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 to do: - https://www.facebook.com/groups/MilkTheFunk/permalink/3292440674117457/ [[File:Brussels brett blend pellicle1.jpg|thumb|Pellicle from The Yeast Bay Brussels Brett Blend; courtesy of Dan Pixley]] A '''pellicle''' is a type of [[Quality_Assurance#Biofilms|biofilm ]] which appears on the surface of beer (the classification of pellicles as a type of biofilm is up for has recieved some debate. See [[Pellicle#Scientific_Terminology|Scientific Terminology]]). It consists of an aggregation of cells, proteins, and polymers <ref name="Branda"></ref><ref name="matt"></ref>. The "bubble" formations are caused by trapped CO2 beneath the pellicle film. Pellicles are often formed by ''[[Brettanomyces]]'', [[Pediococcus]], and [[Lactobacillus]], as well as ''Acetobacter spp'' <ref name="Perumpuli">[http://ir.kagoshima-u.ac.jp/bitstream/10232/21479/4/Diss_Perumpuli+Arachchige_Buddhika+Niroshie_RNK999_2014.pdf.pdf Pellicle of thermotolerant Acetobacter pasteurianus strains: Characterization of polysaccharide and induction patterns. Perumpuli Arachchige Buddhika Niroshie. 2014-09-30.]</ref> , and other gram-negative bacteria such as some species of ''Acinetobacter'', ''Escherichia'', ''Burkholderia'', ''Dickeya'', ''Gluconacetobacter'', ''Pseudomonas'', ''Salmonella'', ''Shewanella'', and ''Vibrio''. Gram-positive bacteria ''Bacillus subtillis'' can also form a pellicle <ref name="Armitano">[http://www.ncbi.nlm.nih.gov/pubmed/25756106 Gram-negative bacteria can also form pellicles. Armitano J, Méjean V, Jourlin-Castelli C. Environ Microbiol Rep. 2014 Dec.]</ref>. It can also be formed by ''[[Saccharomyces]] '' in rare occasions (most likely wild species more so than brewer's yeast), and it might be possible for ''[[Lactobacillus]]'' and ''[[Pediococcus]]'' to form a pellicle (according to Matt Humbard; see reference), however other references have not been found as far as we know <ref name="matt">[http://phdinbeer.com/2015/01/30/beer-microbiology-what-is-a-pellicle/ Beer Microbiology – What is a pellicle? A PhD in Beer blog. Dr. Matt Humbard. 01/30/2015. Retrieved 04/26/2015.]</ref>. Pellicles are not formed by and should not be confused with [[mold]].

Pellicles presumably form when the surface of the beer is exposed to even small amounts oxygen <ref>[http://www.themadfermentationist.com/2009/11/brewing-sour-beer-at-home.html Brewing Sour Beer at Home. The Mad Fermentationist Blog. Michael Tonsmeire. 11/06/2009. Retrieved 02/28/2015.]</ref> Why microbes in sour/funky beer form a pellicle when exposed to oxygen is unknown. The most likely [http://phdinbeer.com/2015/01/30/beer-microbiology-what-is-a-pellicle/ hypothesis by Dr. Matt Humbard is that the formation of a pellicle ] allows the organism (particularly ''[[Brettanomyces]]'', which prefers aerobic fermentation) to access the small amount of oxygen that is present in the headspace of the fermentation vessel. Another hypothesis, and one that may be less accurate according to Dr. Matt Humbard, is that the ''pellicle'' protects the beer from other microorganisms <ref name="matt"></ref>. Yet another hypothesis that is often cited is that the pellicle protects the beer from oxygen itself <ref>[http://jesterkingbrewery.com/pellicle-photos Jester King Blog on Pellicles. Retrieved 09/24/2015.]</ref>; however, however arguments evidence and rationale for this hypothesis are is lacking.

Pellicle formation by microbes found in sour beer such as [[Brettanomyces]] and [[Lactobacillus]] has not been closely studied. However, we We may also be able to glean some insight from studies done on ''Acetobacter'' pellicle formation during vinegar production.

Pellicle formation in ''Acetobacter tropicalis'' has been linked to a gene cluster (polABCDE), and disruption of these gene switched the cells from producing CPS (and pellicle formation) to producing EPS instead <ref name="Perumpuli"></ref>..

The presence of ethanol in concentrations of 1-4% encourages pellicle production in some strains of ''Acetobacter pasteurianus'' (although the presence of ethanol encourages pellicle formation, the amount of ethanol did not make a difference). In some strains of ''A. pasteurianus'', sugar encourages pellicle formation. It has been suggested that CPS production in ''Acetobacter'' is a function of stress tolerance by acting as a barrier around the cell that protects it from acetic acid in the environment. Strains that form a pellicle in the presence of ethanol and/or higher temperatures can fully ferment vinegar whereas those that do not create a pellicle perform poorly in comparison <ref name="Perumpuli"></ref>.

===Identification in Contaminated BeersPellicle Appearance as a Microbe Identifying Indicator===An often asked question [[File:Pichia_pellicle.jpg|thumb|[https://www.facebook.com/groups/MilkTheFunk/permalink/1524191477609061/ Pellicle formed by homebrewers what is can a contaminating microbe likely identified to be identified based on the appearance of a pellicle''Pichia anomala''. This is an impossible exercise for a few reasonsPhoto provided by Nick Rutkowski.]]]

# While different strains of An often asked question by homebrewers is can a contaminating or wild caught microbe be identified based on the same species appearance of microbe (at least in ''Acetobacter'') can create different types of polysaccharides a pellicle. This is a difficult exercise for a few reasons. # There is very little research on pellicles when it comes to beer fermentation, so saying anything definitive about them is difficult, and identifying one or more microbes that make up created a pellicle based on what the pellicle, their chemical make up looks like is more or less very similarcurrently impossible.# Numbers The number and size of bubbles in a pellicle is created by largely dependent on the amount of trapped CO2, and thus is not an identifying feature.# Scientific research on pellicle formation in beer is currently next to none.# The factors that cause pellicle formation are not well understood. Oxygen is said to cause pellicle formation, but how, why, and in what species is not known. Does more oxygen create a thicker pellicle? If so, for what species does this apply to? Does beer composition (more or less proteinsprotein content, residual sugars, etc.) affect pellicle appearance? The lack of answers to these questions and others imply that environmental factors are also not helpful in identifying what species of microbe produced a pellicle.# Identification of microbes microbe species and sometimes even genus under a [[Microscope|microscope ]] based on cell morphology alone is not enough to be certain of that identification(see [http://suigenerisbrewing.blogspot.com/2014/12/brett-trois-riddle-wrapped-in-mystery.html this article on Trois identification] as an example), let alone attempting to identify microbes based on a visual "macro-level" formation such as a pellicle. # DNA analysis of a microbe is the only way to reliably identify the species of a microbe (see . This is why [http://suigenerisbrewing.blogspotbootlegbiology.com/20142015/09/1217/brettlocal-yeast-project-troisstudy-riddlebarrel-wrappedculture-int-mysteryshirts/ Bootleg Biology is launching a program to identify wild caught microbes through DNA analysis]. The above stated, it might be possible to make an educated guess as to the genus of a microbe that produced a pellicle if there was a controlled study (same wort, same incubation conditions, etc.) that examined the visual differences between pellicles formed by different genera of microbes. Unfortunately, such a study has not been performed that we know of. Therefore, it is currently unknown if the genus of a contaminating microbe can be determined based on the visual appearance of the pellicle alone, though it is probably unlikely <ref>[https://www.facebook.html this article com/groups/MilkTheFunk/permalink/1153845284643684/?comment_id=1153891934639019&reply_comment_id=1154095574618655&total_comments=5&comment_tracking=%7B%22tn%22%3A%22R2%22%7D Conversation with Richard Preiss and others on Trois genera identificationMTF. 09/24/2015.]</ref>. * See also [[Wild_Yeast_Isolation#Growing_and_Testing_Without_Plating|Wild Yeast isolation]] as an example)for more information on wild yeast isolation, pellicles, and safety.

Most Many brewers will advise that if it all possible, try not to disturb the pellicle too much when taking a sample, racking, or moving the fermenting fermentation vessel. However, if the pellicle is disturbed, this should be fine as long as the beer is not exposed to too much oxygen. If taking a sample or racking, gently pierce the top of the pellicle with the racking cane or wine thief. If the pellicle does break up, don't worry too much. It will usually reform if oxygen is still present in the headspace. Purging with CO2 might be a good idea if you think that a lot of oxygen got introduced during handling. While packaging the beer, try not to disturb the pellicle too much because clumps of the pellicle on the surface of the beer can fall back into solution when disturbed, and can then get transferred into the bottles or kegs. If the beer is being transferred, if fruits are being added, if new product is being added in a [[Solera]] process, or if it is being used as a starter for another batch, do not worry about disturbing the pellicle. Disturbing the pellicle isn't really a problem; letting in too much oxygen is the problem.

Often a pellicle will form on the surface of the beer inside the bottle shortly after packaging. This is no different than the pellicle forming in the fermentation vessel, and presumably occurs because of the oxygen in the headspace of the bottle. The pellicle will eventually settle out either on it's its own during aging, or when the bottle is refrigeratedor disturbed. Other than for aesthetics, there should be no concern if this happens. Pierre Tilquin of [http://www.gueuzerietilquin.be Gueuzerie Tilquin] suggests that storing bottles horizontally on their side during bottle conditioning will prevent a pellicle from forming in the bottles. This is a common practice among lambic brewers, and when Pierre stored bottles upright he saw the formation of a pellicle in his bottles of lambic <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1210209235673955/?comment_id=1210248462336699&reply_comment_id=1210254505669428&comment_tracking=%7B%22tn%22%3A%22R8%22%7D Conversation with Pierre Tilquin and Raf Soef on MTF. 01/08/2016.]</ref>. Gently tipping the bottles or even just standard handling will also dissolve the pellicle without harming the beer. See also [[Aging_and_Storage#Bottle_Orientation|Bottle Orientation]].

The usage of the term "biofilm" has been used to describe the layer of film that covers sherry known as "Flor" <ref>[http://aem.asm.org/content/76/12/4089.full Ethanol-Independent Biofilm Formation by a Flor Wine Yeast Strain of Saccharomyces cerevisiae. Severino Zara, Michael K. Gross, Giacomo Zara, Marilena Budroni and Alan T. Bakalinsky. 2010.]</ref><ref>[http://femsle.oxfordjournals.org/content/femsle/237/2/425.full.pdf FLO11 is essential for flor formation caused by the C-terminal deletion of NRG1 in Saccharomyces cerevisiae. Mari Ishigami, Youji Nakagawa, Masayuki Hayakawa, Yuzuru Iimura. 2004.]</ref>. The word "pellicle" generally isn't used, although it has appeared on occasion when referring to sherry flor in the 1960's <ref>[http://www.ncbi.nlm.nih.gov/pubmed/5311988 On the pellicle formation by “flor” yeasts. Cantarelli C, Martini A. Antonie Van Leeuwenhoek. 1969.]</ref>, and as least once in brewing science from the 1960's <ref name="Macrae_1964">[https://onlinelibrary.wiley.com/doi/abs/10.1002/j.2050-0416.1964.tb02001.x SIGNIFICANCE OF THE USE OF HOPS IN REGARD TO THE BIOLOGICAL STABILITY OF BEER: I. REVIEW AND PRELIMINARY STUDIES. R. M. Macrae. 1964.]</ref>. More recently, there have been studies that define a "pellicle" like we see in fermentation as a type of biofilm that forms on the air-liquid interface of a liquid (see references: <ref name="Armitano"></ref><ref>[http://www.ncbi.nlm.nih.gov/pubmed/26122431 Motility, Chemotaxis and Aerotaxis Contribute to Competitiveness during Bacterial Pellicle Biofilm Development. Hölscher T, Bartels B, Lin YC, Gallegos-Monterrosa R, Price-Whelan A, Kolter R, Dietrich LE, Kovács ÁT. J Mol Biol. 2015 Jun 26.]</ref><ref>[http://www.ncbi.nlm.nih.gov/pubmed/24035282 Modulation of curli assembly and pellicle biofilm formation by chemical and protein chaperones. Andersson EK, Bengtsson C, Evans ML, Chorell E, Sellstedt M, Lindgren AE, Hufnagel DA, Bhattacharya M, Tessier PM, Wittung-Stafshede P, Almqvist F, Chapman MR. Chem Biol. 2013 Oct 24.]</ref><ref name="Branda">[http://www.horadent.com/uploads/media/2005_Branda.pdf Biofilms: the matrix revisited. Steven S. Branda, Ashild Vik, Lisa Friedman and Roberto Kolter. Jan 2005.]</ref><ref>[http://onlinelibrary.wiley.com/doi/10.1046/j.1365-2958.2003.03877.x/full Genes involved in matrix formation in Pseudomonas aeruginosa PA14 biofilms. Lisa Friedman andRoberto Kolter. Dec 2003.]</ref><ref>[http://aem.asm.org/content/77/15/5079.short Increased Transfer of a Multidrug Resistance Plasmid in Escherichia coli Biofilms at the Air-Liquid Interface. Jaroslaw E. Król, Hung Duc Nguyen, Linda M. Rogers, Haluk Beyenal, Stephen M. Krone1, and Eva M. Top. 2011.]</ref><ref>[https://books.google.com/books?id=PO3QrZJnuGgC&pg=PA7&dq=biofilm+air-liquid+interface&hl=en&sa=X&ved=0CDAQ6AEwBGoVChMIjP--39D5xwIVFDWICh285gg_#v=onepage&q=biofilm%20air-liquid%20interface&f=false Bacterial Biofilms. Tony Romeo. Springer Science & Business Media, Feb 26, 2008. Pg 7.]</ref>). Lebleux et al. (2019) defines biofilm as, "Biofilms are complex associations of single- and multiple- species interconnected cells embedded in a hydrated self-produced matrix established at a solid/liquid or liquid/air interfaces." <ref>[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> However, however those objecting to the definition of a biofilm as including air-liquid interface aggregates say that the use of the term in a few studies does not warrant a change in the textbook definition <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1142388839122662/ Discussion of this wikipage on MTF. 09/06/2015.]</ref>.  The importance of understanding the established terminology of "biofilms" and "pellicles" versus the brewing terminology becomes apparent when trying to research the topic of pellicles in beer. Currently beer pellicles have not been studied scientifically much at all, whereas pellicles of dentistry and microbiology have been studied in depth, as well as biofilms in general. Thus, brewers should take care when reading scientific publications, and understand that brewing terminology does not usually overlap with scientific terminology in regards to pellicles.