Pellicle

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Pellicle from The Yeast Bay Brussels Brett Blend; courtesy of Dan Pixley
Lactobacillus pellicle; courtesy of Matt Humbard

A pellicle is a type of biofilm which appears on the surface of beer that 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, Pediococcus, and Lactobacillus, as well as Acetobacter spp [3] and other gram-negative bacteria [4]. It can also be formed by Saccharomyces in rare occasions [2]. Note that pellicles in brewing have not been scientifically defined, and there have been objections to defining them as a type of biofilm. See Scientific Terminology for more information.

Characteristics

Introduction

Pellicles form when the surface of the beer is exposed to oxygen [5] One theory is that the formation of a pellicle allows the organism to access the small amount of oxygen that is present in the headspace of the fermentation vessel. Another theory, and one that may be less accurate according to Dr. Matt Humbard, is that the pellicle protects the beer from other microorganisms [2]. 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. Another myth 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.

Acetobacter

Pellicle formation by microbes found in sour beer such as Brettanomyces and Lactobacillus has not been closely studied. However, we may 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 [6]) 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].

Handling/Racking

Most 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 vessel. 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. 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.

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

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. Many microbiology textbooks define a biofilm as an aggregate of microorganisms where the cells adhere to each other on a solid surface [7]. 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 [8]." 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 [9]. 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 [9][10]. Pellicles in beer do not attach to a solid surface, they appear on the "air-liquid interface" (the surface of the beer). They are also not colonies within an adhesive [10]. To make matters even more confusing, the two established definitions of a "pellicle" in biology only include the outer boundary of a protozoa cell [11], and the protein film that forms on the surface of teeth [12].

The usage of the term "biofilm" has been used to describe the layer of film that covers sherry known as "Flor" [13][14]. The word "pellicle" generally isn't used, although it has appeared on occasion when referring to sherry flor in the 1960's [15]. 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][16][17][1][18][19][20]), 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 [21].

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. Thus, brewers should take care when reading scientific publications.

Images

See Also

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. ↑ Heteropolysaccharide. Encyclopedia Britannica. Retrieved 09/05/2015.
  7. ↑ e-Study Guide for Brock Biology of Microorganisms, textbook by Michael T. Madigan.
  8. ↑ 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.
  9. ↑ 9.0 9.1 Center For Biofilm Engineering. Montona State University. Retrieved 09/05/2015.
  10. ↑ 10.0 10.1 Conversation about Pellicles on MTF. 08/20/2015.
  11. ↑ Biology of Protozoa. D.R. Khanna. Discovery Publishing House, Jan 1, 2004. Pg 38.
  12. ↑ Wikipedia. Dental Pellicle. Retrieved 08/23/2015.
  13. ↑ 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.
  14. ↑ 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.
  15. ↑ On the pellicle formation by “flor” yeasts. Cantarelli C, Martini A. Antonie Van Leeuwenhoek. 1969.
  16. ↑ 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.
  17. ↑ 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.
  18. ↑ Genes involved in matrix formation in Pseudomonas aeruginosa PA14 biofilms. Lisa Friedman andRoberto Kolter. Dec 2003.
  19. ↑ 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.
  20. ↑ Bacterial Biofilms. Tony Romeo. Springer Science & Business Media, Feb 26, 2008. Pg 7.
  21. ↑ Discussion of this wikipage on MTF. 09/06/2015.
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