Changes

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 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.

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). 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 reported in the literature and the time to reach maximum cell density is much shorter than previous reports for propagation of ''Brettanomyces''.

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 ''BrettBrettanomyces'' 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 ''BrettBrettanomyces'' cells to adapt their metabolism to malt sugars. Preiss reports seeing something similar when growing ''BrettBrettanomyces'': 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 ''BrettBrettanomyces'' exhibit the "second lag phase", and that the chosen ''BrettBrettanomyces'' 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>.