Pellicle

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 (the classification of pellicles as a type of biofilm is up for debate. See Scientific Terminology). It consists of an aggregation of cells, proteins, and polymers . The "bubble" formations are caused by trapped CO2 beneath the pellicle film. Pellicles are often formed by Brettanomyces, Acetobacter , 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 . It can also be formed by Saccharomyces in rare occasions (probably only wild species), and it might be possible for Lactobacillus and Pediococcus to form a pellicle (according to Matt Humbard; see reference) .

Characteristics

Introduction

Pellicles form when the surface of the beer is exposed to even small amounts oxygen 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 . Yet another hypothesis that is often cited is that the pellicle protects the beer from oxygen itself , however arguments for this hypothesis are 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.

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 ) 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 .

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 . 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 ..

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 .

Pellicle Appearance as a Microbe Identifying Indicator

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 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 genre of microbes. Unfortunately, such a study has not been performed that we know of. Thus, it is 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 .

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 fermentation 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.

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 . Gently shaking the bottles so that the pellicle dissolves into the beer will also destroy the pellicle without harming the beer.

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 . 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 ." 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 . 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 . Pellicles in beer do not attach to a solid surface, they appear on the "air-liquid interface" (the surface of the beer) . To make matters even more confusing, the two established definitions of a "pellicle" in biology only include the outer boundary of a protozoa cell , and the protein film that forms on the surface of teeth .

The usage of the term "biofilm" has been used to describe the layer of film that covers sherry known as "Flor" . The word "pellicle" generally isn't used, although it has appeared on occasion when referring to sherry flor in the 1960's . 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: ), 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 .

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, and understand that brewing terminology does not usually overlap with scientific terminology in regards to pellicles.

Images

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See Also

Additional Articles on MTF Wiki

• Pediococcus
• Brettanomyces
• Lactobacillus
• Saccharomyces

External Resources

• Beer Microbiology – What is a pellicle? A PhD in Beer blog. Dr. Matt Humbard. 01/30/2015.
• Discussion on MTF, 09/06/2015.
• Thread on MTF demonstrating the difference in appearance of bottle pellicles between 4 different The Yeast Bay Brett blends. 11/13/2015.