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====Biofilm====
[[File:Chlamydospore Brett.JPG|thumb|First evidence of possible (unconfirmed <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/3118617694833090/?comment_id=3120943487933844 Dr. Bryan Heit. Milk The Funk Facebook group thread on Lebleux et al. (2019) and chlamydospore in Brettanomyces. 12/11/2019.]</ref>) chlamydospore cell structures of ''B. bruxellensis'', found in a biofilm. Photo by [https://www.sciencedirect.com/science/article/abs/pii/S0168160519303952 Lebleux et al. (2019)] <ref name="Lebleux_2019">[https://www.sciencedirect.com/science/article/abs/pii/S0168160519303952 New advances on the Brettanomyces bruxellensis biofilm mode of life. Manon Lebleux, Hany Abdo, Christian Coelho, Louise Basmaciyan, Warren Albertin, Julie Maupeu, Julie Laurent, Chloé Roullier-Gall, Hervé Alexandre, Michèle Guilloux-Benatier, Stéphanie Weidmann, Sandrine Rousseaux. 2019. DOI: https://doi.org/10.1016/j.ijfoodmicro.2019.108464.]</ref>.]]
''Brettanomyces'' has the ability to form a [[Quality_Assurance#Biofilms|biofilm]]. Biofilm formation is a survival mechanism induced by stress whereby the cells adhere to non-living surfaces such as plastic and stainless steel. After adhesion to the surface, the cells produce a protective layer of proteins and polysaccharides that help protect the organism from cleaning and sanitizing agents.
Dimopoulou et al. (2019) studied ''Brettanomyces bruxellensis'' biofilms from each of the genetic branches of ''B. bruxellensis''. They found that for the wine strains biofilms formed more readily when grown in wine must rather than YPD media; however, the beer strains grew biofilms equally well in wine must and YPD media. The biofilms contained a large portion of saturated fatty acids and a smaller portion of monounsatured fatty acids. The amount of exopolysaccharide produced varied widely across the strains tested per cell population, with some wine strains producing little EPS (40 mg/L/OD), beer strains producing moderate amounts, and the other wine group producing a high amount (100 mg/L/OD). Additionally, the different strains displayed a varying degree of negative cell wall charges, with the beer and tequila strains being more negatively charged than wine strains, which could help them adhere to surfaces and form biofilm <ref>[https://europepmc.org/article/PPR/PPR73221?singleResult=true Dimopoulou M., Renault M., Dols-Lafargue M., Albertin-Leguay W., Herry J., Bellon-Fontaine M., Masneuf-Pomarede I. 2019. DOI: 10.1101/579144.]</ref>.
Lebleux et al. (2019) measured biofilm density for 12 strains across 5 of the genetic groups of ''B. bruxellensis''. All of the strains produced a biofilm when in contact with a surface (polystyrene and stainless steel, in the case of this study), and the thickness of the biofilm was proportional to the cell size of each strain. The biofilms contained filamentous cells that started from the base of the biofilm and extended upward, indicating multiple layers. The biofilms also contained exopolysaccharides (EPS), but the make up makeup of the EPS was not analyzed and this was identified as a goal for further study. The average thickness was only 9.45 µm which is much thinner than other biofilm-forming yeast species (''Candida'' and a biofilm -producing strain of ''S. cerevisiae'' <ref>[https://bmcmicrobiol.biomedcentral.com/articles/10.1186/s12866-014-0305-4 Saccharomyces cerevisiae biofilm tolerance towards systemic antifungals depends on growth phase. Bojsen, R., Regenberg, B. & Folkesson, A. BMC Microbiol 14, 305 (2014). DOI: 10.1186/s12866-014-0305-4.]</ref>). They found that one or two strains were less dense (contained fewer cells) than the average. A couple of the strains grew a biofilm a little slower than average. Two of the strains in biofilm form were added to wine; each of the biofilms released cells into the wine, although one strain released more cells than the other. Introduction to the wine first led to cell death for some cells due to the harsh environment of the wine, but after several days the ''B. bruxellensis'' strains began to re-grow in the wine. It was observed that for one of the strains, the cells appeared larger than normal, round, and had thicker cell walls, apparently possibly forming what is known as [https://en.wikipedia.org/wiki/Chlamydospore chlamydospore cell structures]. It was not confirmed in the study whether these cells were actually chlamydospores. Chlamydospore cell structures are known to help certain species of non-yeast fungi survive harsh environments; however, it has not yet been established that yeast with chlamydospore cell structures helps them survive harsh conditions, and this was also identified in the study as an area for further research <ref name="Lebleux_2019" />.
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