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This page documents an experiment performed conducted by Mark Trent to compare different aeration conditions for [[Brettanomyces]] during propagation. It was originally posted by [https://www.facebook.com/groups/MilkTheFunk/permalink/1168024059892473/ Trent on the Milk The Funk Facebook Group]. Significant and relevant follow up comments from other MTF members have been included in this write up.

For brewers there have generally been two approaches to making starters to propagate ''BrettBrettanomyces''. Basically, the first approach is to create a semi-aerobic environment. The second approach is to use a stir plate. Using a stir plate will introduce more oxygen, which can create a a more acetic starter. Usually the starter beer is decanted so that the acetic acid is not added to the beer. See the [[Brettanomyces#Starter_Information|Brett Starter Information]] page for details.

A strain of ''BrettBrettanomyces'' believed to be the species ''B. bruxellensis '' was isolated by Trent from a bottle of Orval in 2012. This ''BrettBrettanomyces'' 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.

The initial inoculum of ''BrettBrettanomyces'' was grown in 3 steps to 300 mL slurry from a single colony. For each treatment 35 mL of the inoculum ''BrettBrettanomyces'' was added to 230 mL of 10°P wort in 500 mL Erlenmeyer flasks for an initial cell density of 112.87 million cells per mL.

Cell counts and pH readings were taken every 24 hours until all treatments showed no or little increase in cell count. Cell counts were done with a hemocytometer. Final gravity for each treatment was recorded at the end of the experiment. The results of this experiment are shown below in figures 1-3.

Because the cell counts were so high and the time to completion was so short, it was thought that these results may be due to the high level of initial cell count. Therefore, another experiment was performed comparing an initial cell count of 30 million cells per mL (low) and 120 million cells per mL (high). This experiment was also The treatments were duplicated. The ''BrettBrettanomyces'' inoculum was prepared as described above and each treatment was prepared in 265 mL of wort and incubated on an orbital shaker at 80 RPM and 26°C. Because "life got in the way", cell counts and pH readings were only taken for the first 2 days, and then again on the 6th day. Both treatments in this second experiment resulted in a final gravity of 2°P on day 6. Figures 4-5 show the results of this experiment.

The results indicate that for this strain of ''BrettBrettanomyces'' a near equal cell density can be achieved with aerated or semi-aerated propagation while anaerobic incubation under agitation or still incubation resulted in less than half the cell density when compared to the former treatments. While the pH decreased with more aeration during propagation, it is interesting that the pH of all treatments were near equal by the end of the experiment. The cell counts reached by the aerobic and semi-aerobic treatments were much higher than I have seen reported in the literature and the time to reach maximum cell density is much shorter than previous reports for propagation of ''Brettanomyces''. In Figure 2, the pH of the starter wort changed throughout the incubation period, with the aerobic and semi-aerobic treatments creating a lower pH from 24 hours to 72 hours. However, after 90 hours the pH for all treatments stabilized around the same pH (~3.4). Trent noted that although the pH of the final starters were nearly the same, the aerated condition tasted more acidic than the other three conditions <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1168024059892473/?comment_id=1168027756558770&reply_comment_id=1168031146558431&total_comments=8&comment_tracking=%7B%22tn%22%3A%22R9%22%7D Conversation with Mark Trent regarding his Brett experiment and sensory analysis. 10/23/2015.]</ref>.  This experiment demonstrated a significantly faster growth rate than many other growth rate studies <ref name="Yakobson">[http://www.brettanomycesproject.com/dissertation/propagation-and-batch-culture-growth/propagation-results/ The Brettanomyces Project. Propagation and Batch Culture Results. Retrieved 11/05/2015.] </ref>. It has been suggested by MTF member and microbiologist [http://www.escarpmentlabs.com/ Richard Preiss] that propagation times vary widely among ''Brettanomyces'' strains, and that the selected strain most likely exhibits faster reproduction than other strains <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1168024059892473/?comment_id=1168034189891460&offset=0&total_comments=20&comment_tracking=%7B%22tn%22%3A%22R4%22%7D Comments by Richard Preiss on MTF regarding Mark Trent's Brett growth experiment. 10/23/2015.]</ref>. Additionally, Trent's experiment did not demonstrate the same "second lag phase" as shown in Chad Yakobson's "The Brettanomyces Project" <ref name="Yakobson"></ref>, including for the lower cell count treatment in the second experiment. Richard Preiss hypothesizes that one possibility to explain this is that the cells had a two step growth process applied in preparation for the experiment, and that this prior exposure to wort allows the ''Brettanomyces'' cells to adapt their metabolism to malt sugars. Preiss reports seeing something similar when growing ''Brettanomyces'': when first grown in [[YEPD|YPD]], then in ~50 mL of wort, the culture then grows to maximum cell density within 24-72 hours (depending on strain). Preiss also notes that not all strains of ''Brettanomyces'' exhibit the "second lag phase", and that the chosen ''Brettanomyces'' strain may be one of these strains <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1168024059892473/?comment_id=1168034189891460&reply_comment_id=1169578973070315&total_comments=2&comment_tracking=%7B%22tn%22%3A%22R9%22%7D Comments by Richard Preiss on MTF regarding Mark Trent's Brett growth experiment. 10/23/2015.]</ref>.  Oxygen levels between the four treatments were not measured with a Dissolved Oxygen meter. However, some observations were made by Trent that indicate a large difference in dissolved oxygen between the four treatments. The aerated (stir plate) treatment formed a significant vortex and the color (before growth) appeared milky due to the amount of agitation and perhaps gas dissolving into the media. Also a thick layer of foam was maintained on the aerated treatment throughout growth while only bubbles were observed on the semi-aerated <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1168024059892473/?comment_id=1168034189891460&reply_comment_id=1169576006403945&total_comments=2&comment_tracking=%7B%22tn%22%3A%22R9%22%7D Comments by Mark Trent on MTF. 10/26/2015.]</ref>. ==Conclusion==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 ''Brettanomyces'' 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 ''Brettanomyces'' 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.  It is recommended that this experiment is repeated with other strains of ''Brettanomyces''. [http://www.omegayeast.com/ Lance Shaner of Omega Yeast Labs] and [http://www.escarpmentlabs.com/ Richard Preiss of Escarpment] Yeast Labs volunteered to independently grow the strain of ''Brettanomyces'' used in the experiment to verify the maximum cell density of this strain (and thus the data collected - results pending).