brettanomyces when compared to other sugars and may correlate with the niche from which the strains have been isolated. The aging of beer or wine in wood barrels containing cellobiose may correlate with isolates having the ability to metabolize this sugar.
47
Figure 12. Number of days required for each of the strains to reach an OD595 >0.5 at (A) 15°C and (B) 22.5°C. Growth of the strains was tested on 7 different carbon sources: Glucose, fructose, maltose, sucrose, maltotriose, cellobiose, and maltodextrin. OD595 readings were taken at various time-points over the course of 5 days to monitor growth progress (see Methods). Each of the numbers represents the mean of biological replicates (n=3). Strains that failed to reach this OD are marked with NR (Not Reached). Shading increases with prolonged incubation.
48 Chapter 4 - Results: Primary, Secondary, and Co-Pitch
Fermentations
4.1: Brettanomyces strain selection impacts secondary fermentation profiles more than co-pitch
The impact of Brettanomyces on a secondary and co-pitch fermentation were tested in lab-scale fermentations at 22°C over a 21-day period. In order to determine fermentation kinetics of each strain primary fermentations were set up with each
Brettanomyces and Saccharomyces strain used in the secondary and co-pitch fermentations. The primary fermentations served as controls to gauge how each strain behaved individually and what impact Brettanomyces had in combination with each
Saccharomyces on the established fermentation kinetics and ethanol and flavour compound production.
The Brettanomyces strains chosen for these and all further experiments were
BSI Drei, BBY011, and PEST IV, based on the results presented in Chapter 3. In order to have a better understanding of the differences displayed by strains from different environments a beer strain (BBY011) and wine strain (PEST IV) were chosen. Both of these strains were strong candidates in their respective isolation groups based on fermentation profiles and flavour compound production shown in Chapter 3. BSI Drei was included as the industry standard, allowing comparison of these novel strains to one that is already established within the industry. The Saccharomyces strains were chosen to represent different flavour profile categories: neutral (Cali, POF -), fruity
(Vermont, POF -), and phenolic (St. Remy, POF +). This would allow for observation of the impact Brettanomyces has on the established Saccharomyces flavour profiles.
49 Typical fermentation profiles were observed for all Saccharomyces and
Brettanomyces strains in the primary fermentation. Saccharomyces fermentations did not experience any lag phase and were exhibiting signs of active fermentation (weight loss and production of bubbles on top of the medium) within the first day of the experiment (Figure 13 Primary). In addition to these visual cues, the fermentations were quickly losing weight indicating fermentation and production of CO2. The CO2 escapes through the fermentation lock causing the weight loss. Cali, Vermont, and St. Remy all appeared to be finished fermenting around day 5 of the experiment, as evidenced by the tapering off of the fermentation curve caused by a lack of CO2 production (Figure 13
Primary). While Saccharomyces strains are known for finishing a fermentation quickly,
Brettanomyces fermentations are characterized by an initial lag phase followed by a slow and steady fermentation. Indeed, the primary Brettanomyces fermentations in this experiment experienced a lag phase during the first few days, after which they began fermenting and exhibiting a gradual weight loss and CO2 evolution over the course of the fermentation (Figure 13 Primary). Overall, by the end of the 21 days the
Saccharomyces and Brettanomyces fermentations lost a similar amount of weight and thereby generated similar amounts of CO2 (Figure 13 Primary). However, the
Brettanomyces fermentation curves did not taper off, as was observed with the
Saccharomyces fermentations, suggesting that the fermentations may have required more time to reach completion.
Brettanomyces was able to complete a secondary fermentation following the initial Saccharomyces fermentation. Saccharomyces was pitched into the wort at the beginning of the experiment and CO2 evolution was observed within the first few days
50 indicating active fermentation (Figure 13 Secondary). The fermentation curves began to taper off around day 6/7 of the experiment suggesting that Saccharomyces was finished its fermentation (Figure 13 Secondary). Brettanomyces was pitched into the fermentation on day 7 and began fermenting after a short lag phase (Figure 13
Secondary). The highest fermentation rates were observed for samples fermented with the St. Remy Saccharomyces strain, while those fermented with Cali appeared to ferment more weakly (Figure 13 Secondary). The Cali and Vermont fermentations appeared to follow a pattern in terms of fermentation efficiency, with PEST IV performing the best, followed by BBY011, and then BSI Drei (Figure 13 Secondary).
The St. Remy fermentations did not follow this pattern; the St. Remy x BSI Drei fermentation achieved the highest CO2 evolution, followed by PEST IV, and then
BBY011 (Figure 13 Secondary).
No significant differences were observed between different strains in the co-pitch fermentation profiles (Figure 13 Co-pitch). Unlike the secondary fermentations, all of the
Saccharomyces and Brettanomyces fermentations appeared to ferment with equal efficiency and displayed very similar fermentation profiles, with ~2.5-3.5 g of CO2 evolution observed by the end of the fermentation. In addition, while differences in the fermentation curves could be seen in the secondary fermentations, there did not appear to be significant differences in any of the curves observed for co-pitch fermentations.
51
52
Figure 13. Fermentation profiles for primary, secondary, and co-pitch fermentation models. Timepoints represent the mean of biological replicates (n=3).
4.2: Secondary and co-pitch fermentations produce higher ethanol concentrations compared to primary
Once the 21-day fermentation was completed the samples were analyzed by
HPLC to determine ethanol content. A higher ethanol content was observed in the secondary and co-pitch fermentations compared to the primary fermentation, potentially due to Brettanomyces’ ability to ferment additional sugars (Figure 14). Indeed, this has been well-documented in the literature, with Brettanomyces producing beers that are considered ‘superattenuated’ with moderately higher ethanol concentrations [135]. This effect was not as evident in the primary Brettanomyces fermentations, likely due to the fermentations not being fully completed by day 21.
When comparing the secondary and co-pitch fermentations to each other, the secondary fermentations had a slightly higher ethanol content compared to the co-pitch
53 fermentations (Figure 14). These results correspond to the fermentation profiles shown in the previous section (Figure 13), where more CO2 evolution was observed in the secondary fermentations. In general, there were no significant differences observed between the different strain combinations in the co-pitch fermentations in terms of ethanol production (Figure 14). Similarly, the different strain combinations in secondary fermentations produced comparable levels of ethanol, with the exception of the St.
Remy x PEST IV combination which had the lowest ethanol concentration. Interestingly, all of the fermentations containing Cali produced higher levels of ethanol than St. Remy x PEST IV, despite having the weakest production of CO2 throughout the fermentation.
This may indicate that St. Remy x PEST IV diverted these resources into producing other metabolites rather than ethanol.
Figure 14. Final ethanol concentrations for primary, secondary, and co-pitch fermentations. The bars represent the mean of biological replicates (n=3).
54 4.3: Secondary and co-pitch ferments produce decreased ester and phenolic compounds compared to primary
The impact of different Brettanomyces strains on the flavour profiles in primary and in secondary and co-pitch fermentations with Saccharomyces strains was tested.
The final concentrations of volatile esters, phenols, higher alcohols, and medium chain fatty acids were measured following the 21-day fermentation. All of the results obtained are summarized in a heatmap (Figure 15). The impact of the various strain combinations on specific flavour compounds is shown in Figure 16. As shown by heatmap clustering the primary Saccharomyces strain is distinguishable despite the influence of Brettanomyces (Figure 15). Clustering of the secondary and co-pitch fermentations based on the primary strain used can be seen (Figure 15). This could indicate that the flavour profile developed by the Saccharomyces strain still plays an important role in the final flavour profile of the beer. Another potential reason for the clustering that was observed is the possibility that there was not enough time in the fermentation for the Brettanomyces flavour profile to develop fully, allowing the flavours produced by Saccharomyces to play a more substantial role in the finished beer product. In general, significant differences are not apparent between different strain combinations in secondary and co-pitch fermentations (Figure 15).
As expected, the Saccharomyces strains produced low, below-threshold levels of
4-vinylguaiacol and negligible amounts of 4-ethylguaiacol and 4-ethylphenol (Figure 16).
In contrast, Brettanomyces produced all three of these phenolic compounds; however, only 4-EG was produced in above-threshold concentrations (Figure 16). The secondary and co-pitch fermentations appeared to be less ‘funky’, as lower levels of 4-EG were
55 detected than in the primary fermentations (Figure 16). 4-ethylphenol production was uniform across all of the Brettanomyces strains in primary, while lower levels of 4- vinylguaiacol and 4-ethylguaiacol were produced by PEST IV compared to BSI Drei and
BBY011 (Figure 16). This pattern was not observed in secondary and co-pitch fermentations.
The ester profile of the secondary and co-pitch fermentations was diminished in comparison to the primary fermentations (Figure 16). Lower levels were observed for a number of compounds, such as ethyl butyrate, ethyl caprylate, ethyl decanoate, ethyl nonanoate, and ethyl lactate (Figure 16). This effect was most pronounced for ethyl caproate with the concentrations for certain secondary and co-pitch fermentations dropping to a level at which they were below the sensory threshold. For other compounds, such as ethyl decanoate and ethyl caprylate levels were lower in secondary and co-pitch fermentations; however, for strains that produced above- threshold concentrations in primary the amount produced in secondary and co-pitch tended to remain above threshold as well. Overall, these results indicate that mixed fermentations are an effective strategy for producing beers with Brettanomyces flavours, albeit at slightly lower levels, which may be favourable in some instances.
56
Figure 15. Heat map and cluster analysis of different strains in primary, secondary (S), and co-pitch (Co) fermentation. Analysis was performed using the Heatmap3 package in R.
57
58
59
Figure 16. Volatile flavour metabolite analysis following a 21-day primary (P), secondary (S), or co-pitch (C) fermentation. Following the fermentation, samples were filtered and analyzed by HS-SPME-GC-MS. Error bars represent SEM (n=3).
60 Chapter 5 - Results: Timecourse Fermentation
5.1: Strains show different rates of sugar consumption and ethanol production
The sugar utilization and ethanol production of BSI Drei and BBY011 were tested in a primary fermentation and a co-pitch fermentation with Cali over the course of a 21- day fermentation. In order to better understand the order and extent of sugar consumption by each yeast samples were collected for analysis on the following days:
1, 2, 3, 4, 5, 7, 10, 14, 17, and 21. This allowed for monitoring of sugar depletion and subsequent ethanol production throughout the course of fermentation, as opposed to one reading at the end of the fermentation. Once the experiment was finished glucose, maltose, maltotriose, and ethanol levels were measured using HPLC. It is important to note that this experiment was done in two batches with two different worts. The initial sugar concentrations of each wort and the fermentations they were used for are summarized in Table 6.
Table 6. Initial sugar concentrations for wort used in timecourse experiment Wort Experiments [Glucose] [Maltose] [Maltotriose]
Batch 1 BSI Drei; Cali; 0.54% 4.12% 1.35% Cali x BSI Drei
Batch 2 BBY011; 0.33% 3.51% 1.19% Cali x BBY011
HPLC analysis of the samples showed that glucose was completely utilized in all of the fermentations by day 21 of the experiment (Figure 17). In fermentations
61 containing the S. cerevisiae strain, Cali, glucose was depleted following the first day of fermentation. In general, glucose was the preferentially utilized sugar as it was the first to be consumed in the BSI Drei, Cali, Cali x BSI Drei, and Cali x BBY011 fermentations.
Following glucose, maltose was the next sugar to be consumed fully by the yeasts.
There was no residual maltose present after the first week in fermentations containing
Cali and after the first 10 days for the BBY011 primary fermentation (Figure 17). The yeasts appeared to struggle more with maltotriose utilization, as only the BBY011 and
Cali x BBY011 fermentations were able to utilize all of the sugar by day 21 (Figure 17), which may indicate a stronger capacity for consumption of this sugar by BBY011.
Residual maltotriose levels were still seen in the Cali, BSI Drei, and Cali x BSI Drei fermentations by day 21 (Figure 17). In general, BBY011 appeared to be a strong fermenter without a preference for a specific type of sugar. Glucose, maltose, and maltotriose were all consumed at similar rates by BBY011 in the primary fermentation, with no residual levels of any of the sugars observed beyond day 10 of the fermentation.
In contrast, BSI Drei appeared to struggle more than the other yeasts in terms of sugar consumption. Maltose and maltotriose were not fully consumed by the end of the fermentation and glucose was only fully depleted near the end of the fermentation
(Figure 17).
HPLC measurement of ethanol concentrations in the samples at each of the timepoints indicated that ethanol production varied significantly between different
Brettanomyces strains and between different Cali/Brettanomyces combinations (Figure
17). Despite appearing to struggle with sugar consumption, BSI Drei produced significant amounts of ethanol (~5.5%). In fact, BSI Drei outperformed BBY011 in both
62 the primary and co-pitch fermentations, with more ethanol being produced than in either fermentation containing BBY011. This suggests that BBY011 is utilizing the sugar it consumes for something other than ethanol production, such as an increase in biomass, production of metabolites like acetic acid, or intracellular storage of these sugars. In general, ethanol levels peaked near the end of the 21-day fermentation for primary
Brettanomyces fermentations and within the first few days for fermentations containing
Cali (Figure 17). Interestingly, the ‘make-accumulate-consume’ phenomenon could be observed in fermentations containing Cali, with ethanol levels peaking within the first few days, followed by a dip in the ethanol concentration, prior to levels rising again
[101].
63
Figure 17. Sugar utilization and ethanol production profiles for Cali, BSI Drei, BBY011, Cali x BSI Drei, and Cali x BBY011 during a 21-day fermentation at 22°C. Samples were collected on the indicated days. Each bar represents the mean of biological replicates (n=3).
64 5.2: Brettanomyces’ production of esters peaks around day 14 of the fermentation
Volatile flavour compounds were measured at each timepoint using HS-SPME-
GC-MS in order to determine the rate and timing of production in primary fermentations compared to the co-pitch fermentations. In general, the co-pitch fermentations appeared to be less ‘fruity’ than the primary Brettanomyces fermentations as evidenced by lower levels of esters, such as ethyl caproate, ethyl butyrate, ethyl isovalerate, and ethyl decanoate (Figure 18). This effect was particularly evident when comparing the primary
BSI Drei fermentation to the Cali x BSI Drei fermentation, where a drastic drop in concentration could be seen for a number of the esters. For both BSI Drei and BBY011 ester production peaked around day 14 of the fermentation and decreased slightly afterwards (Figure 18). It is also interesting to note that fermentations with
Brettanomyces contained significantly higher levels of ethyl acetate compared to the
Cali primary fermentation and lower levels of phenyl ethyl acetate (Figure 18). It has been shown that this is due to the presence of esterases, which are responsible for producing ethyl esters while also degrading acetate esters [136,137]. Phenyl ethyl acetate could be detected in the Cali primary fermentation and the Cali x BSI Drei fermentation, although levels in the latter dropped throughout the course of the experiment suggesting that it was being degraded by BSI Drei’s esterases (Figure 18).
Ethyl acetate was also found in the Cali fermentations but levels were lower than in any of the fermentations containing BSI Drei or BBY011.
65
66
67
Figure 18. Volatile esters at each timepoint during the timecourse fermentations. Samples were collected and filtered at each timepoint and frozen until the end of the experiment. Upon completion of the experiment, the metabolites were measured using HS-SPME-GC-MS. Error bars represent the SEM (n=3).
68 5.3: Phenolic compound production begins at the start of fermentation
Volatile phenols were measured using the same process that was used for esters. As expected, Cali produced 4-vinylguaiacol but no 4-ethylguaiacol or 4- ethylphenol (Figure 19). In contrast, Brettanomyces began producing these compounds at the beginning of fermentation (Figure 19). Hydroxycinnamic acids (HCAs), such as p- coumaric acid and ferulic acid, are precursors for hydroxystyrenes, 4-vinylphenol and 4- vinylguaiacol, respectively [138]. HCAs also serve as antimicrobials and as a result many wild yeasts and bacteria have evolved to possess enzymes to convert HCAs to compounds that will not be toxic to the cells [139, 140]. This may explain why 4-VG production is observed from the beginning of fermentation in all the strains and combinations tested. Brettanomyces is then able to further convert the hydroxystyrenes to ethylphenols through the activity of a VPR enzyme [138]. It is a well-established fact that Brettanomyces experiences a lag phase upon being shifted to an anaerobic environment and there is evidence linking this phenomenon to a redox imbalance caused by a buildup of NADH and a shortage of NAD+ [141]. VPR requires NADH as a cofactor during hydroxystyrene reduction, suggesting that production of ethylphenols early on in the fermentation allows for replenishing of the NAD+ levels thereby alleviating the lag phase [142]. The levels of vinylguaiacol fluctuate throughout the
Brettanomyces fermentations, suggesting that the yeast may be converting it to ethylphenols (Figure 19). In general, the levels of 4-ethylphenol and 4-ethylguaiacol peak towards the end of the experiment (Figure 19), which confirms results obtained by other researchers that observed the highest concentration of these metabolites during late exponential and early stationary phase despite production beginning at the start of
69 fermentation [142, 143]. Fermentations containing BSI Drei appeared to be more
‘funky’, with higher levels of both 4-ethylguaiacol and 4-ethylphenol, particularly in the
Cali x BSI Drei mix, compared to other fermentations (Figure 19). Activity of VPR and the conversion of p-coumaric and ferulic acid has been shown to be strain-dependent
[17, 143].
70
Figure 19. Volatile phenols at each timepoint during the timecourse fermentations. Samples were collected and filtered at each timepoint and frozen until the end of the experiment. Upon completion of the experiment, the metabolites were measured using HS-SPME-GC-MS. Error bars represent the SEM (n=3).
71 Chapter 6 - Results: Nutrient Supplementation 6.1: Impact of various nutrient additions is strain-dependent One of the main challenges with working with Brettanomyces in beer fermentation is the lag phase experienced when it is pitched into an anaerobic environment. This is not ideal for brewers as it lengthens the time needed for the fermentation to finish and ties up production equipment thereby increasing costs.
Solutions for decreasing this lag time that can prove to be viable solutions for a brewer in terms of food safety and accessibility are highly sought after. To this end, nutrient supplementation as a means to decrease the lag phase was tested. Thiamine and three different amino acids (aspartate, glutamate, and glutamine) were added to the wort pre- growth, to the fermentation, or to a combination of the two, along with a control that received no supplementation.
These nutrients were chosen based on positive results on Brettanomyces growth rates in previous experiments. Previous research has shown that thiamine is required by many strains of Brettanomyces for efficient growth [61]. Meanwhile, aspartate, glutamate, and glutamine were found to positively impact the growth rate of
Brettanomyces in an anaerobic environment, with aspartate having the most profound effect of the three [75]. Both of these studies were conducted in a synthetic defined medium and as such there was a need to test the effect of the supplementations in a complex medium, such as wort, in a fermentation setting.
In terms of fermentation rate, thiamine supplementation in both the pre-growth and fermentation in BSI Drei and in the pre-growth in BBY011 and PEST IV resulted in the highest CO2 evolution (Figure 20). This indicates that supplementation does have a positive effect for all of the strains, although this effect is not uniform for all of the
72 strains. Indeed, the order of most effective to least effective treatment varies for all three strains (Figure 20). Differences can also be seen in ethanol production for some strains based on treatment that was administered (Figure 21). The ethanol concentration was the highest for the control sample of BBY011 that received no nutrient addition, followed by the sample that received thiamine in the fermentation only. This suggests that while thiamine addition may result in a quicker and higher fermentation rate, it hinders ethanol production by potentially diverting resources towards biomass or alternative metabolite production. PEST IV produced the highest amounts of ethanol when thiamine was added to the fermentation only, while the other treatments and the control all produced similar levels of ethanol (~2%). BSI Drei’s production of ethanol appeared to be unaffected by thiamine addition as all three treatment samples contained the same level of ethanol as the control. The BSI Drei and BBY011 final products had no residual maltose and maltotriose present indicating complete utilization of both sugars by both strains regardless of nutrient supplementation (Figure 21). In contrast, PEST IV was not able to consume either maltose or maltotriose fully by the end of the 28-day fermentation (Figure 21). The following amounts of maltose and maltotriose, respectively, were consumed in each fermentation by PEST IV: 47% and 24% for w/ w/;
52% and 25% for w/ w/o; 77% and 36% for w/o w/; 43% and 26% for w/o w/o. This indicates that PEST IV that received thiamine supplementation in the fermentation was most successful at utilizing the sugars.
Overall, thiamine supplementation at any stage during the experiment did not affect flavour metabolite concentrations in the final product (Figure 22). The concentrations measured in the three treatment samples did not differ significantly from
73 the control, with the following exceptions (p<0.05): an increase in ethyl caproate for
PEST IV w/ w/o; an increase in ethyl caprylate for PEST IV w/ w/; a decrease in ethyl decanoate for PEST IV w/ w/; a decrease in ethyl nonanoate for BSI Drei w/ w/; a decrease in 4-EG for BSI Drei w/ w/; an increase in 4-EP for BSI Drei w/ w/o; s decrease in ethyl nonanoate for BBY011 w/ w/ (Figure 22). It appears as though thiamine supplementation has less of an effect on flavour compound production in
BBY011 compared to BSI Drei and PEST IV as there was only one instance where there was a significant change in metabolite concentration between the treatment and control group.
Figure 20. Fermentation curves for BSI Drei, BBY011, and PEST IV with thiamine supplementation in pre-growth and fermentation (w/ w/); with thiamine supplementation in pre-growth only (w/ w/o); with supplementation in fermentation only (w/o w/); and a control containing no supplementation (w/o w/o). The data points represent the SEM (n=3).
74
Figure 21. Final ethanol, maltose, and maltotriose concentrations in thiamine supplementation fermentations. Concentrations were determined through HPLC analysis and error bars represent the SEM (n=3).
75
Figure 22. Volatile flavour metabolite analysis following a 28-day thiamine supplementation fermentation. Error bars represent SEM (n=3). Statistical significance was calculated using an unpaired t-test (* represents p<0.05).
76 A previous study observed that Brettanomyces readily assimilates glutamine, glutamate, and aspartate as a nitrogen source in anaerobic conditions and experiences an increase in growth rate in these conditions [75]. Based on these results, the addition of these amino acids in the pre-growth and fermentation steps was tested to determine whether it would lead to an increase in fermentation rate in wort medium. Aspartate had the strongest effect for BSI Drei and BBY011, with all samples that contained supplementation at either the pre-growth step, fermentation step, or both producing greater amounts of CO2 by the end of the fermentation compared to the control (Figure
23). The effect of this supplementation was particularly evident in BSI Drei, with the sample that received supplementation in both pre-growth and fermentation experiencing a shorter lag phase and beginning to ferment quicker than the other samples (Figure 23).
This effect was not observed in PEST IV, where the control finished fermenting with almost double the amount of CO2 produced when compared to the other samples (Figure
23). The sample that received aspartate in the fermentation experienced the shortest lag phase; however, it was not able to produce as much CO2 as the control. In terms of glutamate addition, the same effect can be seen for all of the strains. BSI Drei and
BBY011 appear to benefit from the supplementation, with the samples that received supplementation at both stages producing the most CO2 and the control producing the least or the same amount as the next lowest sample (Figure 23). Glutamate did not appear to benefit PEST IV, as the control produced the highest amount of CO2 by the end of the fermentation and experienced the shortest lag phase. Glutamine addition offers some benefit to both BSI Drei and BBY011, with the samples that received the amino acid in the fermentation experiencing the shortest lag phase. In addition, the control performed the
77 worst or on par with another sample for both strains. Glutamine had the strongest effect on
PEST IV of the tested amino acids. While the control sample produced the highest amount of CO2 by the end of the fermentation, the sample that had glutamine added to the pre- growth and the fermentation exhibited the shortest lag phase and produced the second highest amount of CO2 (Figure 23). This indicates that glutamine addition may still be a potential route of accelerating PEST IV fermentation. In addition to the positive effects observed in fermentation rate, addition of these amino acids did not hinder ethanol production by any of the strains. The levels of ethanol that were present in samples fermented with nutrient addition at some stage were very similar to those found in the control (Figure 24).
Figure 25 shows some flavour metabolites that were produced during the fermentation and indicates any instance where the treatment sample contained a significantly different amount of the metabolite compared to the control. Ethyl nonanoate and ethyl phenol appeared to be the least affected by the nutrient addition, with only PEST
IV supplemented with glutamine at some stage and BBY011 supplemented with aspartate in the fermentation showing significant differences in the production of the former and latter metabolite, respectively. 4-vinylguaiacol production was also only minorly affected, with a few BSI Drei samples and one BBY011 sample showing significant differences compared to the control (Figure 25). BSI Drei production of ethyl caproate and ethyl caprylate was not significantly affected by addition of any of the amino acids, contrary to
BBY011 and PEST IV which both had instances of significant decreases in the production of these esters with certain treatments (Figure 25). Ethyl caproate production by BBY011 was diminished with the addition of aspartate or glutamate at any stage of the pre-growth
78 or fermentation process. PEST IV production of this ester decreased with the addition of glutamate in the fermentation and addition of glutamine during pre-growth or during fermentation, but not both. Aspartate addition at any point during the pre-growth or fermentation stages also resulted in decreased production of ethyl caprylate. In addition, ethyl caprylate production was reduced with glutamate addition in the w/ w/ and w/o w/ fermentations, indicating that this amino acid affects production of this ester if it is present during fermentation. Glutamine addition had the greatest impact on PEST IV’s production of ethyl caprylate, with all experimental fermentations showing decreased production compared to the control. The production of ethyl decanoate and 4-ethyl guaiacol was affected to some degree in all of the strains depending on the level of amino acid supplementation, suggesting that the production of these metabolites was the most susceptible to changes in media composition. This could indicate that amino acid additions repress flavour metabolites to some extent. Previous work on Saccharomyces has shown that higher nitrogen levels result in lower ester concentrations in the final beer product
[144].
79
Figure 23. Fermentation curves for BSI Drei, BBY011, and PEST IV with amino acid supplementation in pre-growth and fermentation (w/ w/); with amino acid supplementation in pre-growth only (w/ w/o); with supplementation in fermentation only (w/o w/); and a control containing no supplementation (w/o w/o). The data points represent the SEM (n=3).
80
Figure 24. Final ethanol concentrations for BSI Drei, BBY011, and PEST IV with amino acid supplementation of aspartate (A), glutamate (G), or glutamine (GL) in pre-growth and fermentation (w/ w/); with supplementation in pre-growth only (w/ w/o); with supplementation in fermentation only (w/o w/); and a control containing no supplementation (w/o w/o). Error bars represent the SEM (n=3).
81
82
83
84
Figure 25. Final concentrations of volatile flavour metabolites following a 28-day fermentation with aspartate (A), glutamate (G), or glutamine (GL) supplementation in pre-growth and fermentation (w/ w/); pre-growth only (w/ w/o); fermentation only (w/o w/); compared to a control that received no supplementation (w/o w/o).
85 Chapter 7 - Discussion
The brewing industry has been growing exponentially in the past decade and there is a need to provide unique and quality products in order to stand out from the competitors [135]. One of the ways in which breweries are producing these novel products is through the use of non-conventional brewing yeast [135]. The most popular of these yeasts is Brettanomyces bruxellensis, which despite diverging from
Saccharomyces cerevisiae nearly 200 mya shares many of the same characteristics that make it well suited for a brewing environment [102]. This is indicative of convergent evolution that has occurred between these two yeast species [7]. Prior to this it was regarded as a spoilage organism in the wine industry and this status hampered research on its biology and fermentation capabilities [145]. As a result, there is a need for research on how it performs in a brewing environment in order to better inform brewers on how to work with this organism.
Selecting strong candidates for further experiments
In the first part of my thesis a diverse group of eight novel B. bruxellensis strains was explored in temperature controlled primary fermentations to determine their suitability in terms of fermentation efficiency and flavour compound production. Distinct attenuation capabilities were observed among the isolates and between the two temperatures, indicating that B. bruxellensis wort fermentation rates are temperature- dependent and strain specific. While all isolates attenuated at the higher fermentation temperature, variations in duration of the lag phase, the fermentation rate, and the final level of attenuation were observed between strains during the 28-day fermentation. BSI
Drei and BBY011 fermented quickly, while EBY010, BBY011, and BBY028 attenuated
86 slower but ultimately reached a similar final attenuation after 28 days. The remaining isolates attenuated poorly at the warmer temperature. By contrast, all the strains showed slower fermentation rates and lower final levels of attenuation at 15°C. S. cerevisiae strain Cali outperformed all of the B. bruxellensis strains at the lower temperature; however, BBY024, EBY010, and PEST IV reached final fermentation levels similar to BSI Drei, suggesting that they are strong fermenters at the lower temperature.
Carbon utilization profiles also differed amongst the strains and the two different temperatures, with a marked difficulty in using the majority of the sugars at 15°C. These data corroborate the fermentation trial findings where the isolates also struggled to ferment at the lower temperature. It is therefore clear that warmer temperatures are more conducive to Brettanomyces-based fermentations; however, attenuation capacities vary in a strain-dependent manner at both temperatures. This suggests that careful strain selection can potentially assist in achieving custom attenuation targets for
Brettanomyces-impacted beers.
As expected, the volatile flavour compound profile produced by B. bruxellensis in wort was shown to be different than that of the S. cerevisiae control, specifically in the levels of certain volatile esters and phenols produced. Notably, there was above- threshold production of ethyl caprylate (pineapple aroma), ethyl caproate
(apple/pineapple aroma), ethyl decanoate (red apple aroma), 4-ethyl guaiacol (clove aroma), and 4-ethyl phenol (barnyard aroma) in the B. bruxellensis fermentations, with a high degree of variability in ester production. Previous research has shown that the production of volatile esters is higher at warmer temperatures for ale yeasts [146], which
87 was confirmed by this data. The majority of the Brettanomyces beer isolates produced higher levels of esters ethyl caproate, ethyl caprylate, and ethyl decanoate at 22.5°C thereby contributing to the ‘fruity’ profile associated with Brettanomyces beers [147]. In addition, BBY011 and EBY010 produced higher levels of esters than BSI Drei. Also, both these strains showed good attenuation which could further support the development of the fruity character of Brettanomyces beers using these two strains.
Interestingly, the Brettanomyces wine isolates showed much lower levels of esters compared to the beer isolates. There are several potential explanations for the increased presence of ethyl esters in Brettanomyces-fermented beers compared to
Saccharomyces. While the enzymes responsible for the production of these metabolites have not been elucidated in Brettanomyces it is possible that there are duplications present in the genes for these enzymes or that the enzymes themselves are more active. In addition, it has been shown that ethyl ester diffusion into the medium by
Saccharomyces is dependent on the composition of the ester, with smaller molecules diffusing more rapidly than larger ones [148]. It is possible that the diffusion of these esters is more efficient in Brettanomyces compared to Saccharomyces. It is known that many volatile flavour active compounds are captured as non-volatile glycosides that require enzymatic hydrolysis by β-glucosidases to be released for the development of beer and wine flavour [89, 149]. To this end, a variation in phenotypic profiles based on the functionality of β-glucosidases was previously reported for Brettanomyces strains isolated from different niche environments [62]. The exact molecular and genetic underpinnings of this phenomenon are currently unclear.
88 While ester production is clearly linked to temperature, a dramatic link between phenol production and fermentation temperature could not be discerned. Production of
4-EG and 4-EP were similar between the two temperatures and between most isolates.
Only BBY028 and EBY010 showed a small, but statistically significant, increase in 4-EG at 22.5°C. Interestingly, BBY011, BBY028, EBY010, and PEST IV produced slightly higher levels of 4-EG than BSI Drei at 22.5°C. Prior research has indicated that while volatile phenol production is delayed at lower temperatures, it is not inhibited, and final volatile phenol concentrations are similar between colder and warmer fermentations
[109]. Importantly, these phenolic compounds were barely present in the Cali control, which supports prior research and the notion that these compounds give Brettanomyces beers a unique taste and aroma [89].
In characterizing these eight strains of Brettanomyces, a high degree of strain- dependent variability in fermentation efficiency and flavour metabolite production were observed. This may be a result of varying degrees of use of each of these strains in beer fermentation until now, resulting in greater adaptation among some strains to these conditions. In particular, this can be seen with the wine-isolated PEST strains, which have only recently been subjected to a beer fermentation environment, causing them to have lower fermentative capabilities compared to the beer isolates. Based on the results of these experiments, it was decided that further experiments would be conducted at a warmer temperature as Brettanomyces is a poor attenuator at lower temperatures for the most part. In addition, the strains were narrowed down to BSI Drei, BBY011, and
PEST IV for further experiments in order to have a control and a representative from a
89 beer and wine environment. BBY011 and PEST IV were chosen based on their fermentation efficiencies and positive flavour profiles amongst the two isolation groups.
Benefits to the Industry
Despite the increasing use of Brettanomyces in industrial beer fermentations, its impact and contribution towards different types of fermentation is not well characterized
[150]. There is a need for this research to better inform brewers on the best practices and methods to achieve a desired outcome when fermenting with this yeast. Currently,
Brettanomyces fermentations can be highly unpredictable causing these fermentations to be a gamble for brewers [150, 151]. Due to the longer fermentation times associated with Brettanomyces breweries must be willing to occupy fermenters for longer periods of time, which translates to a higher cost of production. Through investigating
Brettanomyces in primary, secondary, and co-pitch fermentations and mapping sugar consumption and ethanol and flavour compound production over a 21-day fermentation period, data was generated that will allow brewers to have a better understanding of how Brettanomyces will perform in each of these situations and what flavour profiles can be expected.
My data showed that Brettanomyces is capable of completing a secondary fermentation following the initial alcoholic fermentation carried out by Saccharomyces.
Brettanomyces’ impact was more clearly observed in the secondary fermentation curves compared to the co-pitch fermentations. While the co-pitch curves all appeared fairly uniform, regardless of the strain combination, differences were evident between different strain combinations at the end of the secondary fermentation. In general, fermentations that utilized St. Remy as the primary strain performed the best. Since
90 Brettanomyces was not pitched until day 7 of the secondary fermentation the differences that were observed may be due to the shorter acclimatization and fermentation time that it had compared to co-pitch, where it was present in the fermentation from the beginning. As expected, secondary and co-pitch fermentations were found to have a higher ethanol content than primary Saccharomyces fermentations, which confirms previous research [152]. Brettanomyces is a “super attenuator”, capable of fermenting sugars, such as cellobiose and dextrins, that
Saccharomyces cannot utilize, resulting in the increased ethanol concentrations that were observed [152].
In terms of flavour profiles, secondary and co-pitch fermentations were found to be less fruity and less phenolic with decreased levels of both esters and phenols observed. In addition, the influence of the primary Saccharomyces strain was still evident, likely due to the fermentation time being insufficient to allow Brettanomyces flavours to fully develop. Together this data indicates that mixed fermentations are an effective approach to producing beers that have Brettanomyces flavours and aromas on a shorter time frame thereby freeing up fermenters more quickly. While the flavour profile is slightly less pronounced in these mixed fermentations compared to primary the beers still have “Brett character”. Since “Brett character” can be a polarizing flavour profile if it is present in high quantities [153], achieving some of these flavours in lower concentrations through mixed fermentations may make these beers more appealing for a wider range of consumers.
The timecourse data revealed that Brettanomyces’ contribution towards the flavour profile of the beer peaks around day 14 in terms of esters and near the end of
91 fermentation for phenolic compounds. The levels of esters present in the medium may decrease after day 14 due to evaporation or further metabolization by the yeast.
Previous research has shown that Brettanomyces possesses esterases, which are capable of breaking down esters present in the medium thereby decreasing the ester profile of the beer [115]. This information is helpful for brewers that are looking to produce a more specific flavour profile in their beers, particularly if Saccharomyces is used to complete the alcoholic fermentation and Brettanomyces is used mainly to contribute to the flavour profile. Brewers can tailor their fermentation times to target production of specific flavour compounds.
Media customization is necessary for optimal Brettanomyces fermentations
One of the major hindrances in working with Brettanomyces is its lengthy lag phase upon being transferred to an anaerobic environment, such as the one that it encounters in fermentation [117]. This problem was identified decades ago and studies seemingly ‘solved’ this issue through the addition of various compounds to the medium
[121, 122]. Unfortunately, this research was conducted prior to the intentional use of
Brettanomyces in beer production and therefore none of the compounds used are viable solutions for the brewing industry due to toxic, carcinogenic, or off-flavour properties.
More recent research has also aimed to identify key nutrients that are necessary for
Brettanomyces growth [61, 154, 155, 156]; however, a majority of them do not focus specifically on the impact they have on fermentation or use wort or beer as the medium.
As a result, this remains an underexplored area of potential benefit to the industry.
The addition of thiamine or amino acids (aspartate, glutamate, or glutamine) was tested on three Brettanomyces strains in a fermentation setting. Positive results were
92 seen for all of the strains with the addition of thiamine in terms of fermentation rate, although the degree of supplementation that produced the best outcomes differed among the strains. BBY011 and PEST IV benefited the most from thiamine supplementation in the pre-growth only, while BSI Drei had the best results when thiamine was added to both the pre-growth and the fermentation. This corroborates previous research that also saw a positive impact with thiamine supplementation [61].
Thiamine is a cofactor for enzymes that are involved in carbohydrate metabolism, such as those that would be active during fermentation of sugars to ethanol and other by- products [125]. The precursor for thiamine is synthesized through an NAD+-dependent reaction [125], thereby suggesting that because of the redox imbalance experienced by
Brettanomyces upon transfer to an anaerobic environment thiamine synthesis is delayed and the carbohydrate metabolizing enzymes are unable to function properly causing a lag phase to occur. Based on the positive results observed here, it is likely that the addition of thiamine to the media is allowing the enzymes to function unhindered and shortening the lag phase. The amount of ethanol that was produced was only affected by the thiamine addition in BBY011, with the highest levels being produced during those fermentations that received no thiamine addition at any stage, followed by thiamine addition only in fermentation. This suggests that thiamine addition may hinder the production of ethanol by this strain. If the primary goal of fermentation with this strain is ethanol production, the benefit in fermentation rate may be rendered inconsequential by its effect on ethanol levels. Thiamine addition does not affect the flavour profiles of any of the strains to a large degree, with only a handful of significantly different concentrations of compounds observed. In fermentations where the primary
93 goal is the development of the Brettanomyces flavour profile within a shorter amount of time, thiamine addition may be useful.
Amino acid supplementation was explored as a way to decrease the characteristic lag phase experienced by Brettanomyces in fermentation. A previous study found positive results with the addition of various amino acids in both aerobic and anaerobic environments in terms of growth rate, with amino acids being divided into three groups based on their level of assimilation [75]. Based on the results of that study, aspartate, glutamate, and glutamine were selected for testing in a fermentation, as they had the greatest impact on Brettanomyces growth in an anaerobic environment. Amino acid supplementation proved beneficial for both BSI Drei and BBY011, with all of the amino acids having a positive effect to some degree. Of the three that were tested aspartate had the greatest effect in both strains, indicating a higher need for this amino acid in an anaerobic fermentation setting. This is in agreement with the previous study, where the growth rate for Brettanomyces was highest when the medium was supplemented with aspartate [75]. In contrast, PEST IV did not respond to aspartate and glutamate addition, and had only a moderately positive response to glutamine, with supplementation in pre-growth and fermentation producing the quickest fermentation rate and resulting in the second highest final CO2 evolution following the control. This suggests that PEST IV might have higher nutritional requirements in a fermentation than the beer isolates, thereby resulting in decreased fermentation efficiency. The amino acid supplementation did not affect ethanol production with very similar production observed among the treatment and control samples. Conversely, the supplementations had more of an impact on the production of flavour compounds, with significant differences
94 observed when comparing the treatment groups to the control. This is in accordance with previous research on Saccharomyces, where it was found that there were lower levels of esters in beers that had a higher nitrogen content compared to those with a low nitrogen content [144]. My results suggest that the same phenomenon may be occurring here. As a result, amino acid supplementation is a good avenue to explore if achieving ethanol production by Brettanomyces on a shorter time frame is the primary goal, with the knowledge that this may impact the flavour profile that is produced to some extent.
Overall, the major take-away from this experiment proves to be that no two strains are exactly identical in their nutrient requirements. It is impossible to fit all
Brettanomyces strains into a mold and create a standard nutrient cocktail that will equally benefit each individual strain. While a mix of certain nutrients may offer a benefit overall, customization of media and nutrients is necessary in order for each strain to perform at its maximum potential. It is important for brewers to know their strains prior to fermenting with them and to understand the nuances between the various
Brettanomyces strains they may be using. When beginning to use a new strain in fermentations it is unwise to assume that it has the same requirements as previously used strains or that it will perform in the same way that other strains have in the same medium.
This is not the end of the road
While the data found in this thesis has helped to answer some questions regarding Brettanomyces in a brewing environment, more research is still needed to fully understand this organism and address some shortcomings in the current research methods. The fermentations that were performed throughout my thesis were done on a
95 small scale, which may cause variation in the fermentation curve data due to evaporation over time at a warmer temperature. Controls were used in order to normalize the data; however, it is not certain if this is enough to account for possible variation and water loss that could occur at a small scale. As a result, weighing as a means to approximate fermentation vigor may not be optimal. We are moving away from this method and have started using larger volumes for fermentations and densitometer readings to determine the rate of sugar consumption over time in a fermentation. This allows for a more precise measurement of the fermentation rate and is not impacted by possible water loss throughout lengthy fermentations at warm temperatures.
The flavour profiles that were constructed in these experiments are comprised solely of volatile metabolites. Consequently, any flavour metabolites that cannot be volatilized have not been measured in the present research. Metabolites that are important to the characteristic flavour profile of Brettanomyces may have been overlooked as a result. Additionally, the current scope of flavour compound analysis does not include off-flavours. Further characterization of these metabolites, through broadening of the analysis performed through HS-SPME-GC-MS or through nuclear magnetic resonance (NMR) spectroscopy, will provide a better understanding of the complete flavour profile that is produced by this yeast.
Expanding upon this work and scaling up the fermentations to industry volumes is a natural next step. Fermenting in larger volumes that are typical of those used within the industry will help to determine whether the results obtained in small-scale lab fermentations are representative of those that are achieved in larger volumes.
96 Additionally, expanding upon the primary, secondary, and co-pitch fermentation experiment to include bacterial species is a potential follow-up experiment. Many of the traditional styles that utilized Brettanomyces, such as lambics, also included bacteria
[156, 157]. Understanding how these organisms interact with one another to affect the fermentation capacity and flavour development of the beers is an interesting potential area of research.
The genetic makeup of Brettanomyces has been the focus of a number of research papers [8, 9, 19, 158]; however, there are still areas that are underexplored.
As whole genome sequencing becomes more affordable we will gain better insights into the inner-workings of different strains of Brettanomyces, such as identifying currently unknown ester synthesizing enzymes and potential nutrient auxotrophies that exist. This will help immensely in tailoring media composition for individual strains so that they are performing optimally during growth and fermentation. Discovering the enzymes that are responsible for ester synthesis in this yeast will not only provide a better understanding of how this process is carried out but will open up opportunities to exploit the synthesis of these compounds as a means of producing fruitier beers.
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