Leo Chan, Nexcelom Bioscience, Lawrence, MA, U.S.A.
Coauthor(s): Jason Bolton,
University of Maine,
University of Maine,
Yeast, Fermentation, and Microbiology
In recent years, there has been increasing interest in utilizing Brettanomyces spp. in the brewing industry due to their novel flavor and aroma compounds, which are used to create complex flavors for specialty beer products. Currently, several breweries have been performing 100% Brettanomyces fermentation for their beverage products, such as Midnight Brett from Allagash Brewing Company. One of the challenges when working with Brettanomyces spp. is the formation of pseudohyphae, which can increase the difficulty of the traditional yeast enumeration method during the fermentation process. The current cell counting method involves manual counting of methylene blue-stained yeasts in a hemocytometer using light microscopy. However, the method can be time-consuming and has high operator-dependent variations. More importantly, subjectivity of what should be counted in the pseudohyphae makes enumeration of Brettanomyces cells extremely difficult. Therefore, it is important to develop a rapid, robust, and nonsubjective method for the quantification of Brettanomyces spp. Numerous breweries have employed the use of automated fluorescence-based image cytometers with acridine orange (AO) and propidium iodide (PI) fluorescent stains to overcome issues from manual Saccharomyce counting. In this work, we demonstrate a novel cell concentration and viability detection method for Brettanomyces using an image cytometer (Nexcelom Bioscience, Lawrence, MA). First, the automated cell counting method was developed by measuring the yeast propagation of three yeast strains: Brettanomyces bruxellensis, B. clausenii, and B. lambicus, where the counting results were validated against the manual counting method. Finally, two fermentation batches of B. clausenii and B. lambicus were monitored for 42 days, where cell concentration, viability, and budding/pseudohyphae percentages were measured throughout the fermentation. In the propagation experiment, B. clausenii took the longest to start growing, but reached the highest cell concentration on day five at a concentration of 7.87 x 109 cells/mL. B. lambicus showed the quickest propagation, requiring only 24 hours to reach its maximum cell concentration of 4.21 x 109 cells/mL. Finally, B. bruxellensis reached 5.78 x 109 cells/mL after two days, but took an additional two days to reach peak cell concentration of 6.74 x 109 cells/mL. Brettanomyces viabilities were measured simultaneously. In the fermentation experiment, the two 100% Brettanomyces fermentations, B. clausenii and B. lambicus, exhibited two very different growth curves. The results showed that B. clausenii exhibited an extended lag phase of approximately 12 days before increasing substantially to ~1.7 x 108 cell/mL from day 12 to 15 during the log phase. In contrast, B. lambicus exhibited an extended log phase with a steady increase in cell concentration from day 3 to 14 with substantial fluctuation. The proposed novel image cytometric analysis method can provide a simple and nonsubjective automated counting method for Brettanomyces, which can replace the traditional counting methods to improve efficiency and consistency during the fermentation process.
Leo Chan currently serves as the technology R&D manager at Nexcelom Bioscience LLC, in Lawrence, MA. His research involves the development of instruments and applications for the Cellometer image cytometry system for detection and analysis of yeasts used in the brewing and biofuel industries. He is a member of the Master Brewers Association of the Americas and American Society for Brewing Chemists. He received his B.S., M.S., and Ph.D. degrees in electrical and computer engineering from the University of Illinois at Urbana-Champaign (2000–2008).