This study has shown that Brettanomyces yeasts are facultatively anaerobic and capable of pure culture fermentation. The importance of media in culturing and identifying these yeasts can’t be overlooked in the brewery setting due to the possibility of cross contamination. The media recommended for use and found to be the most beneficial to this study were MYPG, WLN, and CuSO4. Additionally it was found that storing cultures at room temperature in a nutrient rich liquid substrate maintained the integrity of cells longer and can be used for repeated propagations. This yeast genus is generally slower growing then normal brewers yeast and propagation methods must be tailored to support proper growth for use in pure culture fermentations. Adequate growth of cells required 7-8 days to reach stationary phase in all of the strains used in this study with mid exponential phase observed between days 3-5 in most of the strains. The use of glucose as the sole carbon source during propagation appears to be more beneficial to cell growth during batch culture but any advantages during fermentation are unknown.
Pure culture fermentations were conducted and it was found that some of the Brettanomyces strains were capable of completing fermentation within 35 days. Esters appear to be the major by-products produced during fermentation along with acids not identified during this study. The esters most crucial to the aroma and flavor of the fermentations were ethyl acetate, ethyl caproate and ethyl caprylate while isoamyl acetate, isobutyl acetate and ethyl butyrate do not appear in any significant quantities. 4-vinylguaiacol was produced in all of the fermentations and it’s probable that the ethyl phenol derivatives make an important contribution to the flavors associated with Brettanomyces yeast. Higher alcohols were not produced in significant quantity possible due to low growth during anaerobic conditions. The results attained from multiple pitching rates showed high variability between strains and more research is needed to make any significant conclusion to the effects of pitching rate on fermentation performance. The acidification of wort using lactic acid appears to have a beneficial effect on most Brettanomyces strains used in this study especially with regards to apparent attenuation, and sugar utilization. Whether lactic acid or a low pH is responsible for a change in metabolic activity is not known but the results attained show that there is a draw back as important aromatic compounds saw a decrease as the concentration of lactic acid was increased.