Pellicle

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Pellicle from The Yeast Bay Brussels Brett Blend; courtesy of Dan Pixley

A pellicle is a type of biofilm which appears on the surface of beer (the classification of pellicles as a type of biofilm has recieved some debate. See Scientific Terminology). It consists of an aggregation of cells, proteins, and polymers [1][2]. The "bubble" formations are caused by trapped CO2 beneath the pellicle film. Pellicles are often formed by Brettanomyces, Acetobacter [3], 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 [4]. 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 Dr. Matt Humbard; see reference and this opinion piece by Dr. Bryan Heit on MTF on why it is difficult to find any scientific sources for pellicles); however, this has not been scientifically proven as far as we know [2]. Pellicles are not formed by and should not be confused with mold.

Characteristics

Introduction

Pellicles presumably form when the surface of the beer is exposed to even small amounts oxygen [5] Why microbes in sour/funky beer form a pellicle when exposed to oxygen is unknown. The most likely 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 [2]. Yet another hypothesis that is often cited is that the pellicle protects the beer from oxygen itself [6]; however, evidence and rationale for this hypothesis is lacking.

Popular thought is that the formation of a pellicle is not indicative of the quality of the sour beer that is being produced; it is only an indication that oxygen has entered the fermentation vessel and that the microbes are reacting to that exposure. One myth about pellicles is that the sour beer will be ready to package once the pellicle falls out; there is actually no correlation between the maturity of the beer and pellicle formation or dissipation. Some sour beers never form pellicles, and turn out fine as well, so the formation of a pellicle has no correlation with the quality of the beer.

Another less common but occasionally discussed myth about pellicles is that sunlight might cause them. There is no evidence to suggest that sunlight has an effect on pellicle growth in beer. A more probable explanation for pellicles appearing in fermenters that have had sunlight on them is that when the sunlight hits the fermenter, the fermenter warms up. As it cools, a vacuum is created and air is sucked into the fermenter. Mark Trent demonstrates how readily air can suck into a fermenter when the fermenter is cooled (temperature shifts in general and atmospheric pressure changes can cause this):

See also;

Yeast

Pellicle formation by microbes found in sour beer such as Brettanomyces and Lactobacillus has not been widely studied. One report by De Roos et al. (2018) reported that in lambic casks that were studied, a pellicle formed at around 6 months into spontaneous fermentation. This pellicle formation was attributed to by Brettanomyces and oxidative yeasts, and this corresponded with a decline in acetic acid bacteria populations. The researchers hypothesized that the formation of the pellicle was the cause of the reduction in acetic acid bacteria populations [7].

Acetobacter

We may also be able to glean some insight from studies done on Acetobacter pellicle formation during vinegar production.

Acetobacter spp. produce homo and heteropolysaccharides (polysaccharides consisting of one type of sugar or more than one type of sugar, respectively [8]) that attach to the surface of the cells (capsular polysaccharides - CPS), as well as polysaccharides that are secreted into the medium in which they live (extracellular polysaccharides, or exopolysaccharides - EPS). CPS is the mechanism that allows pellicle formation in Acetobacter as the cells tightly associate to one another via CPS [3].

The exact composition of the CPS polysaccharides within Acetobacter pellicles varies between not only species of Acetobacter and another acetic acid bacteria genus called Gluconacetobacter, but also strains within species. For example, Gluconacetobacter xylinus produces a homopolysaccharide pellicle consisting of cellulose, A. pasteurianus subsp. Lovaniensis produces a heteropolysaccharide pellicle consisting of glucose and rhamnose, and A. tropicalis produces a heteropolysaccharide pellicle consisting of glucose, galactose, and rhamnos. The ratios of the different sugars in heteropolysaccharides was shown to vary from strain to strain of A. pasteurianus. As pellicle formation increases, the structure of the polysaccharides that make it up do not change [3]. The fact that different species/strains use different types of sugars and different ratio or sugars for pellicle formation might partly explain some of the visual differences between pellicles.

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 [3].

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 [3].

Pellicle Appearance as a Microbe Identifying Indicator

Pellicle formed by what is likely identified to be Pichia anomala. Photo provided by Nick Rutkowski.

An often asked question by homebrewers is can a contaminating or wild caught microbe be identified based on the appearance of a pellicle. This is a difficult exercise for a few reasons.

  1. 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 created a pellicle based on what the pellicle looks like is currently impossible.
  2. The number and size of bubbles in a pellicle is largely dependent on the amount of trapped CO2, and thus is not an identifying feature.
  3. 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 protein 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.
  4. Identification of microbe species and sometimes even genus under a microscope based on cell morphology alone is not enough to be certain of that identification (see 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.
  5. DNA analysis of a microbe is the only way to reliably identify the species of a microbe. This is why 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 [9].

Handling/Racking

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 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.

Pellicle Formation In Bottles

Pellicle formation in a bottle a few days after packaging; courtesy of Dan Pixley

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 its own during aging or when the bottle is refrigerated or disturbed. Other than for aesthetics, there should be no concern if this happens.

Pierre Tilquin of 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 [10]. Gently tipping the bottles or even just standard handling will also dissolve the pellicle without harming the beer.

See also Bottle Orientation.

Scientific Terminology

As with some things in science that are not greatly explored, terminology isn't always agreed upon or fully established, and thus researching such a topic without a lot of prior knowledge can be challenging. Pellicles are a good example of this.

It is sometimes stated that a pellicle is a type of biofilm. However, there have been objections made to defining a pellicle as a type of biofilm. Biofilms are well studied and understood in science. Many microbiology textbooks define a biofilm as an aggregate of microorganisms where the cells adhere to each other on a solid surface [11]. Pellicles do not adhere to a solid surface, and are said to form at the "air-liquid interface" (surface of beer). As such they do not fit the definition of a biofilm in the strictest sense. However, the IUPAC, which is an international federation of National Adhering Organizations that works to standardize nomenclature in chemistry and other fields of science, defines a biofilm as an "aggregate of microorganisms in which cells that are frequently embedded within a self-produced matrix of extracellular polymeric substance (EPS) adhere to each other and/or to a surface [12]." According to the IUPAC, the "and/or" part of the definition allows for pellicles to be defined as biofilms since the cells adhere to each other.

Biofilms are extremely diverse and abundant in nature. Examples of biofilms in the classical sense include dental plaque and the green film produced by algae that covers stones in water streams [13]. Biofilms are encountered in brewing in the form of contaminating microorganisms and poor cleaning/sanitation techniques. Biofilms are a common source of persistent brewhouse infections and can be resistant to the actions of many cleaning and sanitizing agents [13][14]. Pellicles in beer do not attach to a solid surface, they appear on the "air-liquid interface" (the surface of the beer) [14]. To make matters even more confusing, the two established definitions of a "pellicle" in biology only include the outer boundary of a protozoa cell [15], and the protein film that forms on the surface of teeth [16].

The usage of the term "biofilm" has been used to describe the layer of film that covers sherry known as "Flor" [17][18]. The word "pellicle" generally isn't used, although it has appeared on occasion when referring to sherry flor in the 1960's [19], and as least once in brewing science from the 1960's [20]. 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: [4][21][22][1][23][24][25]). 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." [26] 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 [27].

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.

See also:

Images

See Also

Additional Articles on MTF Wiki

External Resources

References

  1. ↑ 1.0 1.1 Biofilms: the matrix revisited. Steven S. Branda, Ashild Vik, Lisa Friedman and Roberto Kolter. Jan 2005.
  2. ↑ 2.0 2.1 2.2 Beer Microbiology – What is a pellicle? A PhD in Beer blog. Dr. Matt Humbard. 01/30/2015. Retrieved 04/26/2015.
  3. ↑ 3.0 3.1 3.2 3.3 3.4 Pellicle of thermotolerant Acetobacter pasteurianus strains: Characterization of polysaccharide and induction patterns. Perumpuli Arachchige Buddhika Niroshie. 2014-09-30.
  4. ↑ 4.0 4.1 Gram-negative bacteria can also form pellicles. Armitano J, Méjean V, Jourlin-Castelli C. Environ Microbiol Rep. 2014 Dec.
  5. ↑ Brewing Sour Beer at Home. The Mad Fermentationist Blog. Michael Tonsmeire. 11/06/2009. Retrieved 02/28/2015.
  6. ↑ Jester King Blog on Pellicles. Retrieved 09/24/2015.
  7. ↑ 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.
  8. ↑ Heteropolysaccharide. Encyclopedia Britannica. Retrieved 09/05/2015.
  9. ↑ Conversation with Richard Preiss and others on MTF. 09/24/2015.
  10. ↑ Conversation with Pierre Tilquin and Raf Soef on MTF. 01/08/2016.
  11. ↑ e-Study Guide for Brock Biology of Microorganisms, textbook by Michael T. Madigan.
  12. ↑ Pure and Applied Chemistry. Volume 84, Issue 2, Pages 377–410, ISSN (Online) 1365-3075, ISSN (Print) 0033-4545, DOI: 10.1351/PAC-REC-10-12-04, January 2012.
  13. ↑ 13.0 13.1 Center For Biofilm Engineering. Montona State University. Retrieved 09/05/2015.
  14. ↑ 14.0 14.1 Conversation about Pellicles on MTF. 08/20/2015.
  15. ↑ Biology of Protozoa. D.R. Khanna. Discovery Publishing House, Jan 1, 2004. Pg 38.
  16. ↑ Wikipedia. Dental Pellicle. Retrieved 08/23/2015.
  17. ↑ 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.
  18. ↑ 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.
  19. ↑ On the pellicle formation by “flor” yeasts. Cantarelli C, Martini A. Antonie Van Leeuwenhoek. 1969.
  20. ↑ SIGNIFICANCE OF THE USE OF HOPS IN REGARD TO THE BIOLOGICAL STABILITY OF BEER: I. REVIEW AND PRELIMINARY STUDIES. R. M. Macrae. 1964.
  21. ↑ 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.
  22. ↑ 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.
  23. ↑ Genes involved in matrix formation in Pseudomonas aeruginosa PA14 biofilms. Lisa Friedman andRoberto Kolter. Dec 2003.
  24. ↑ 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.
  25. ↑ Bacterial Biofilms. Tony Romeo. Springer Science & Business Media, Feb 26, 2008. Pg 7.
  26. ↑ 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.
  27. ↑ Discussion of this wikipage on MTF. 09/06/2015.
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