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→Source of Microbes
Professor Chris Curtin from Oregon State University presented the results of a study by his team where 50 barrels of three different vintages of spontaneously fermented, lambic-inspired beer was sampled three times over a 9 month period. These 50 barrels represented three batches of the same recipe brewed in 2013, 2015, and 2016. With the exception of a small number of barrels, they all contained the same or similar ''Brettanomyces bruxellensis'' yeast. There was no significance between the three different batches. Two of the 2015 barrels had a different species of ''Brettanomyces'' (''claussenii''). Two of the 2016 barrels had more ''Saccharomyces cerevisiae''. This study indicated that barrel to barrel variation as far as yeast goes is fairly small at this brewery. Bacteria populations clustered much closer based on the vintage (but not the barrel). The 2013 batch was dominated by ''Gluconobacter'' and ''Acetobacter'', while the 2015 batch was dominated only by ''Acetobacter'', and the 2016 batch was dominated by ''Lactobacillus''. This indicated that the vintage of the batch plays a major role in determining which bacteria will be the dominant bacteria during maturation, but individual barrels generally do not (although a small number of barrels matured faster than the majority of the barrels and were dominated by bacteria that represented the more mature vintage). Despite the lack of major variation between barrels, Curtin determined that some barrels can introduce microbial variation, perhaps due to insects transferring microbes or differences in the oxygen ingress between different barrels, and the ability of yeast and bacteria to live within the inner surface of the barrels and potentially survive cleaning procedures <ref name="curtain_asbc_2018" /> (~29 minutes in)<ref name="Curtin_2021" />.
Various brewer anecdotes from experiments appear to contradict the published scientific literature. James Howat of Black Project Spontaneous Ales reported conducting an experiment that showed a similar fermentation profile between a barrel fermented spontaneous beer and samples taken from the coolship and aged in glass flasks <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1977772602250944/?comment_id=1977929225568615&reply_comment_id=1978520655509472&comment_tracking=%7B%22tn%22%3A%22R%22%7D James Howat. Milk The Funk thread on the source of ''Brettanomyces'' in lambic. 02/05/2018.]</ref>. The spontaneous beers at Black Project were produced for a few years by placing a kettle on the roof outside of the brewery to collect microbes from the air and then racked directly to barrels, so there was no influence of microbes living within the brewery (Black Project now uses a coolship inside the brewery). This led James to conduct this experiment to see how much the barrels were influencing the microbiome of the beers even after they were steamed to the [[Barrel#Sanitizing|point of possible pasteurization]]. James reported that the sensory characteristics between the barrel aged beers and the flask-aged beers were very similar, other than the obvious differences that the oak would have provided, which led him to believe that air inoculation provides a significant contribution to the microbial load of spontaneously fermented beers. Additionally, Brasserie-Brouwerij Cantillon (ref needed) and Oud Beersel <ref>[https://soundcloud.com/craftbeerbrew/podcast-episode-21-new-belgiums-wood-cellar-director-blender-lauren-limbach Lauren Limbach. Craft Beer and Brewing Magazine Podcast. Episode 21. 02/16/2018.]</ref> (~42 minutes in) are known to steam clean their barrels which might be enough to [[Barrel#Sanitizing|sanitize them]], although former Cantillon brewer and saison expert Yvan De Beats maintains that Cantillon barrels are not heated enough to be pasteurized <ref>[https://www.crowdcast.io/e/saison-ale-myths-yvan-baets/1 De Beats, Ybsn. Doug pipers Crowdcast. 08/26/2021.]</ref>. Pierre Tilquin reported that different worts brewed and cooled by different lambic brewers present different fermentation and flavor profiles when barrel fermented in his blendery, particularly when he began steam cleaning emptied barrels <ref>[https://beerandbrewing.com/podcast-episode-234-pierre-tilquin-of-gueuzerie-tilquin-makes-lambic-and/ Pierre Tilquin. Interview on Craft Beer & Brewing Podcast. Episode 234. 04/15/2022.]</ref>(~15 and ~27 mins in). Mitch Ermatinger of Speciation Artisan Ales reported moving wort cooled over night overnight in a coolship to a stainless fermenter, and the wort began showing signs of visual fermentation four days later <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/3012223975472463/?comment_id=3012239425470918&reply_comment_id=3012428892118638 Mitch Ermatinger. Milk The Funk Facebook thread on sources for microbes in spontaneous fermentation. 10/28/2019.]</ref>. Such anecdotes deserve further investigation using the full scientific process.
Other sources for the microbes include the ceilings of the breweries which are often not cleaned in Belgian lambic breweries, and the coolship itself. Some breweries have opted to craft wooden overhangs to simulate having a relatively low ceiling above the coolship (see [[Coolship#Overhangs_and_Other_Innovations|Coolship]]). As the wort cools overnight, vapor rises and condenses on the wooden ceiling or overhang above, and then drips back down into the coolship carrying any microbes that might be living on the surface of the wood. The air also carries some amount of microbes, although it is rare to find wild ''Brettanomyces'' in the air (see [[Brettanomyces#Environment_and_Survival|''Brettanomyces'' environment]]). The sources for various microbes are unknown and potentially many, and probably vary from producer to producer. Additionally, no single source is likely to be required to have a successful spontaneous fermentation.