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Although spontaneous ales have a common pattern of fermentation by groups of genera of microbes, the diversity in specific species is large across different lambic producers and American spontaneous ale producers (although data for American spontaneous ale producers is limited). In American spontaneous ale producers, ''Klebsiella'' spp., ''Enterobacter'' spp.,'' Pectobacterium carotovorum'', and ''Serratia ureilytica'' have been found. In Belgian lambic producers, ''Enterobacter'' spp., such as ''Enterobacter aerogenes'', ''Enterobacter cloacae'', ''Enterobacter hormaechei'' and ''Enterobacter kobei'', ''Klebsiella aerogenes'', ''Klebsiella oxytoca'', ''Klebsiella varicola'', ''Escherichia coli'', ''Hafnia alvei'', ''Hafnia paralvei'', and ''Citrobacter freundii'', have been found in lambic, with ''E. cloacae'' and ''K. aerogenes'' as the most frequently found ones. Although these enterobacteria contribute little in terms of gravity drop over the first month of fermentation (they mostly consume sucrose in the wort), they do contribute aroma and flavor compounds and precursors during the initial stages of spontaneous fermentation, particularly acetoin, 2,3 butanediol, acetic acid, lactic acid, succinic acid, DMS, acetaldehyde, long-chain fatty acids (these play a role in both flavor impact and providing nutrients for yeast later in the fermentation process), and small amounts of glycerol, ethyl acetate, and higher alcohols which might form esters in the later stages of fermentation. Enterobacteria can also contribute to the production of [https://en.wikipedia.org/wiki/Biogenic_amine biogenic amines] in fermented foods and beverages, including spontaneously fermented beers. Enterobacteria usually disappear after 30-40 days of fermentation due to the increase in ethanol, decrease in pH, and a decrease in food availability <ref name="Martens et al., 1992" /><ref name="Roos_2018">[https://www.ncbi.nlm.nih.gov/pubmed/30246252?dopt=Abstract Microbial acidification, alcoholization, and aroma production during spontaneous lambic beer production. Jonas De Roos and Luc De Vuyst. 2018. DOI: 10.1002/jsfa.9291.]</ref>, although one study by Curtin et al. reported finding at least small populations of enterobacteria as late as up to 4.5 months <ref name="curtain_asbc_2018">[https://www.asbcnet.org/lab/webinars/webinars/Pages/funkyFermentationsWebinar.aspx Chris Curtin. ASBC webinar: "Funky Fermentations". 12/12/2018. Retrieved 01/03/2019.]</ref>(~25 minutes in), as well as a significant population of ''Komagataeibacter'', a genera normally found in kombucha, after 135 day <ref name="Curtin_2021">[https://www.tandfonline.com/doi/abs/10.1080/03610470.2020.1795607?journalCode=ujbc20 Avi Shayevitz, Keisha Harrison & Chris D. Curtin (2021) Barrel-Induced Variation in the Microbiome and Mycobiome of Aged Sour Ale and Imperial Porter Beer, Journal of the American Society of Brewing Chemists, 79:1, 33-40, DOI: 10.1080/03610470.2020.1795607.]</ref>.
Acetic acid bacteria (AAB) are also present during the first stage of fermentation before alcoholic fermentation begins. These consist of a large diversity of species from ''Acetobacter'' and ''Gluconobacter'', with different species thriving more than others at different points during the long fermentation of lambic and some species found being different in different casks <ref name="De_roos_AAB_2018">[https://journals.asm.org/doi/10.1128/AEM.02846-17 Temporal and Spatial Distribution of the Acetic Acid Bacterium Communities throughout the Wooden Casks Used for the Fermentation and Maturation of Lambic Beer Underlines Their Functional Role. ASM Journals. Applied and Environmental Microbiology. Vol. 84, No. 7. DOI: https://doi.org/10.1128/AEM.02846-17.]</ref>, including two species that were first described by studies researching lambic (''Acetobacter lambici'' and ''Gluconbacter cerevisiae'' sp. nov. <ref>[https://www.microbiologyresearch.org/content/journal/ijsem/10.1099/ijs.0.057315-0 Acetobacter lambici sp. nov., isolated from fermenting lambic beer. Spitaels, Freek and Li, Leilei and Wieme, Anneleen and Balzarini, Tom and Cleenwerck, Ilse and Van Landschoot, Anita and De Vuyst, Luc and Vandamme, Peter. International Journal of Systematic and Evolutionary Microbiology. 2014. https://doi.org/10.1099/ijs.0.057315-0.]</ref><ref>[https://www.microbiologyresearch.org/content/journal/ijsem/10.1099/ijs.0.059311-0 Gluconobacter cerevisiae sp. nov., isolated from the brewery environment Free. Spitaels, Freek and Wieme, Anneleen and Balzarini, Tom and Cleenwerck, Ilse and Van Landschoot, Anita and De Vuyst, Luc and Vandamme, Peter. International Journal of Systematic and Evolutionary Microbiology. 2014. https://doi.org/10.1099/ijs.0.059311-0.]</ref>, as well as ''Acetobacter cerevisiae'' and ''Acetobacter faborum'' <ref name="Roosa_2024">De RoosaRoos, J., Vermotea, L., Cnockaertb, M., Vandammeb, P., Weckxa, S., & De Vuysta, L. WOODEN BARRELS HELP TO STEER THE LAMBIC BEER FERMENTATION AND MATURATION PROCESS.</ref>). Acetic acid bacteria are able to grow for the first few weeks because oxygen is available from filling the casks. Once alcoholic fermentation begins, oxygen becomes limited, and the acetic acid bacteria population greatly decreases. Acetic acid bacteria appear again after the alcoholic fermentation phase <ref name="Bongaerts_2021" /><ref name="Roosa_2024"/>. For example, Curtin et al. (2018) reported that acetic acid bacteria came and went at various random points within a 0-4.5 month period of fermentation <ref name="curtain_asbc_2018" />(~26 minutes in). De Ross et al. (2018) reported finding small amounts of acetic acid bacteria in lambic during the first few days of fermentation, which then disappeared once alcoholic fermentation began. AAB then reappeared in the casks in greater numbers at week 7 of fermentation, and continued to be isolated in gradually decreasing cell counts for 24 months, the end of which AAB was no longer isolated <ref name="De_roos_AAB_2018" />.
Acidifying the wort to a pH below 4.5 before cooling and exposing to ambient microbes in a coolship can partially eliminate the enterobacteria phase of spontaneous fermentation and thus avoid or limit biogenic amine production, which is a common practice for some lambic breweries <ref name="Spitaels et al., 2015" /><ref name="Roos_2018_2" />. While enterobacteria and oxidative yeasts are not considered to be a part of the core microbes in spontaneous fermentation, it has been shown that ''Saccharomyces cerevisiae'' is metabolically stimulated when co-fermented with some of these species, allowing the ''S. cerevisiae'' to consume more glucose and nitrogen and to more quickly replicate <ref name="Roos_2018" />. De Roos et al (2018) reported significant populations of the enterobacteria species ''Klebseilla variicola'', ''Klebsiella oxytoca'', and the yeast species ''Hanseniaspora uvarum'', ''Saccharomyces cerevisiae'' during the first week or two of lambic fermentation that was pre-acidified (see [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6252343/figure/F3/?report=objectonly Figure 3]). Landschoot et al (2015) sampled lambic wort that was pre-acidified to a pH of 4 after being housed in a coolship overnight and during the early weeks of fermentation and found no ''Enterobacteriaceae'' in the samples <ref name="Landschoot_2015">[https://www.academia.edu/22769494/The_microbial_diversity_of_an_industrially_produced_lambic_beer_shares_members_of_a_traditionally_produced_one_and_reveals_a_core_microbiota_for_lambic_beer_fermentation?email_work_card=view-paper Spitaels, F., Wieme, A. D., Janssens, M., Aerts, M., Landschoot, A. V., Vuyst, L. D., & Vandamme, P. (2015). The microbial diversity of an industrially produced lambic beer shares members of a traditionally produced one and reveals a core microbiota for lambic beer fermentation. Food Microbiology, 49, 23–32. https://doi.org/10.1016/J.FM.2015.01.008.]</ref>. Oxidative yeasts are also present during the first stage of fermentation, including species of ''Rhodotorula'', ''Candida'', ''Cryptococcus'', ''Hanseniaspora'', and ''Pichia'', some of which might survive pre-acidification <ref name="Bokulic et al., 2012" />. Zach Taggart reported that in a spontaneously fermented beer at his commercial brewery this initial stage also corresponded with a pH drop from 5.0 to 4.5 in 6 days and the aroma went from sweet-smelling wort to phenolic and a light burnt rubber character during this time in one batch of spontaneous fermentation <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/2360399550654912/ Zach Taggart (using his wife's Facebook account). Milk The Funk Facebook group post on analysis of spontaneous fermentation at 42 North Brewing Co. 11/09/2018.]</ref>.
Landschoot et al. (2015) attempted to recover yeasts and bacteria sampled from the brewery walls, ceilings, and coolship in an industrial brewery in West-Flanders that also produces lambic. They were unable to recover any yeasts or bacteria from these surfaces. The team did recover several species of microbes from the air of the coolship room such as ''Klebsiella oxytoca'', ''Bacillus'', and ''Staphylococcus'', but these microbes were not found in the wort after cooling in the coolship nor in the foeders during the sample times of 1, 2 and 3 weeks. ''S. cerevisiae'', ''S. pastorianus'', ''D. bruxellensis'', ''P. damnosus'', and a diverse range of acetic acid bacteria were the dominate species of microbes found during the fermentation of lambic in this brewery. After boiling, the brewery cooled its wort to 40°C using an industrial heat exchanger before pumping it into the coolship to reside overnight for 24 hours. The foeders in this brewery are cleaned with a pressure washer and are no attempt was made to pasteurize or sanitize the foeders with heat or chemicals. The inside surfaces of the foeders were sampled and these samples were the only samples that contained microbe species that were found during the fermentation of the lambic. The authors concluded that the microbes fermenting lambic at this brewery are introduced into the wort via the wooden inner surfaces of the foeders <ref name="Landschoot_2015"/>.
De Roos et al. (2024) reported finding a very diverse set of species in wort sampled from coolships after cooling (100-200 mL samples). Using DNA sequencing techniques and in order of abundance, they found ''H. uvarum'', ''S. cerevisiae'', ''Penicillium roqueforti'', ''Acinetobacter guillouiae'', ''Lactococcus raffinolactis'', ''Geotrichum candidum'', ''Chryseobacterium bovis'', ''Flavobacterium hibernum'', ''Yarrowia lipolytica'', and ''Trichococcus flocculiformis''. However, the only species from the coolship samples that were found during fermentation in two Italian wine barrels were ''H. uvarum'' and ''S. cerevisiae''. The authors concluded that the wooden casks themselves, despite heavy cleaning methods, contributed the majority of impactful microbe species during fermentation and aging, including ''Brettanomyces'' <ref name="Roosa_2024"/>. The authors of the study write(see also [https://www.facebook.com/groups/MilkTheFunk/posts/7935877029773775/ comments by Dr. Bryan Heit this MTF post]):
<blockquote>
''The present study further contributed to the role of wooden barrels in the spontaneous inoculation of the fermenting wort and maturing beer during lambic beer production. Although shotgun metagenomics revealed that the cooled wort had the highest diversity and evenness, its harboring species did not contribute to the wort fermentation and beer maturation in the casks.'' <ref name="Roosa_2024"/>