Changes

A strain of ''Brettanomyces'' believed to be the species ''B. bruxellensis '' was isolated by Trent from a bottle of Orval in 2012. This ''Brettanomyces'' strain was subjected to four different propagation treatments: aerobic (stir plate set to high with a foil cover), semi-aerobic (orbital shaker set to 80 RPM's with a foil cover), anaerobic with constant agitation (Co2 purged and then set on an orbital shaker set to 80 RPM's with an airlock), and still (foil cover, no agitation). All treatments were duplicated.

This experiment supports prior starter recommendations (see [[Brettanomyces#Two_Approaches_to_Starters|Brettanomyces Starters]]) and indicates that oxygen exposure and agitation increase the cell growth rate of ''Brettanomyces'', and that the amount of oxygen required for maximum growth is minimal, although more oxygen decreases the time to achieve maximum cell density. Also, higher levels of oxygen can possibly lead to more acetic acid production, although this was only measured sensorily and not with any kind of chemical analysis, and acetic acid was never identified as the cause of the acidic taste of the aerated treatment. This supports the results of several published studies that show that level of oxygen has a significant impact on level of acetic acid produced (see references: <ref>[http://www.ncbi.nlm.nih.gov/pubmed/12655458 Brettanomyces bruxellensis: effect of oxygen on growth and acetic acid production. Aguilar Uscanga, Délia1, and Strehaiano. 2003.]</ref><ref>[http://onlinelibrary.wiley.com/doi/10.1002/(SICI)1097-0010(199712)75:4%3C489::AID-JSFA902%3E3.0.CO;2-9/abstract Role of oxygen on acetic acid production by Brettanomyces/Dekkera in winemaking. Maurizio Ciani and Luisa Ferraro. April 1999.]</ref><ref>[http://link.springer.com/article/10.1023%2FA%3A1014927129259 Acetic acid production by Dekkera/Brettanomyces yeasts. S.N. Feer. April 2002.]</ref>). Agitation also appears to induce ''BrettBrettanomyces'' growth as shown in Figure 1; the anaerobic treatment with agitation (80 RPM) showed slightly better growth than the treatment with a tin foil cover and no agitation (labelled "Still" in Figure 1).

Based on these findings and others, it is recommended that ''BrettBrettanomyces'' is grown either on a stir plate with tin foil and decanted before pitching, or grown in a semi-aerobic condition. Semi-aerobic conditions can be met ideally by using an orbital shaker at 80 RPM. Those without an orbital shaker can use a stir plate set to the lowest setting, or an initial blast of pure oxygen followed by frequent manual agitation throughout the incubation time period.