Changes

''Brettanomyces'' has been thought to occur naturally on the skins of fruit such as apples and grapes. However, there are only a handful of reports of ''Brettanomyces'' being identified on the skins of fruit, and in some cases where ''Brettanomyces'' has been found, its abundance is extremely minimal <ref>[https://onlinelibrary.wiley.com/doi/full/10.1002/jib.154 Lentz, M., Putzke, T., Hessler, R. and Luman, E. (2014), Genetic and physiological characterization of yeast isolated from ripe fruit and analysis of fermentation and brewing potential, J. Inst. Brew., 120: 559– 564. DOI: 10.1002/jib.154.]</ref><ref name="Comitini">[https://www.frontiersin.org/articles/10.3389/fmicb.2019.00415/abstract Occurrence of Brettanomyces bruxellensis on grape berries and in related winemaking cellar. Francesca Comitini, Lucia Oro, Laura Canonico, Valentina Marinelli, Maurizio Ciani. 2019. DOI: 10.3389/fmicb.2019.00415.]</ref><ref name="Renouf_2007">[https://www.sciencedirect.com/science/article/pii/S0944501306000231?via%3Dihub Development of an enrichment medium to detect Dekkera/Brettanomyces bruxellensis, a spoilage wine yeast, on the surface of grape berries. Vincent Renouf, Aline Lonvaud-Funel. 2007. DOI: https://doi.org/10.1016/j.micres.2006.02.006.]</ref>. In contrast, there are also studies that indicate ''Brettanomyces'' only being found during or after food processing, which indicates that the processing equipment may be the primary source for the ''Brettanomyces''. In addition, ''Brettanomyces'' has been isolated in abundance from the surfaces of equipment/processed materials in wineries and breweries <ref name="smith_divol_2016" /><ref name="Schifferdecker" /><ref name="Loureiro_2003">[https://www.ncbi.nlm.nih.gov/pubmed/12892920 Spoilage yeasts in the wine industry. Loureiro V, Malfeito-Ferreira M. 2003.]</ref><ref name="Steensels" /><ref name="Barata_2008">[https://www.ncbi.nlm.nih.gov/pubmed/18077036 Survival patterns of Dekkera bruxellensis in wines and inhibitory effect of sulphur dioxide. f Barata A, Caldeira J, Botelheiro R, Pagliara D, Malfeito-Ferreira M, Loureiro V. 2008.]</ref> (Table 1). For example, an ongoing survey of wild yeasts in most of the US which isolated nearly 2,000 isolates with 262 unique species has not yet found a single occurrence of ''Brettanomyces'' in the wild (so far they have only surveyed non-human inhabited wild areas of the US and Alaska; substrates sampled included leaves, soil, bark, moss, mushrooms, needles, pine cones, twigs/wood, and other plant matter) <ref>[https://www.biorxiv.org/content/10.1101/2021.07.13.452236v1 Substrate, temperature, and geographical patterns among nearly 2,000 natural yeast isolates. William J. Spurley, Kaitlin J. Fisher, Quinn K. Langdon, Kelly V. Buh, Martin Jarzyna, Max A. B. Haase, Kayla Sylvester, Ryan V. Moriarty, Daniel Rodriguez, Angela Sheddan, Sarah Wright, Lisa Sorlie, Amanda Beth Hulfachor, Dana A. Opulente, Chris Todd Hittinger. bioRxiv 2021.07.13.452236; doi: https://doi.org/10.1101/2021.07.13.452236.]</ref>. It is therefore unclear that ''Brettanomyces'' found on grape skins originated there or from the industrial processing where it is more abundant. It is also thought to disperse via fruit-flies (called "vectors" in the scientific literature), similar to how ''Saccharomyces'' travels, although direct evidence for this has only been reported rarely and only on fruit-flies in wineries that are likely to come into contact with equipment/processed material that is already contaminated with ''Brettanomyces'' <ref>[https://youtu.be/G2nhUM5PIrg?t=309 Dr. Bryan Heit. BotB - Where (Do) The Wild Brettanomyces Roam?. ~5 mins in. Retrieved 07/10/2022.]</ref><ref name="Renouf_2007" /><ref name="Steensels">[http://www.sciencedirect.com/science/article/pii/S0168160515001865 Brettanomyces yeasts — From spoilage organisms to valuable contributors to industrial fermentations. Jan Steensels, Luk Daenen, Philippe Malcorps, Guy Derdelinckx, Hubert Verachtert, Kevin J. Verstrepen. International Journal of Food Microbiology Volume 206, 3 August 2015, Pages 24–38.]</ref><ref name="Barata_2008" /><ref name="Loureiro_2003" />. ''Brettanomyces'' is known to be difficult to grow in a lab due to slow growth, specific nutrient requirements, or perhaps because of a "VBNC" state (see [[Wild_Yeast_Isolation#Wild_Brettanomyces|Wild ''Brettanomyces'']] for more information), which may account for the lack of evidence for fruit being the primary natural habitat for ''Brettanomyces''. More recently, techniques have been invented to more easily isolate and grow ''Brettanomyces'' <ref name="Renouf_2007" /><ref name="Comitini" />. There is also significant evidence that the natural habitat of ''Brettanomyces'' might actually be the root systems of certain plants, known as the [https://www.nature.com/scitable/knowledge/library/the-rhizosphere-roots-soil-and-67500617/ "rhizosphere"]. The rhizosphere refers to the complex symbiotic community of microbe populations that live on and around the root system of plants. Wild strains of ''Brettanomyces'' have been found in the root systems of dill, common beans, sunflowers, maize, corn, jute, cassava, and grey mangroves found in the estuaries of Indonesia <ref>[https://onlinelibrary.wiley.com/doi/abs/10.1111/aab.12309 Weisany, W., Raei, Y., Salmasi, S., Sohrabi, Y. and Ghassemi-Golezani, K. (2016), Arbuscular mycorrhizal fungi induced changes in rhizosphere, essential oil and mineral nutrients uptake in dill/common bean intercropping system. Ann Appl Biol, 169: 384-397. https://doi.org/10.1111/aab.12309.]</ref><ref>[https://archive.aessweb.com/index.php/5003/article/view/3333 I.O, S. ., & G.P, O. . (2012). Diversity of Fungal Populations in Soils Cultivated With Cassava Cultivar TMS 98/0505. Journal of Asian Scientific Research, 2(3), 116–123. Retrieved from https://archive.aessweb.com/index.php/5003/article/view/3333.]</ref><ref>[https://www.ajol.info/index.php/swj/article/view/149513 Rhizosphere and non-rhizosphere soil mycoflora of Corchorus olitorius (Jute). G.S. Olahan, I.O. Sule, T Garuba, Y.A. Salawu. Science World Journal. 2016.]</ref><ref>[https://ojs.unud.ac.id/index.php/jbb/article/view/36023 NOERFITRYANI, Noerfitryani; HAMZAH, Hamzah. THE EXISTENCE OF ENTOMOPATHOGENIC FUNGI ON RICE PLANTS RHIZOSPHERE. International Journal of Biosciences and Biotechnology, p. 12-24, dec. 2017. ISSN 2655-9994. doi: https://doi.org/10.24843/IJBB.2017.v05.i01.p02.]</ref><ref>[https://www.sciencedirect.com/science/article/abs/pii/S2452219818300259 Marcela Sarabia, Saila Cazares, Antonio González-Rodríguez, Francisco Mora, Yazmín Carreón-Abud, John Larsen, Plant growth promotion traits of rhizosphere yeasts and their response to soil characteristics and crop cycle in maize agroecosystems, Rhizosphere, Volume 6, 2018, Pages 67-73, ISSN 2452-2198, https://doi.org/10.1016/j.rhisph.2018.04.002.]</ref><ref>[https://www.sciencedirect.com/science/article/abs/pii/S1049964419303238 Nivien A. Nafady, Mohamed Hashem, Elhagag A. Hassan, Hoda A.M. Ahmed, Saad A. Alamri. The combined effect of arbuscular mycorrhizae and plant-growth-promoting yeast improves sunflower defense against Macrophomina phaseolina diseases. Biological Control. Volume 138, 2019, 104049. ISSN 1049-9644, https://doi.org/10.1016/j.biocontrol.2019.104049.]</ref><ref>[http://ejurnal.its.ac.id/index.php/sains_seni/article/view/5613 Isolation and Characterization of Yeast from Rhizosphere Avicennia Marina Wonorejo. Sitatun Zunaidah, Nur Hidayatul Alami. 2014. DOI: 10.12962/j23373520.v3i1.5613.]</ref>. See Dr. Bryan Heit's video [https://www.youtube.com/watch?v=G2nhUM5PIrg "Where (Do) The Wild Brettanomyces Roam?"] and [https://www.facebook.com/groups/MilkTheFunk/posts/5940213029340195 his comments in Milk The Funk], as well as [https://www.youtube.com/watch?v=BrR7G_YyfmA "Philip Poole. Plant Control of the Rhizosphere Microbiome"]. For documented isolation attempts from plant rhizospheres, see [[Wild_Yeast_Isolation#Wild_Brettanomyces|Wild Yeast Isolation]].

The occurrence of ''Brettanomyces'' has been more commonly identified in industrial food processing areas (wine, beer, kombucha, soft drinks, dairy products, tea, sourdough, etc.) <ref name="Crauwels_2016">[https://academic.oup.com/femsyr/article-abstract/17/1/fow105/2670560/Fermentation-assays-reveal-differences-in-sugar?redirectedFrom=fulltext Fermentation assays reveal differences in sugar and (off-) flavor metabolism across different Brettanomyces bruxellensis strains. Fermentation assays reveal differences in sugar and (off-) flavor metabolism across different Brettanomyces bruxellensis strains. Sam Crauwels, Filip Van Opstaele, Barbara Jaskula-Goiris, Jan Steensels, Christel Verreth, Lien Bosmans, Caroline Paulussen, Beatriz Herrera-Malaver, Ronnie de Jonge, Jessika De Clippeleer, Kathleen Marchal, Gorik De Samblanx, Kris A. Willems, Kevin J. Verstrepen, Guido Aerts, and Bart Lievens. 2016]</ref>. For example, ''B bruxelensis'', ''B. anomala'', and ''B. custersianus'' have mostly been isolated from wine or beer production, while ''B. naardenensis'' has mostly been isolated from soda production <ref name="Tiukova_2019">[https://www.mdpi.com/2076-2607/7/11/489 Assembly and Analysis of the Genome Sequence of the Yeast Brettanomyces naardenensis CBS 7540. Ievgeniia A. Tiukova, Huifeng Jiang, Jacques Dainat, Marc P. Hoeppner, Henrik Lantz, Jure Piskur, Mats Sandgren, Jens Nielsen, Zhenglong Gu, and Volkmar Passoth. 2019. DOI: https://doi.org/10.3390/microorganisms7110489.]</ref>. ''Brettanomyces'' is not considered to be airborne; however, one study has demonstrated a very small amount of cells in the air at wineries where wine with ''Brettanomyces'' in it was being handled (most of the yeasts found in the air were ''Aureobasidium'' and ''Cryptococcus'', which aren't considered spoilage organisms in beer and wine). This set of studies also determined that very specific methodology was needed in order capture ''Brettanomyces'' from the air, and indicated that the yeast was "stressed". While it is possible for ''Brettanomyces'' to be briefly carried by gusts of air, it only happens in the vicinity where the ''Brettanomyces'' beer or wine is being bottled (more so) or is actively fermenting (less so) <ref>[http://www.sciencedirect.com/science/article/pii/S0956713513002284 Screening of yeast mycoflora in winery air samples and their risk of wine contamination. E. Ocón, P. Garijo, S. Sanz, C. Olarte, R. López, P. Santamaría, A.R. Gutiérrez. Food Control Volume 34, Issue 2, December 2013, Pages 261–267.]</ref>. Good cleaning and sanitation and cold temperatures should be employed to keep ''Brettanomyces'' from contaminating other equipment; however, flying insects are also a potential cause for contamination of ''Brettanomyces'' (although evidence for this is lacking).

''Brettanomyces'' is commonly isolated from the surface of wood structures within breweries, wineries, and sometimes cideries (although the median occurrence of ''Brettanomyces'' in barrels may be very low to none within a given winery or brewery depending on their hygiene and other factors <ref>[https://link.springer.com/article/10.1007/s00217-011-1523-8 Guzzon, R., Widmann, G., Malacarne, M. et al. Survey of the yeast population inside wine barrels and the effects of certain techniques in preventing microbiological spoilage. Eur Food Res Technol 233, 285–291 (2011). https://doi.org/10.1007/s00217-011-1523-8.]</ref><ref>[https://agris.fao.org/agris-search/search.do?recordID=IT2007601151 Fontanot, S.; Ninino, M.E.; Comi, G.; Elimination of Dekkera/Brettanomyces from barriques of the Italian CDO Isonzo area. Controlled Designation of Origin; Friuli-Venezia Giulia. 2006.]</ref>). These include structures such as wooden fermentation vessels, walls of the building, as well as the inside surface of wood barrels and actually buried within the wood of barrels. ''Brettanomyces'' has been easily cultured from within the wood of oak barrels up to 4 mm into the wood, and occasionally as deep as 5 to 8 mm, depending on the age and variety (slightly higher populations tend to survive in French oak over American oak, and one study found that the ''Brettanomyces'' was able to penetrate the French oak barrels up to 8 mm, while only penetrate American oak barrels up to 4 mm) of the barrel <ref name="Agnolucci_2017" /><ref name="Cartwright_2018">[http://www.ajevonline.org/content/early/2018/05/23/ajev.2018.18024 Reduction of Brettanomyces bruxellensis Populations from Oak Barrel Staves Using Steam. Zachary M. Cartwright, Dean A. Glawe, Charles G. Edwards. 2018. DOI: 10.5344/ajev.2018.18024.]</ref>, with the highest concentration of surviving cells being at the top staves where oxygen is more accessible (although Cartwright et al. found the opposite was true, perhaps due to methodology of sampling or a difference in SO₂ concentrations). Some strains are able to utilize the cellulose of the wood as a carbon source, and occasionally form pseudohyphae within the wood which expands the surface area of the cells allowing them more access to nutrients and allowing them to survive in nutrient deficient environments <ref name="Cartwright_2018" />. Ozone gas has been shown to be an effective way to kill ''Brettanomyces'' that is buried in the wood of oak barrels, but the ozone must be applied for an adequate time to allow for the ozone to diffuse into the oak. Ozone has also been shown to be an effective way of greatly reducing but not completely eliminating the number of ''Brettanomyces'' on wine grapes. Liquid ozone has been shown to be less effective at eliminating ''Brettanomyces''. Heating the inside of the oak barrels to 60°C for 20 minutes with hot water or steam has also been found to be an effective way of killing ''Brettanomyces'' within the wood of barrels (see [[Barrel#Sanitizing|Barrel Sanitation]] for information on pasteurizing barrels) <ref>[https://www.ncbi.nlm.nih.gov/pubmed/25989358 Heat inactivation of wine spoilage yeast Dekkera bruxellensis by hot water treatment. Fabrizio, Vigentini, Parisi,Picozzi, Compagno, Foschino. 2015.]</ref><ref>[https://www.sciencedirect.com/science/article/pii/S1466856417310068 Control of Brettanomyces bruxellensis on wine grapes by post-harvest treatments with electrolyzed water, ozonated water and gaseous ozone. Francesco Craveroa, Vasileios Englezos, Kalliopi Rantsiou, Fabrizio Torchio, Simone Giacosa, Susana Río Segade, Vincenzo Gerbi, Luca Rolle, Luca Cocolin. 2018. DOI: https://doi.org/10.1016/j.ifset.2018.03.017.]</ref>. Although the role of ''Brettanomyces'' appears to be limited in distillation, it has been isolated during the fermentation process of tequila making. It has also been isolated from drains, pumps, transfer hoses, and other equipment that is difficult to sanitize. The survivability of ''Brettanomyces'' has also partly been attributed to its ability to form a [[Quality_Assurance#Biofilms|biofilm]] (in particular ''B. bruxellensis''). Microorganisms that can form a biofilm are more resistant to chemical cleaning agents and sanitizers than those that don't. ''Brettanomyces'' has therefore been identified as a significant contaminate for breweries and wineries. Oak barrels from wineries with unsanitary practices, in particular, have been identified as common contamination sites for ''B. bruxellensis''. ''Brettanomyces'' is also commonly found in sherry, and is found (although only rarely) in olive production, lemonade, kombucha, yogurt, pickles, and soft drinks. ''B. anomalus'' and ''B. bruxellensis'' are generally found much more commonly than the other three species of ''Brettanomyces'' <ref name="smith_divol_2016">[http://www.sciencedirect.com/science/article/pii/S0740002016302659 Brettanomyces bruxellensis, a survivalist prepared for the wine apocalypse and other beverages. Brendan D. Smith, Benoit Divol. June 2016.]</ref>.

The genetic diversity of ''Brettanomyces'' is particularly wide. For example, one study that analyzed the whole genomes of 53 strains of ''B. bruxellensis'' found that the overall genetic diversity between different strains of ''B. bruxellensis'' was higher than strains of ''S. cerevisiae'' (however, the entire gene set, known as the ''pangenome'', of all the genes among all of the strains of ''B. bruxellensis'' is much smaller than the entire gene set of ''S. cerevisiae'') <ref name="Gounot_2019" />. Some studies have indicated that strains of ''B. bruxellensis'' have adapted to specific environments. For example, one study found that strains of ''B. bruxellensis'' isolated from wine had 20 genes involved in the metabolism of carbon and nitrogen, whereas strains isolated from beer did not. This indicated that ''B. bruxellensis'' strains living in wine have adapted to the harsher environment of wine <ref name="smith_divol_2016"></ref>. Another study found that one out of the two strains tested that were isolated from soda could not ferment maltose, and only strains isolated from wine were able to grow in wine and the beer/soda strains did not. The wine strains were also more resistant to sulfites, which are commonly used in the wine industry to prevent microbial contamination <ref name="Crauwels_2016" />. The whole genome sequencing of one strain of ''B. naardenensis'' and lambic strains of ''B. bruxellensis'' found that they are missing the genes associated with nitrate utilization, indicating that the assimilation of nitrates is not required to survive in beer, perhaps because of the abundance of nitrogen from other sources found in beer <ref name="Tiukova_2019" /><ref name="colomer_2020_genome" />.