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Oxygen levels are an important factor to consider when deciding which of the above two methods to use for a ''Brettanomyces'' starter. ''Brettanomyces'' creates acetic acid in the presence of oxygen, potentially leading to higher levels of ethyl acetate, which is considered an off flavor in higher amounts. As the amount of oxygen increases, cell growth increases, but so does acetic acid production. The amount of acetic acid produced is species/strain dependent, so some strains may benefit from more aeration without having the negative effect of creating too much acetic acid. Other strains may need a less aerobic starter (semi-aerobic) in order to produce the highest cell count with minimal acetic acid <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>. In addition to acetic acid production, it has been observed that some ''Brettanomyces'' strains grown under aerobic conditions continue to produce THP when transferred to anaerobic conditions. See [[Tetrahydropyridine#Brettanomyces|THP]] for details.
This presents a sort of "catch 22" when growing ''Brettanomyces'' in a starter. The brewer must weigh the pros and cons of how much aeration to provide. If the ''Brettanomyces'' is going to be used in a [[Brettanomyces_Fermentation|100% Brettanomyces Fermentation]], for example, then a stir plate with foil covering the flask is the best choice. If the ''Brettanomyces'' is instead being pitched in secondary with the intention of long aging, then having a high cell count isn't as necessary and the risk of adding more acetic acid/ethyl acetate to an aging beer is greater. If a lot of acetic acid is produced during the starter, then they can opt to cold crash and decant the starter. ''Brettanomyces'' can have a difficult time flocculating and settling out, even when cold crashed. The brewer may need to allow a few days for the cells to fully sediment <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1099473923414154/?comment_id=1099522943409252&offset=0&total_comments=25&comment_tracking=%7B%22tn%22%3A%22R%22%7D Conversation with Richard Preiss of Escarpment Yeast Labs on MTF. 6/26/2015.]</ref>. Additionally, ''Brettanomyces'' that is cold crashed may be slower to begin fermentation. If the brewer believes that the amount of acetic acid produced was insignificant, then cold crashing can be skipped and the entire starter can be pitched. Even if the starter has a lot of acetic acid, the amount of acetic acid in the volume of a starter is fairly insignificant once diluted into a full batch of wort or beer. If the starter is not going to be used within a month, then an aerobic starter is not the best option since the presence of a lot of acetic acid will slowly kill the ''Brettanomyces'' over time. In this case, the starter should be lightly shaken (or occasionally manually stirred), and an airlock put in place on the flask in order to keep out most of the oxygen.
Although more experiments are probably needed, agitation is believed to be an important factor for any species of microbe (yeast and bacteria). Gentle stirring on a stir plate or orbital shaker, or frequent gentle manual agitation leads to faster growth and a higher number of organisms. Agitation keeps the microbes in solution. It also maximizes the microbes' access to nutrients and disperses waste evenly. In a non-agitated starter, the microbes are limited to the diffusion rate of nutrients, leading to a slower and more stressful growth <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1168024059892473/?comment_id=1174865305875015&reply_comment_id=1176092372418975&total_comments=1&comment_tracking=%7B%22tn%22%3A%22R9%22%7D Conversation with Bryan of Sui Generis Blog about starters and agitation. 11/09/2015.]</ref>.
====Pitching Rate Calculators====
Current yeast pitching calculators for brewers are not adequate for determining ''Brettanomyces'' pitching rates based on starter volume size because the maximum cell density of ''Brettanomyces'' per mL of wort is 3 to 6 times the cell density of ''Saccharomyces''. For example, a given ''Saccharomyces'' strain may reach a cell density of 130 million cells per mL in a 1.040 wort (different ''Saccharomyces'' strains can have different cell densities as well, although they are a lot lower than ''Brettanomyces'' overall). Different ''Brettanomyces'' strain cell densities have been reported to be 600 to 885 million cells per mL in 1.040 wort depending on the species/strain <ref name="Yakobson_Propagation">[http://www.brettanomycesproject.com/dissertation/propagation-and-batch-culture-growth/propagation-results/ Yakobson, Chad. The Brettanomyces Project. Propagation and Batch Culture Results. Retrieved 2/17/2015]</ref><ref name="MarkTrent">[https://www.facebook.com/groups/MilkTheFunk/permalink/1114254011936145/ Conversation with Mark Trent and Lance Shaner on MTF regarding Brett pitching rates. 07-21-2015.]</ref>. Since yeast calculators are based on ''S. cerevisiae'' or ''S. pastorianus'' cell density, using one of these tools for ''Brettanomyces'' starters will create an unexpectedly high cell count in reality. There is not currently enough data to accurately determine starter volumes for ''Brettanomyces'', particularly because each strain and species have a different maximum cell density per mL of wort. However, pitching around 500-600 mL of a ''Brettanomyces'' starter for 5 gallons of 1.060 SG wort will achieve a pitching rate that is similar to lager yeast pitching rates, which has been recommended for [[Brettanomyces_Fermentation|100% Brettanomyces Fermentation]]. [[Omega Yeast Labs]] is currently working on a project to create a more accurate ''Brettanomyces'' pitching rate calculator (it will also contain pitching rate calculations for specific strains of ''Saccharomyces'', which is something that current yeast pitching calculators do not include) <ref name="MarkTrent"></ref>.
Given this information, many brewers historically have been using the lager pitching rate settings in online yeast pitching calculators for ''Brettanomyces'' starters (around 2000 mL for 5 gallons, for example). Effectively, this means they have been pitching around 4 to 5 times the amount of ''Brettanomyces'' cells that they thought they were pitching. However, if this very high pitching rate is giving good results for brewers, it should continue to be used. Exploration of ''Brettanomyces'' pitching rates for 100% Brett fermentations is something to be desired once we know what our pitching rates actually are, and many brewers have been pitching 4-5 times the pitching rate for lagers if they use an online yeast pitching rate calculator instead of counting the cells under a [[Microscope|microscope]].
* [[Brettanomyces secondary fermentation experiment]]
* [[Brettanomyces Storage Survival Experiment]]
* [[Brettanomyces_Fermentation|100% Brettanomyces Fermentation]]
* [[Crooked Stave Artisan Beer Project]]
* [[Scientific Publications]]