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.