Brettanomyces Propagation Experiment
This page documents an experiment performed by Mark Trent to compare different aeration conditions for Brettanomyces during propagation. It was originally posted by Trent on the Milk The Funk Facebook Group.
Purpose
For brewers there have generally been two approaches to making starters to propagate Brett. 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 Brett Starter Information page for details.
It is thought that oxygen will encourage growth for Brettanomyces. To test this hypothesis, Mark Trent executed the following controlled experiment. This experiment aims to determine how aeration and agitation affects Brett' cell growth and pH of the starter wort.
Procedures
A strain of Brett believed to be B. bruxellensis was isolated by Trent from a bottle of Orval in 2012. This Brett 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 Brett was grown in 3 steps to 300 mL slurry from a single colony. For each treatment 35 mL of the inoculum Brett 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. 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 duplicated. The Brett 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. Figures 4-5 show the results of this experiment.
Results
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Discussion
(In progress)
The results indicate that for this strain of Brett 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 maximum cell density is much shorter than previous reports for propagation of Brettanomyces.