In order to obtain yeast cells in large enough quantities, which were physically ready to begin fermentation, a propagation method slightly unique to Brettanomyces spp. was developed. Batch culture conditions were chosen based on the findings by Aguilar Uscanga et al. (2003) which showed maximum cell growth was attained with a semi aerobic aeration rate of 60 l/hr (0.1 vvm). Based on the general time it took colonies to form distinct morphologies on media agar a method was devised to look at the length of propagation necessary for cells to reach early stationary phase. Assuming that Brettanomyces spp. physiology was similar to Saccharomyces cerevisiae, early stationary phase growth was determined the appropriate time to harvest and pitch cells for fermentation. From the mean growth curve produced during batch culture propagations, it was observed that 192 hours (8 days) was needed for cells to reach the latter part of the early stationary phase. This is similar to the findings of Aguilar Uscanga et al. (2000) who found the growth of B. bruxellensis to take 90 to 180 hours in batch culture.
During the study, 2-phase and 3-phase growth patterns were observed indicating diversity in the metabolic and physiological behavior of the strains. In 1984, Wijsman et al. showed resting cells of a Brettanomyces sp. grown aerobically had different phases of metabolic activity in which glucose was first dissimilated into ethanol, acetic acid, and equivalent amounts of CO2. A second phase was observed where ethanol was converted to acetic acid, following a long lag phase in which the acetic acid produced in the first and second phases was dissimilated to CO2. From these observations, Wijsman et al. (1984) concluded that the TCA-cycle did not play an important role during the first two phases of growth, and the Custers effect was not restricted to stationary cells, which resulted from the excess formation of NADH from NAD+ due to acetic acid formation.
Similar diauxic growth has also been reported by Barbin et al. (2008) when they categorized four types of growth with 23 strains of B. bruxellensis. Diauxic growth has been described by Gilis (1999) for glucose + sucrose and by Blondin (1982) when glucose + cellobiose were used as the carbon sources, with glucose being consumed first (Barbin, 2008). Sequential consumption of sugars was not monitored during this study only the final concentration of sugars, which found all glucose, fructose and sucrose to be completely consumed during semi-aerobic batch culture propagations. It is possible that a growth phase associated with each fermentable sugar exists, with variations in growth curves being due to the time needed for each strain to re-adapt its metabolism before resuming growth.
While it’s also possible sugars are competing over the same transporter, Silva et al. (2004) demonstrated the co-existence of two glucose transport systems, one of which was for the facilitated diffusion of both glucose and fructose while the other was an H+ dependent transport for glucose and galactose that was subject to glucose repression. Given this information it seems possible that the second or third growth phases were observed due to enzymatic adaptation necessary for the consumption of maltose and maltotriose. While four of the strains consumed minor amounts of maltose and maltotriose during semi-aerobic batch culture propagation, a greater degree of fermentation occurred with B. lambicus (WLP653) as only limited amounts of maltose and maltotriose were left in solution. This is similar to the finding of Scheffers (1961, 1966, 1979) in which anaerobic fermentation and growth was stimulated by minute amounts of oxygen (0.1 – 0.5% O2). While all batch cultures were conducted simultaneously under identical conditions it’s possible the metabolism of B. lambicus (WLP653) adapted consuming fermentable sugars more rapidly then the other strains. Ciani and Ferraro (1997) found semi aerobiosis to be the best condition for alcoholic fermentation associated with lower levels of acetic acid production when compared to full aerobiosis.
Brettanomyces strains initially cultured in MYPG substrate before final propagation in wort substrate had two fold higher cell counts after seven days of batch culture when compared to the same strains cultured in wort substrate only. Cells initially cultured in MYPG substrate appear to be better adapted for growth in wort due to the lack of defined growth phases observed in cells grown in wort. It’s not understood why an initial culture substrate consisting of glucose as the sole carbon source would produce cells more adapt for growth in a substrate containing complex sugars and nutrients, while cells initially cultured in the wort substrate would not observe such strong growth.
The propagation method employed in this study was successful in culturing cells with 96+% viability and of adequate quantities comparable to cell counts seen in Saccharomyces cerevisiae after batch culture propagations. Worth noting was the observation that stationary cells maintained high viabilities even eight days after reaching maximum growth (data not shown). This could be due to the slow consumption of sugars by stationary cells observed in previous studies as Brandam et al. (2008) reported stationary cells were capable of utilizing sugars to form ethanol and acetic acid. This might explain the ability of Brettanomyces spp. to survive in minimal nutrient conditions for extended periods.
Summarizing Custers 1940’s thesis, Skinner (1947) stated that when grown aerobically B. bruxellensis and B. claussenii only formed ethanol, CO2, and acetic acid. These findings concur with Wijsman et al. (1984) who found acetic acid to be the only acid produced along with ethanol and CO2. In this study ethanol, acetic acid, and CO2 production during semi-aerobic batch cultures were not analyzed, instead secondary volatile compounds were measured to observe the degree of fermentation type compounds produced. Volatile compound analysis carried out on batch culture propagations found only trace amounts of secondary compounds formed. There were a few compounds present in high enough concentrations to be considered a major by-product during semi aerobic batch cultures, but it was not consistent for all strains. Even in analysis from B. lambicus (WLP653), which under went a considerable degree of fermentation, not a single volatile compound was observed near threshold levels. The only compounds found above threshold value were ethyl acetate present in two strains and diacetyl in one other. It could be assumed considerable amounts of acetic acid were produced by B. bruxellensis (CMY001 & BSI-Drie) given the levels of ethyl acetate produced. These finding are in agreement with Brandam et al. (2008) who reported no secondary products were produced in any significant quantity by B. bruxellensis. The lack of esters and other volatile compounds during aerobiosis has been explained by Boulton and Quain (2008). They point out that oxygen has a molecular signaling function in yeast. The transcription of genes responsible for ester synthase is subject to repression by oxygen, therefore in semi-aerobic batch cultures the enzymes responsible for volatile compound production would be greatly repressed. Further, it could be assumed the lack of an active TCA-cycle shown by Wijsman et al. (1984) would cause less compound metabolites to be present. It should be noted that organic acids, the precursors for some of the volatile compounds might also be absent due to little or no amino acid metabolism during aerobic and semi-aerobic batch cultures.