The Brewing Science of Brettanomyces

The British Fungus

Brettanomyces is a genus of non-Saccharomyces yeast of importance to the brewing industry. It was named by NH Claussen, the Director of the Carlsberg laboratory, in a paper published in the Journal of the Institute of Brewing in 1904¹. He had identified from English stock ales the organism responsible for “both the condition of these beers and their flavour”. He named it Brettanomyces (British fungus) due to its close connection to the British brewing industry. Stock ales underwent a long maturation and for them to condition a true secondary fermentation, by a secondary yeast, was necessary. Using Hansen's pure yeast culture method for producing stock ales obtained poor results as the pure cultures were free of Brettanomyces. Demand for stock ales was however in terminal decline by the time of Claussen's paper. Mild or running beers served soon after brewing replaced them, and like the cask beers of today they came into condition though the action of Saccharomyces on residual or added priming sugars without the need for a secondary fermentation by Brettanomyces.

Adding Brettanomyces to bring about a secondary fermentation was however used in Courage Imperial Russian Stout, and in Belgium, the method continues to be used in the production of the Trappist beer Orval. It also plays an important role in Belgian sour beer production, and recently interest in using this yeast has grown, to the extent that some breweries now use it for primary fermentation. 

Orval Brett.

Current Classification

As with most micro-organisms the Brettanomyces species have been reclassified as scientific understanding has advanced. Brettanomyces had only been known to reproduce asexually, but the discovery of ascopsore formation in Brettanomyces by Van der Walt and Kerken² led to its reclassification of the genus as Dekkera. Species have also been reclassified which adds to the difficulty of keeping up with current nomenclature. At the present time there are four species³ in the genus, two of which have been seen to form spores:

The spore formers are Dekkera anomala (into which the species claussenii has been merged) and Dekkera bruxellensis (into which the species lambicus has been merged) and the non-spore-formers are Brettanomyces custersianus and Brettanomyces naardenensis. As it is likely that the genus will be reclassified back to Brettanomcyes⁴, and it is the most commonly used term by those that brew with these organisms, I shall continue to use it in this article. Genetically Brettanomyces shows a marked degree of diversity, which will no doubt keep the taxonomists busy. Researchers looking at B. bruxellensis have found large variation in chromosome size and number⁴ and that it has a core diploid genome however triploid strains are common⁵. 

Vegetative Brettanomyces cells

Spore forming D.bruxellensis cells

Non-Saccharomyces yeast

As a non-Saccharomyces yeast its metabolism differs from that of normal brewing yeast strains. It is able to use a wider range nitrogen source, which gives it an advantage over Saccharomyces spp. in nutritionally depleted environments such as beer⁴. This has also been used as the basis for selective media such as lysine agar⁶.

Brettanomyces will ferment glucose faster in the presence of oxygen than it will anaerobically. This was named the Custers effect after the researcher that discovered this in 1940⁷. It will also produce acetic acid at the same time – one of the reasons that Brettanomyces is often associated with sour beer. However in an anaerobic environment this does not occur so it is perfectly possible to make beers using Brettanomyces that are not sour. As many beers fermented using Brettanomyces are aged in wooden barrels or vats, it is worth noting that the larger the vessel, the less oxygen ingress there is over time per unit volume of beer⁸, which perhaps explains the fondness the old porter brewers had for giant vats.

Carbon source

One of Brettanomyces best known characteristics is its ability to attenuate beer further than normal brewing yeast by utilising dextrins that they cannot ferment. Andrews and Gilliland of the Guinness laboratory described how a secondary attenuation limit was determined after fermentation with Saccharomyces by using a culture of Brettanomyces⁹. It can produce both an intra- and extra-cellular α-glucosidase, both of which can potentially hydrolyse dextrins with greater than 9-12 glucose units, producing glucose and the next lower dextrin¹⁰. In a research project carried out at Heriot-Watt University, Chad Yakobson found that glucose levels could even increase during fermentation as this dextrin breakdown occurs¹¹.

Sugar utilisation is highly variable within the genus^9,11. The slow growth of Brettanomyces means that in mixed fermentations it is out competed by Saccharomyces and cannot take full advantage of the wort glucose and maltose. In pure culture Brettanomyces fermentations it is found that most strains can however utilise both these sugars, though this does to some extent repress dextrin utilisation and limit super-attenuation¹². Some strains produce a β-glucosidase which allows them to ferment the wood sugar cellobiose. β-glucosidase is also involved in the bio-transformation of hop compounds leading to the release of glycosidically-bound flavour active volatile compounds¹³.

Esters and esterases

Researchers looking into the complex world of lambic fermentations have found that all the Brettanomyces isolates they examined showed esterase activity not found in Saccharomyces. The esterases found in Brettanomyces do not only break down esters, they also have ester-synthesising activity¹⁴. In lambic beers after Brettanomyces growth, high levels of ethyl acetate (fruity, solvent flavour) and ethyl lactate (fruity, creamy) were found, but very low levels of iso-amyl acetate (banana). A similar situation is found in beers that have a secondary Brettanomyces fermentation after primary fermentation with Saccharomyces. The ester profile of the beer is changed, something that will not happen if Saccharomyces is used for conditioning. Other esters, such as ethyl caproate (pineapple) and ethyl caprylate (fruity, winey, waxy) are also associated with the flavour of lambic beers and are likely to be produced by Brettanomyces¹⁵.

Phenolic compounds

Some of the characteristic flavour compounds produced by Brettanomyces are phenols¹⁶. Flavour active compounds produced include 4-vinylguiacol (clove flavour), 4-vinylphenol (barnyard, medicinal, plastic) and 4-vinylcatechol (plastic, bitter, smoky). Unlike Saccharomyces, Brettanomyces has the enzyme vinylphenol reductase which will reduce these compounds to their corresponding ethyl derivatives³ : 4-ethylguiacol (spicy, clove), 4-ethylphenol (medicinal) and 4-ethylcatechol (medicinal, barnyard). In beer the concentration of 4-ethylphenol is lower than 4-ethylguaicol, though in wine the situation is reversed¹⁷.

Other flavour compounds

Mousy off-flavours can be caused by Brettanomyces due to production of ETHP (2-ethyltetrahydropyridine) and ATHP (2-acetlytetrahydropyridine). The amount produced is strain specific, though the presence of oxygen stimulates their production⁴. During anaerobic growth, Brettanomyces will produce a number of fatty acids, some of which have cheesy or goaty flavours such as isovaleric acid, caproic acid and caprylic acid^3,11,15.

As can be seen not all the flavours produced by Brettanomyces are desirable. A number of factors influence which flavours are produced, these include the strain, pitching rate, wort composition and fermentation conditions^11,12. Flavours can also change over time, as for example fatty acids are esterified. When Brettanomyces beers are matured for a long time they will require regular sampling to determine if the desired flavour profile has developed. Some breweries will then use pasteurisation when packaging to prevent further flavour changes¹².

Fermentation

Brettanomyces can be used to carry out fermentations in a number of different ways. When using B. clausenii (WLP645) for secondary fermentations in a way similar to that first described by Claussen, I found that the gravity of the beer would drop by another two degrees Sacch (half a degree Plato) and the fermentation would be complete after two months at room temperature. As many brewers will be aware, a very small amount of Brettanomyces can have a large effect and the recommended pitching rate for secondary fermentations ranges from 100 to 2,000,000 cells per ml!¹² I used approximately 500,000 cells per ml. 

Pure Brettanomyces fermentations are slow to start due to its inability to produce glycerol⁴ but will still be completed within weeks with most strains giving an apparent attenuation of 80-90%. The lack of glycerol production can also make pure Brettanomyces beers taste thinner than normal. As the Brettanomyces has easier access to nutrients and is less stressed when no competing organisms are present, the flavour development has been reported as more muted than that found when it is used for secondary fermentations – though in my experience this is not always the case. Pitching rates similar to that of Saccharomyces are recommended for primary fermentation, and some brewers oxygenate as normal whereas others to restrict oxygenation to stress the yeast and increase production of flavour compounds. Some brewers ferment at around 20°C, though others will allow it to rise as high as 27-28°C to promote ester formation¹².

In the “spontaneous” fermentations of lambic and similar beers it can be eight months before the Brettanomyces starts to out-compete the Saccharomyces, but once established, it can continue to grow for years¹⁸. Growing in mixed cultures with lactic acid bacteria leads to increased attenuation^9,19, as does adding lactic acid to the wort¹¹.

Despite its long history in brewing, the full potential of making beer with Brettanomyces is only now being found and there is still much to learn about this interesting organism.

1. Claussen, NH. (1904). On a Method for the Application of Hansen's Pure Yeast System in the Manufacturing of Well-Conditioned English Stock Beers. Journal of the Institute of Brewing, Vol 10, Issue 4, 308-311.
2. Van der Walt, JP and Kerken, AE. (1960). The Wine yeasts of the Cape part IV – Ascospore formation in the genus Brettanomyces. Antonie van Leeuwenhoek International Journal. Vol 26, Issue 1, 292-296.
3. Crauwels, S. et al. (2015). Brettanomyces Bruxellensis, Essential Contributor in Spontaneous Beer Fermentation Providing Novel Opportunities for the Brewing Industry. Brewing Science. Vol 68, 110-121.
4. Steensels, J. et al. (2015). Brettanomyces yeasts - From spoilage organisms to valuable contributers to industrial fermentations. International Journal of Food Microbiology, issue 206, 24-38
5. Borneman, A.R. et al. (2014). Insights into the Dekkera bruxellensis Genomic Landscape: Comparative Genomics Reveals Variations in Ploidy and Nutrient Utilisation Potential amongst Wine Isolates. PLOS genetics. Vol 10, Issue 2, 1-11.
6. Morris, E.O. and Eddy, A. A. (1957). Method for the measurement of wild yeast infection in pitching yeast. Journal of the Institute of Brewing. Vol 63, Issue 1, 34–35.
7. Custers, M.J.T. (1940). Onderzoekingen over het Gistgeslacht Brettanomyces. PhD thesis, University of Delft.
8. Sparrow, J. (2005). Wild Brews: beer beyond the influence of brewer's yeast. Brewers Publications, Colorado. p197.
9. Andrews, J. and Gilliland, R.B. (1952). Super-attenuation of beer: a study of three organisms capable of causing abnormal attenuation. Journal of the Institute of Brewing. Vol 58, Issue 3, 189-196.
10. Shantha Kumara, H.M.C. et al. (1993). Localisation and characterisation of α-glucosidase activity in Brettanomyces lambicus. Applied and Environmental Microbiology. Vol 59, no. 8, 2352-2358.
11. http://brettanomycesproject.com/
12. Tonsmeire, M. (2014). American Sour Beer. Brewers Publications, Colorado.
13. Daenen, L. et al. (2007). Screen and evaluation of the glucoside hydrolase acitivity in Saccharomyces and Brettanomcyes brewing yeasts. Journal of Applied Microbiology, Vol 104, 478-488.
14. Spaepen, M. and Verachtert, H. (1982). Esterase activity in the genus Brettanomcyes. Journal of the Institute of Brewing. Vol 88, issue 1, 11-17.
15. Spaepen, M. et al. (1978). Fatty acids and esters produced during the spontaneous fermentation of lambic and gueuze. Journal of the Institute of Brewing. Vol 84, issue 5, 278-282.
16. Licker, J.L. et al. (1999). What is “Brett” (Brettanomcyes) flavor?: a preliminary investigation. In Chemistry of Wine Flavor; Waterhouse, A. et al. ACS Symposium Series. Washington.
17. Schifferdecker, A.J. et al (2014). The wine and beer yeast Dekkera bruxellensis. Yeast, 31: 323-332.
18. Van Oevelen, D. et al. (1977). Microbiological aspects of spontaneous wort fermentation in the production of lambic and gueuze. Journal of the Institute of Brewing, Vol 83, issue 6, 356-360.
19. Martens, H. et al. (1997). Microbiological aspects of a mixed yeast-bacterial fermentation in the production of special Belgian acid ale. Journal of the Institute of Brewing, Vol 103, issue 2, 85-91.

This article apperared in the December 2016 issue of Brewer and Distiller International magazine. It was based on at talk I gave at the Carnivale Brettanomyces. Thanks to my colleagues Chris Rice and Chris Raleigh for the pictures of the cells, the picture of the D.bruxellensis is the first one I've seen of this bug when it's formed spores.

The UK's first craft beer pub

An invitation to the opening of the UK's first craft beer pub was something I could not turn down. I've always wanted to time travel, and going back to when this momentous event occurred would be fascinating. Though come to think of it you'd probably have to go back a further ten years so you could trash out exactly what you mean by craft beer first anyway.

Sadly no TARDIS was involved, the invitation was to the opening of Goose Island's (ABInBev) first pub in London, the Vintage Ale house in Balham. It's more a bar than a pub, having no hand pumps or carpet, and being in a shop unit sized space. Interestingly, the guy who founded Goose Island was there and he said they normally have a cask ale on at their brewpub in Chicago. It's nice to think that even in barbarian lands it's possible to receive the sacrament of the one true living beer. I've always quite fancied a visit to Chicago, though admittedly mainly to visit the Martyrs' Monument.

With no cask beer available in Balham I stared on the keg IPA, which was the best I've had in a long while. It seemed to have got back some of the zing it's been missing since production moved to a bigger brewery. I discussed this with one of the Goose Island people and he said to try and match the flavour they actually had to increase the hopping rate when they upscaled production to a new plant.

Next we were given a beer cocktail, a mimosa, made with Sofia, orange juice and (I think) Gran Marnier. I was very nice, but tasted nothing like beer, and I was wary about how strong it might be so didn't finish it. I wanted to save as much of my sadly reduced alcohol capacity for the coming main event, a five course beer and food matching meal:

The beer and food matching was by all accounts a great success, with the bloke who founded Goose Island even coming over to tell the guy who'd organised it how it was one of the best beer dinners he'd been to. It was mostly wasted on me though.

I mean the food was great, and there was some nice beer, but I'd have quite happily stuck with the beers I liked best (the IPA and Matilda) thoughout the evening. Matilda is Goose Island's take on Orval, and very good it is too. As Brettanomyces is one of my obsession it was interesting to hear the Goose Island people talk about, as it featured in the Matilda and some of the other beers. They didn't get much wrong either. Intentional use of Brett. in breweries is clearly growing. And come to think of it unintentional probably is too.

It was a cracking do Goose Island put on, but as I was ligging I've no idea how much it would cost to go there as a paying punter. Probably more than I'd fork out to be honest, I am quite tight.

SIBA SE AGM 2016

It was the SIBA South Eastern AGM last week. There were a lot of people at the meeting, including the managing director Mike Benner and the operations director Nick Stafford. The meeting was well attended, and there was a lot to discuss so at times it felt a bit rushed and chaotic.

Nick Stafford spoke on the recent drop in the money that Enterprise Inns will pay under SIBA's Beerflex scheme. Rather than the negotiated price drop they mentioned in emails it was clear that the price drop was imposed by Enterprise, and also that he himself didn't think the prices paid by Beerflex were worth it. He said Beerflex now only accounted for 5% of the sales from his own brewery.



Mike Benner also spoke, a slightly surreal intervention particularly sticks in mind. After saying that he was not here to influence decisions taken by the members he argued at length against a proposal for members to have control over SIBA's decisions that affect them financially, leading one brewer to interject with the impressively sarky comment "That's you sitting on the fence then?". Brenner's arguments were that all decisions were overseen by 24 brewing representatives. This got me thinking about the gap between small and very small breweries, which Hardknott Dave sums up very well on his blog. He also points out why most SIBA members' interests are different from that of the Small Brewer Duty Reform Coalition (SBDRC), so I was quite surprised to see a proposal from Hogsback's Rupert Thompson backing their case get voted in. Turkey's voting for christmas I thought.



So far SIBA seem very opposed to the SBDRC and it will be interesting to see how things pan out at the national AGM. Now the practical benefits of SIBA membership are much diminished their main use seems to be for political lobbying.

Continuous Beer Stabilisation

After the excitement of discussing deliberately hazy beer  the talk at the IBD section AGM was on Continuous Beer Stabilisation. To go with their new cross flow filter Fuller's have got a new bit of kit for removing haze forming polyphenols from beer.

Non-biological hazes in beer are mainly caused by the presence of proteins and polyphenols.The polyphenols can be removed by treating the beer with PVPP. In traditional systems the PVPP is added to the beer as a powder and filtered out in a batch process. The Continuous Beer Stabilisation system uses PVPP in cassettes so there is no movement of the PVPP, and no need for cleaning filter screens. The cassettes hold the PVPP but allow beer to pass. They are stacked in columns (three or six) and the beer flows through the cassettes and into a central shaft until the PVPP needs regenerating by cleaning with caustic.

Behold the beer stabilisationmatron

The number of columns means the system can work continuously as some columns can be working whilst others are on standby or being cleaned. The degree of polyphenol removal can be controlled by adjusting the flow rate of the beer. It can also be used as a batch system (as indeed it is at Fuller's). The ability of operate continuously means the equipment is smaller in size than if it was designed for working in batches.

Gripping stuff, eh? And after the thrill of the talk there was still time for the all important networking. Which reminds me, I got a tantalising hint of gossip about ... but I will say no more. Unless it's revealed that what I suspect is true, in which case I will say "knew that".

Pub crawl of East London

On Saturday I went on one of Jimmy's ticking expeditions. It started in East Central London, worked its way further East and then back to Waterloo.

First stop was the Hoop and Grapes. This was a Shep's pub, not my favourite brewery but some of their beers are growing on me. I went for a Whitstable Bay Pale Ale, which was perfectly pleasant.  

Sadly it was about the only good thing about the pub.

After I'd arrived they turned on some awful music, and there weren't really any redeeming features about the pub, so using the scientific scoring system it gets just one point.

 The next stop was the Old Red Cow, and altogether more pleasant place.

 I had a pint of Twickenham's bitter in here.

Jim was confused by the board saying they had a Session IPA at 4.7% ABV, as something at that strength is clearly not a session beer. I had to explain that Americans had imported the British term session beer and adapted it to their own market by making it stronger. Some British brewers had now adopted the American usage which doesn't make any sense in Britain, as let's face it you can't have a session on beer of that strength.

The Old Red Cow was an all together much better place than the Hoop and Grapes, and scored a respectable five:

On the way to the next pub was passed a sign advertising "The Brewery". At first I wasn't sure if this was an old closed brewery, or a new recent start up. When we got closer I saw it was the former, and the penny dropped about where we must be. "Hang on a minute, are we on Chiswell Street?" I said, and sure enough we were, standing opposite the old Whitbread brewery.

Not that there was any need to locate myself as I was just following Jim round, but this is possibly the closest I've come to beer nerdery actually being useful for anything.

 We were heading to the Crown and Shuttle, a large pub with a two story smoking shelter made of scaffolding built out the back. 

I had the Dark Star best. 

We quite liked this one, shame the bogs smelt of piss.

Williams Ale and Cider House was next. At first I went for the mild, as you don't see milds often. Sadly it had a touch of the Sarsons about it so I had to switch it for a porter.

 I liked some of the old adverts they had on the walls. Advertising that you have equipment is a rather odd way to promote your brewery, which I guess is why all breweries decided to settle on saying they use the finest malts and hops instead.

 The adverts brought the score up slightly, but only to a lowly two:

The King's Stores followed, which had a customer possibly the best beard of the day. The rise of bearded youths has led to the scientific scoring system having to be adjusted slightly, but that's science for you. To score "Beard or weird" you really need a customer with a big enough beard to look like god, or a proper pub weirdo, not just a kid with a goatee. In future it's likely the category will therefore be re-named "God or odd".

 I had the By The Horns bitter.

I wasn't overly taken with the pub but it managed to get a four:

There was a woman outside in a coat with umpteen pockets being photographed, though by the time we got there the photographer had switched from her proper camera to using her phone. If they'd been inside I'm sure it would have counted as a special feature.

I don't think the situationists would approve

Though they might have liked thes

 It was an old favourite of mine next, the Pride of Spitalfields.

The ladder's still in the bog, though I'm sure when I were a lad it was propped up in the corridor outside.

And Mary's Pantry is still there, despite the fact I've never seen them sell food and Mary's been dead for years.

The Pride was as good as every, though I didn't stick with the name and had an ESB. 

It was the first pub we'd been to that had a carpet, which not only helps deaden the sound but adds to the cosy atmosphere that's so important in a pub.

Why don't more pubs have these?

The pub was so good we stopped for two here, following up the ESB with a Truman's porter. I was a bit suspicious this calling for a second beer meant my friends were having fun. I had to make clear we were on a scientific research trip and not here to enjoy ourselves. Despite a well deserved nine for the Pride it was time to move on.

The next pub on the list, the Dispensary, was shut as the management clearly prefer to spend Saturdays having conjugal relations with a close relative rather than opening the pub. So we went on to Turner's Old Star.

We were asked by a bloke there why the four of us were drinking halves, as he'd never seen that before. We did explain the mission we were on, but he was drinking wine anyway so was a fine one to talk. 

After that we were close to the river at the Town of Ramsgate. I'd been to this one before and wasn't that taken with it.

 But it still scored a very respectable six.

Our next port of call, the Draft House in Seething was not so good though. In fact it had all the charm of a ferry waiting room.

I know we had to be there for research purposes, but I can't for the life of me think why you'd go there by choice. Each to their own I suppose. Still, the beer was fine, Great Heck Voodoo Mild if I remember rightly.

 Then it was on to the Old King's Head.

It had a ladder on the way to the bogs, but I don't think it counts as a special feature like it does in the Pride.

A slight detour to The Rake followed, as one of us needed to tick it.

It was a salted caramel milk stout here. I'm not sure why caramel has to be salted nowadays, but the beer was fine. 

The Rake has the special feature of having brewers' autographs on the walls, so here's some pictures I prepared earlier:

It was a curry after that, then we were into the home straight.

The Lord Clyde was a great pub, and could really have done with a beardy weirdy to boost its score, a very solid six.

Last stop was the King's Arms for an Adnams bitter. 

It's a bit of an old favourite, and obviously not just with me, as it got an impressive eight.

It was definitely home time after that.