A Ph.D. in Beer - A Study of Beer and Fermentation Science

Beer Microbiology – What is a pellicle?

Advertisements

I am a microbiologist by trade, I have a Ph.D. and have been brewing for over 10 years at this point. A large portion of this blog is dedicated to my own homebrew efforts but sprinkled throughout are articles like this one, where I isolate a biological or chemical aspect of the beer and explain it and if possible analyze it myself. This post is going to be about pellicles, what causes them, what organisms can make them, and what exactly they are and possibly their function. As with all my science posts, I hope you find this helpful or interesting, otherwise there will be a saison post in a few days for your digestion. Any comments, additions or corrections, leave a comment at the bottom. Whenever I publish one of these someone comes along and fills in important gaps I’ve left. I will edit the article and give you credit for the edit. I look forward to your input.

What is a pellicle?

A pellicle is the term brewer’s use for the layer that forms on top of the wort / beer in the fermenter (liquid-air interface). It can appear to be uneven or appear to have “bubbles” in it. It can also appear ropey or like a spider web. I’ve seen ones that are slimy looking or looks like small dry patches of while floating on top of beer. I’ve included a few photos of pellicles from my own fermentations found in various places within this blog. You can use google to find all the permutations a pellicle can take.

Brettanomyces pellicle

If you are a brewer that leans toward the funky end of the spectrum (you brew with Brettanomyces or lactic acid bacteria), you probably have seen a pellicle forming in your fermenter. So, what is it? In the parlance of microbiologists, it is a biofilm. A biofilm is basically a community of microorganisms that are connected to one another through the use of an extracellular matrix (material outside of the cell). The extracellular matrix can be composed of just about anything…proteins, long chains of sugars, live (or dead) cells, or other materials found in the surrounding environment. This network of cells and materials is there to support a structure. In the case of the pellicle so organisms can “float” on top of the liquid. Other biofilms can form on solid surfaces such as teeth or medical equipment, the majority of scientific research about pellicles or biofilms focus on those forms. A pellicle is a subtype of biofilm, not as stable as the solid surface types but they employ the same mechanisms of attachment to one another (at the cell level). There is some debate about the purpose of a pellicle in fermentation but it is undeniable that it occurs at the liquid-air interface. The leading hypothesis is so the cells can undergo oxidative metabolism, to access the little oxygen present in the atmosphere of the fermenter. This is by far the most reasonable explanation I’ve heard, obviously the cells are trying to get at the atmosphere. It is widely believed that the formation of a pellicle is a sign that oxygen is getting into the fermenter somehow, although most of these stories are speculation and/or anecdotes (believable though, more below). If that is the case, then bacteria or yeast species capable of running respiratory processes would be producing a different profile of metabolites than those individuals that are within the beer/wort itself. In the case of many yeast and bacteria, exposure to oxygen would allow them to make acetic acid in low quantities, which would have a profound effect on sourness of a beer. I’ve also heard the hypothesis that the pellicle protects the beer from oxygen. I think this is likely false. While the organisms would prevent the oxygen in the head space from getting to the beer, it isn’t protecting. Those organisms will use the oxygen to produce various chemicals that may negative affect the beer, so you might be safe from oxidation but not from acetic acid.

As a result of the hypothesized roles and products, pellicles are generally seen by the professional brewing and wine industry as a sign of spoilage (for non-Brettanomyces and sour beers). The majority of literature concerning pellicles is directed at wine producers, basically how to recognize them and what to do when you encounter them, with little else written about them. Brettanomyces bruxellensis is a common wine “spoilage” organisms that can form pellicles within barrels…the general advice for vintners when this happens is to waste the wine and burn the barrel. Some beer makers have a more enlightened view of B. bruxellensis and pellicles in general.

Lactobacillus pellicle.

I’ve included two photos above of beers that I’ve made in the past few years that both formed pretty substantial pellicles. One was a saison that contained B. bruxellensis and the other was a berlinerweiss that had no Brettanomyces in it but contained a Saccharomyces strain and a Lactobacillus species. In this case, it is likely that the Lactobacillus species was forming the pellicle and not the Saccharomyces.

So which organisms can form pellicles? When talking about bacteria, many bacteria that are not strict anaerobes (cannot grow in the presence of oxygen) can form some sort of pellicle. But for the purposes of brewing we are usually talking about only a few different kinds of bacteria.

It is also possible that pellicles are a mixture of species either “intentionally” interact or just get caught up in the super-structure of the pellicle.

Can Saccharomyces form a pellicle?

This question was the ultimate inspiration for this post because I think there is a mild misconception about pellicles in the community in general. Most homebrewers, and probably professional brewers, would immediately say “no” to this question. This is likely due to the highly selected strains used by amateurs and professionals in the brewing process. The reality seems from the literature, that there is nothing stopping Saccharomyces from forming a pellicle. This specifically came up with the recent controversy about WLP644 not being Brettanomyces but rather Saccharomyces. You can read about that controversy here, I will not cover it in this blog. Some people thought that since that strain was forming a pellicle, it couldn’t be Saccharomyces, or at least that was speculated.

And I am not including that tweet to call either of those people out, I respect both of those individuals, but rather to illustrate that this is a question that is out there and this was only one example of someone asking it. Asking questions is how people learn and fortunately there is an answer to this question in particular.

There are a set of genes in yeast called “flocculation” genes or FLO for short. Expression of these genes is what generally controls how well (or poorly) a yeast cell will floc out of solution. And Saccharomyces has them. More specifically, Saccharomyces cerevisiae has the gene FLO11, the gene that is required for pellicle formation. Strains of Saccharomyces cerevisiae that are forced through engineering to over-expresses FLO11 started forming pellicles on glucose media at the liquid-air interface. FLO11 is a cell surface protein. It is decorated by a series of polysaccharides (chains of sugar molecules) that stick out of the cell into the surroundings. FLO11 is required for “pseudohyphal formation” formation which is morphologically distinct cell shape that looks like filaments. It is also believed that FLO11 is required for cell-cell contact, an important feature of a pellicle.

Scanning electron microscopy of yeast colonies forming extracellular matrix (from “Domestication of wild Saccharomyces cerevisiae is accompanied by changes in gene expression and colony morphology” in 2003)
Cells expressing FLO11 (left) and cells not expressing FLO11. FLO11 expressing cells form a pellicle in the test tube (from “FLO11-Based Model for Air-Liquid Interfacial Biofilm Formation by Saccharomyces cerevisiae” 2005)

The expression of this gene alone is enough to form a pellicle and since Saccharomyces cerevisiae species have this gene, they can definitely form a pellicle under conditions where that gene is expressed, which brings me to my last point. If Saccharomyces CAN form a pellicle (or at least has the genetic ability to do so) why DOESN’T it form a pellicle under “normal” use? This is likely the fault of the brewing industry. Pellicles, as stated above, are generally seen as a spoilage state of beer and wine. And since yeast strains have been cultivated by the alcohol manufacturing industry for centuries (millions of generations of yeasts), we have selected for strains that do not express this gene…we have unintentionally evolved Saccharomyces away from this ability. Looking at the current expression patterns for that gene, it is heavily repressed by the presence of glucose. And since wort has a lot of glucose in it, pellicle formation in wort would be rather difficult. Strains with this property would be visibly appealing to brewers.

Bottom line, a lot of micro-organisms can form a pellicle because all that is required to do so is the ability to build this extracellular space to capture other cells. The majority of bacteria and yeast can do this because even microorganisms survive better in communities than as individuals.

So what can we do with this information?

Pellicles are a cool side effect of brewing sour / funky beers. I got a pellicle when I made my hot sauce a few months back, that was fun to see (photo below). Seeing a pellicle makes me smile for some reason. But they are mostly an aesthetic property of the brew rather than specifically indicative of anything expect perhaps a little oxygen in the fermenter. There are a few experiments someone (perhaps me) can do someday to examine if pellicle formation can be completely blocked even in an organism that may naturally form a pellicle such as a Brettanomyces species. One thing that always springs to mind when I see a pellicle is “has oxygen gotten into the fermenter?” People claim that plastic fermenters (buckets or better bottles as examples) are more permeable to oxygen than glass carboys…it is believable but people aren’t actually measuring dissolved oxygen at the homebrew level (are they?) so it is all based on people’s assumptions, prejudice, and taste perceptions. It might be a worthwhile experiment to brew the same beer and put half in a glass carboy, half in a plastic carboy and purge both with CO2 and see if a pellicle forms in one or both of the carboys. Sound doable. Would sparging with CO2, or actually activating all the oxygen out of a glass jar prevent pellicle formation? An anecodote (which isn’t evidence but nevertheless), I decided to take some WLP644, the Saccharomyces that forms a pellicle, and put it into a 6 gallon glass carboy. I sparged the liquid with CO2 and blew out the headspace with more CO2 and put an airtight cap on it. It has yet to form a pellicle after two weeks. This wasn’t a very well thought out test or experiment but I’ll think of a better way to do it and try again in the future.

Author’s Note

I have updated I’ve received a lot of feedback through Facebook, twitter, and in the comments section of this blog about this post and I wanted to express my appreciation to everyone that helped make this post more accurate and helpful. It is always my goal to be as accurate as possible and to provide interesting and useful information that might not be covered in the “beer blog” community.

One suggestion I got that I did not address was to include a section on flor, as in sherry flor. This is another reason why I love this blog, in danger of revealing my ignorance, I had never heard of this. After doing some research on that phenomenon, I decided it would be too off topic to include here but I learned so much reading about that. So thanks for reading my blog and writing me back about the post. I hope the more I do this the better I get but I never want to stop getting your feedback. Thanks again. Please continue to email me errors or omissions.

 

Matt

 

 

“Under the most rigorously controlled conditions of pressure, temperature, volume, humidity, and other variables, any experimental organism will do as it damn well pleases.” –The Harvard Law

Red Sauce Pellicle

 

 

Affiliate Links

 

Click on Product pictures to be directed to Amazon.com to help support the blog.

Advertisements