Differential contributions of Saccharomyces cerevisiae and Brettanomyces claussenii to a Belgian strong beer

Parallel 49 Brewing Company has become an award-winning microbrewery at the heart of British Columbia’s growing beer culture. Wild Ride, one of their most successful beers, is brewed with a co-culture of Saccharomyces cerevisiae and Brettanomyces claussenii (synonym B. anomalus). While the use of co-cultures in brewing represents a profitable niche market, Parallel 49 has ceased production of Wild Ride as these fermentations are technically challenging and difficult to reproduce. In order to support the development of alternative production methods, Parallel 49 needs to gain an understanding of the genomic profiles of the two yeast strains, profile yeast metabolites in relation to gene expression, and understand the genetic and metabolic interactions during co-culture fermentation. I hypothesize that, during co-culture fermentation, the “omic” profiles of the two yeast strains will be altered, and that there will be detectable interactions between the two strains. In order to test this hypothesis, a S. cerevisiae mono-culture brew, a B. claussenii mono-culture brew, and a Wild Ride co-culture brew were carried out. Fermentations proceeded for twenty-two days according to Parallel 49’s recipe, and specific gravity, dissolved oxygen and pH were monitored. Daily samples were taken for metabolite analysis via heated headspace gas chromatography coupled to a flame ionization detector and a mass spectrometer, transcriptomic analysis via RNA-seq on an Ion S5 System, and proteomic analysis via Waters Synapt G2 high definition mass spectrometry (Q-TOF MS) coupled to a nanoAcquity ultra performance liquid chromatography system. The genomes of both yeast cultures were sequenced on an Ion S5 System. To date, the day 7 transcriptomic profile of the Wild Ride co-culture has been sequenced, producing 9,734,188 Q20 reads and >= 1,620,889,603 Q20 bases. The Wild Ride reads were mapped to the Saccharomyces cerevisiae S288C reference genome at 17.65%, with 76.93% of the reads mapping to rRNA regions located on chromosome XII. Through metabolic analysis, 33 compounds were resolved and identified by mass spectrometry in the three fermentations; of those, production kinetics for 10 were monitored using flame ionization detection. From whole genome sequencing. 6,281,650 Q20 reads and >= 1,887,285,253 Q20 bases were produced for Saccharomyces, while 5,804,220 Q20 reads and >= 1,655,746,215 Q20 bases were produced for Brettanomyces. From this, a contaminant was detected in the two yeast cultures that shared 99.98% average nucleotide identity with the bacterium Cellulosimicrobium cellulans strain NEB113; the average nucleotide identity between NEB113 and the two contaminants sequenced in the two yeast cultures was >99.9%. Currently, proteomic analysis of samples is underway at the University of iii Regina. In the future, additional metabolites will be identified in the liquid phase of fermentation cultures, and quantities of each compound in all three brews will be determined. Additional transcriptome sequencing will be carried out so that multiple sample days from all three brews can be analyzed, and yeast abundance in the Wild Ride co-culture will be monitored by qPCR. Thegenomes of S. cerevisiae and B. claussenii will be sequenced at a greater depth of coverage so that they may be used as reference genomes for transcriptome mapping. The possibility of C. cellulans contamination will be eliminated by re sequencing the genomes using new cultures, and differentrRNA removal techniques will be implemented. The mono- and co-culture fermentations will be repeated so that all aspects of the experiment can be replicated.