Yearly Archives: 2014

ITS-PCR of WLP644 Brettanomyces bruxellensis trois

A couple of days ago, Omega Yeast Labs posted an update on their facebook page, where they stated that they’ve done 5.5S rDNA sequencing on White Labs’ WLP644 Brettanomyces bruxellensis trois strain, and results showed that it was actually a strain of Saccharomyces cerevisiae. Since I have some pure cultures of the yeast strain myself, and am currently doing lots of ITS-PCR for work-related things, I decided to test WLP644 myself. Note, I haven’t done any sequencing, just amplification of rDNA (ITS1, 5.8S and ITS2) using the primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′), and then digestion of amplicons using the HaeIII restriction enzyme. The amplicon size of Saccharomyces yeasts is 840-880 bp, while the amplicon size of Brettanomyces yeasts is around 500 bp. Digestion of the amplicon with HaeIII results in a four-band pattern for Saccharomyces cerevisiae (320, 225, 180, 140 bp), and a two-band pattern for Brettanomyces bruxellensis (380, 100 bp). Anyways here is my gel:


First lane 100 bp ladder, second lane WLP644 amplicon, third lane HaeIII-digested WLP644 amplicon, fourth lane S. cerevisiae A62 amplicon, fifth lane HaeIII-digested S. cerevisiae A62 amplicon, sixth lane negative control, seventh lane 100 bp ladder. I unfortunately didn’t include any positive control for Brettanomyces (500 bp amplicon; 380 and 100 bp HaeIII-digested fragments), as I didn’t have any easily and rapidly accessible, but the results should still speak for themselves. It seems very likely that WLP644 is in fact a strain of Saccharomyces (cerevisiae) and not a Brettanomyces yeast (or at least contains Saccharomyces yeast in case WLP644 is a blend, and the single colony I’ve purified and extracted DNA from was Saccharomyces). This is basically a confirmation of Omega Yeast Labs’ results.

Edit: For more (great) information, check out these posts on Sui Generis and Embrace the Funk!

Pham T, Wimalasena T, Box W, Koivuranta K, Storgårds E, Smart K & Gibson B (2011) Evaluation of ITS PCR and RFLP for differentiation and identification of brewing yeast and brewery ‘wild’ yeast contaminants. J Inst Brew 117: 556-568. Full-text available here.

Visiting Rodenbach Brewery

Here is the short report I promised to write of my visit to Rodenbach Brewery in the end of October (as a part of the YSS2014 conference). I took notes on my phone, so I have to apologize that a) I didn’t manage to capture all the details, b) there may be some inaccuracies in my text. Anyways, we arrived at the brewery in the evening after a 1.5 hour bus ride from Gent. Here, Site Manager / Brewmaster Rudi Ghequire welcomed us and took us on a tour around the brewery.


We began by visiting their new brewhouse (finished in 2001), which was supplied by Meura. It is a 250 hL brewhouse, which is capable of doing both decoction and infusion mashing. Below are some pictures of the boil kettle (I think) and the mash filter. We have a similar mash filter in our pilot brewery here at work, but it is of course much smaller (1 hL brewhouse). Rodenbach beers are quite lightly hopped, and the brewery uses hop extract in the boil. After the boil, the wort is cooled and aerated with sterile air. After this it is transferred onwards for primary fermentation.




Rodenbach carries out their primary fermentation in 1 of 10 of their stainless steel cylindroconical vessels (I didn’t manage to capture the size). Primary fermentation is carried out for one week at 20C, after which the beer is cooled to 8C. The wort is pitched with a blend of Saccharomyces yeast and lactic acid bacteria (I’m not sure if they pitch any Brettanomyces?). After fermentation, some of the beer is transferred to wooden vats for aging (more of that later), while the rest is used for blending. The ‘regular’ Rodenbach is 75% fresh beer and 25% aged beer, while the Rodenbach Grand Cru is 33% fresh beer and 66% aged beer. Rodenbach Vintage is 100% aged beer. Below you can see pictures of the fermentation vessel and their airlocks (slightly bigger than the homebrew versions!).



Next we moved on to the bottling hall. Rodenbach is today owned by Palm Breweries, which means distribution is probably better than it has been previously. Rodenbach also contract brews for other breweries (which ones, I do not know). While we were there, we got to see Tripel Karmeliet (Brouwerij Bosteels) getting put into corked 75 cl bottles. Really fascinating to see these traditional beers getting packaged with modern automated technology. They had quite an impressive wall of empty beer cases waiting to be filled in the hall.



After this we made a quick tour through the old brewhouse (which I think is some kind of museum now), after which we walked through the vat cellar. Rodenbach apparently produced geuze previously, by cooling their wort in a coolship, but they stopped doing this in the 1970s. I unfortunately don’t remember the reason, but maybe they though they had more control over their brewing process with their current fermentation methods. Anyways, after primary fermentation, a portion of the fresh beer is transferred to one of 294 wooden vats, made from French oak, where it is allowed to mature for 2 years. During this time, the beer develops stronger lactic and acetic acid notes. The vats stand vertically (which was pointed out as an important characteristic for the flavour), and are not filled completely. This is to allow some air in the headspace, which promotes the growth of acetic acid bacteria. The vat cellar is kept at 15C, which I guess keeps metabolic activity of the microbes at a lower rate. Rudi told us that the beer develops a thick pellicle in the vats, and that a thick layer of beer stone also builds up along the inside of the vat with time. This means that the flavor contribution from the wood decreases with vat age. I don’t remember the life-time of the vats, but I think it might have been around 50 years if I’m not completely mistaken. A batch of Rodenbach beer is blended from several different vats, since they can taste very different. New vats are made at the brewery in their wood-working shop. Before they are used for beer, they are ‘wetted’ for three months to proof the wood.






After the tour, Rodenbach were kind enough to serve us dinner and beer (thank you!). I’m normally not that big a fan of sour beer, but I thought both the regular Rodenbach and the Grand Cru were really refreshing and nice (not too acidic)! No surprise that the great Michael Jackson once said that Rodenbach Grand Cru is “The most refreshing beer in the world”. Thank you Rudi (standing in the background behind my glass in the picture below) for your hospitality!


Back from Gent

I was in Gent during the week attending the 4th Young Scientists Symposium on Malting, Brewing & Distilling, and I had some great fun. There were lots of interesting lectures and I met lots of interesting people. I’ll post some thoughts about the current trends in brewing research in a later post. We also visited Rodenbach brewery, where brewmaster Rudi Ghequire guided us around and was kind enough to invite us to dinner (thank you!). I will also post some pictures and notes I took during the tour in a later post. While in Gent, I also had time for a quick visit to De Hopduvel, where I found some real gems:


All in all, a really nice trip!

Small update

Sorry for not having posted in a while. I’ve been quite busy with other things and have also been abroad (Berlin again). Nothing new on the homebrewing front, since we’re still in the middle of moving to a new brewing location. We have lots of nice plans though, including an update to our brewing kettle and the addition of a whirlpool kettle. More about those once we make them. In Berlin, I didn’t do much beer-related, other than a quick visit to Getränkefeinkost in Friedrichshain. I can definitely recommend a visit as they had a nice selection of German microbrews and craft beer imports (USA, Denmark, UK, Italy etc.). Oh, and they had most the craft beer in coolers! I bought a total of 12 bottles, some of which I had during the trip already. Tomorrow I’m off abroad again, this time to Gent in Belgium for a conference (Young Scientists Symposium on Malting, Brewing & Distilling). The social programme includes a visit to the Rodenbach brewery, which should be very interesting. I will try to post some kind of report from the visit, and will hopefully post more homebrewing-related posts next month as well!

Hops Growing Update

A couple of weeks ago, my first-year hop plants finally started producing cones, and today they were looking like this:



We were hit by some really cold weather (temperature dropped to 0C the previous night), so the cones might not develop properly. If they do develop, I will be brewing a fresh hopped ale at some point in the middle/end of October.

Homebrew: Hopfenschiessen – German Pale Ale

This weekend we will be brewing an American Pale Ale / IPA using only German hops. The German hop growers have been releasing lots of new interesting varieties the last couple of years, and I am really looking forward to trying Hüll Melon and Hallertauer Blanc. I bought some earlier this year, and will debut them in this beer. We decided to blend in some more traditional varieties as well, to balance the fruity tones with some more herbal hop tones. We have a lot of Vienna malt in our inventory, so we decided to use it as our base malt. These will be joined by some Pilsner, Carapils and Crystal 100 malt. We will be trying a new yeast strain as well, Wyeast’s 1217 West Coast IPA, which has been getting lots of good reviews (good attenuation and flocculation, together with a really clean flavour profile). Hopefully everything will go smoothly and the beer will taste good!

[beerxml recipe= metric=true cache=-1]

Homebrew: Sour Ale – Bottling and addition of berries

Yesterday, the Sour Ale I brewed in October 2013 had been in the primary fermenter for almost 11 months. I didn’t have enough patience to wait the full year, so yesterday I also decided to add berries to the beer. I had originally planned to only use blueberries, but I ended up adding 2.2 kg of blueberries and 0.8 kg of raspberries. Before adding the berries, I bottled 5 bottles of the plain base beer, so I can use it for later comparisons. The gravity of the base beer had fallen to 1.010, while its ABV% was 4.2%, and its pH was 3.74. Compared to the pH of other commercial sour ales, this beer had a slightly higher pH (I’ve read that first generation homebrewed sours typically don’t get very acidic). This was reflected in the flavour as well, which was acidic and tart, but quite mild compared to the 3 Fonteinen Oude Geuze I had during the bottling session (I pitched the dregs of the bottle into the homebrew as well). Otherwise the flavour was quite clean (i.e. no major off-flavours such as solvents, higher alcohols, diacetyl, chlorophenolics, etc.) and quite cider-like, with hints of apple and funk. It will be interesting to follow how this one evolves and compares with the ‘berry version’. I was quite surprised at the high FG and hopefully I won’t get any bottle bombs (the beer had been untouched in the fermenter for so long that one would think that fermentation would have ‘finished’). I ended up adding around 300 g berries / liter of beer, so they will hopefully contribute with a lot of flavour! I plan to bottle the ‘berry version’ in around 3 months time.

Here is a picture of the pellicle prior to bottling around 1.7 liters of the base beer:


The beer had cleared really nicely under the pellicle. Unfortunately it was lacking carbonation and was warm for the taste sampling, but despite this it was tasting promising.


I used frozen Finnish berries. I added them frozen directly to the beer.


Here is the 3 Fonteinen Oude Geuze I had while bottling. I added the dregs from the bottle to the homebrew.


Here I am adding the berries through a sanitized funnel.


This is how it looked once all the berries had been added.


Physiology of Finnish Baker’s Yeast (Suomen Hiiva)

While ale yeast and baker’s yeast belong to the same species, Saccharomyces cerevisiae, they have over time been adapted to different functions. Brewing with baker’s yeast is not a very common practice, nor generally recommend amongst brewers, mostly because these strains might not exhibit favorable fermentation characteristics, such as production of desirable flavour compounds, adequate attenuation, and flocculation. Of the recognized beer styles, it is more or less only in Northern European Traditional ales (such as Sahti, Gotlandsdricka and Kvass) that it acceptable to ferment with baker’s yeast. As there is little information published on the physiological characteristics of Finnish baker’s yeast in brewing, I thought I’d post some recent results from my related research.


I performed a series of mini-fermentations (30 ml of wort) on a range of yeast strains earlier this spring for screening purposes, and I included the baker’s yeast from Suomen Hiiva, as well as two common homebrewing strains WLP002 (English Ale) and WLP380 (Hefeweizen IV). These mini-fermentations were performed at two different temperatures, 15 and 20 °C. Fermentation progress was monitored by weight loss, and after fermentation the resulting beer was analyzed for ABV% (Anton Paar Alcolyzer ME), Extract (Anton Paar DMA 5000 M), pH (Anton Paar pH ME), aroma compounds (HS-GC/FID), diacetyl (HS-GC/ECD), and phenolics (HPLC/PAD). A 15 °Plato (OG 1.060) wort was used for all fermentations, and the yeast was pitched at a rate of 2.5 g/L (~ 10 million cells/ml).

What first surprised me was how well the baker’s yeast performed during fermentation. Compared to the other two reference strains presented here, it fermented faster at both temperatures and reached a higher final attenuation. It managed to reach a final attenuation of 85% in just 5 days when fermented at 20 °C. Not bad for an all-malt wort at this strength. So the baker’s yeast can definitely give rise to adequate attenuation. Note, a pure culture of the yeast was used (starting from a similar package as in the picture above), so there was no risk of any lactic acid bacteria contamination, which otherwise is probable when using the yeast directly. The pH values of all beers were quite similar irrespective of yeast strain and temperature, and it seems like the baker’s yeast acidified the wort slightly less than the other two reference strains. In the table below you can find a summary of the Extract, ABV% and pH of the beers. In the figure below you can find a plot of the fermentation progress over time (20 °C squares; 15 °C circles).



I have never actually used baker’s yeast for beer fermentation myself, as I’m not that big of a Sahti fan, but all beers (well mainly Sahtis) I’ve tried have had quite a ester-dominated aroma. Especially 3-methylbutylacetate (isoamyl acetate), with its prominent banana aroma, is very pronounced in, and also an integral part of, Sahti. Hence, it comes as no surprise that, compared to the other two reference strains, the baker’s yeast produced more higher alcohols and esters. It was not only 3-methylbutylacetate that was produced in large amounts (even more than the WLP380 Hefeweizen strain), but also the ethyl esters. Ethyl acetate, with its solvent-like aroma, is typically unwanted at higher concentrations in beer, but the other ethyl esters may contribute a fruity aroma to the beer. In the figure below you can see a summary of the flavour impact (i.e. the concentration of the compound in the beer divided by its flavour threshold. An impact above 1 should affect flavour, while an impact between 0.5 and 1 may affect flavour) of the various higher alcohols, esters, and acetaldehyde in the beers fermented with the different yeast strains (20 °C solid; 15 °C striped). As can be seen from the figure, only 3-methylbutanol (the precursor to the banana ester) of the higher alcohols is close to the flavour threshold. As should come to no surprise, less higher alcohols and esters were produced at a lower fermentation temperature. From these aroma compound results, it is clear that the baker’s yeast will produce a fruity, and maybe even slightly solvent-like, beer. Hence, the yeast would probably be suitable for Hefeweizens and Belgian-style beers, and a slightly lower fermentation temperature is probably recommended.sh_aroma


Finally we arrive at two other important aroma compounds in beer, diacetyl (butter-like aroma) and 4-vinylguaiacol (clove-like aroma; 4-VG). Diacetyl is always considered an off-flavour (well in some cases in might be acceptable, but in my opinion it is just a sign of poor fermentation practices), while 4-VG is acceptable (and even required) in some styles (such as Hefeweizens and Belgian-style beers). Diacetyl levels decrease towards the end of fermentation, and are highly dependent on fermentation dynamics, so the concentration that was measured here at the end of fermentation doesn’t say that much. The diacetyl concentration was above the flavour threshold (50 ppb) for all strains at both temperatures, which is not that surprising since the measurements were made 120 hours after pitching the yeast, and the lowest flavour impact was observed in the beer fermented with the baker’s yeast at 20 °C. This result again suggests that baker’s yeast is a good candidate for beer fermentations (and especially more rapid ones). The baker’s yeast is also POF+ (i.e. positive for producing ‘phenolic off-flavours’; the yeast produces a phenylacrylic acid decarboxylase enzyme, that decarboxylates ferulic acid (and other phenolic acids) from the wort into 4-vinylguaiacol (and other phenolic compounds)), since 4-VG was observed in the beer. The baker’s yeast produced slightly less 4-VG than WLP380 (the table below displays the percentage of ferulic acid converted into 4-vinylguaiacol; 77% is the theoretical maximum), but it still produced concentrations above the flavour threshold in an all-barley wort (worts made from wheat malts contain more ferulic acid). This slightly limits the beer styles that the baker’s yeast could potentially be used for, but again it could be used for Hefeweizens and Belgian-style beers.


To conclude, it is evident that the Finnish baker’s yeast is perfectly usable for beer fermentations, and it comes to no surprise that it has successfully been used for traditional beer fermentation in the form of Sahti. Of the two reference strains, the baker’s yeast was closer to WLP380 (Hefeweizen IV), and it can be described as a faster-fermenting and fruitier version of it. As a side-note, it can be mentioned that the baker’s yeast flocculated very poorly, which is also similar to the behavior of WLP380. I would not recommend the use of the baker’s yeast for beer styles calling for a clean yeast aroma and no spicy phenolics, but it would be perfectly suitable for a Hefeweizen, Belgian Blond and why not even Belgian Strong Ale (Sahtis are brewed strong, so the baker’s yeast should be quite tolerant to alcohol levels reaching up to 10% ABV). It fermented quite fast at both 20 °C and 15 °C, and I would recommend a lower temperature if you are after a cleaner finish. As mentioned previously, the yeast packages found in the supermarket are all almost certainly contaminated with lactic acid bacteria, so the use of a pure culture is definitely recommended. Good luck with the brewing!

Feel free to contact me if you have any questions!

Reference for flavour thresholds:

  • Meilgaard, M., Prediction of Flavor Differences between Beers from Their Chemical Composition. Journal of Agricultural and Food Chemistry 30 (1982) 1009-1017.

Article on the differences in diacetyl formation between lager yeast strains and the role of the ILV6 gene

I’ve been researching diacetyl formation and removal by brewing yeasts the past year(s), and we recently did some screening of various lager yeast (Saccharomyces pastorianus) strains for diacetyl production and subsequent transcriptional analysis of key genes in the isoleucine-valine catabolic pathway (diacetyl is formed as an indirect by-product of valine biosynthesis in yeast; more information available here). We did this in order to identify what genes contribute most to the differences in wort total diacetyl concentration we observed between the different strains. We observed that particularly the ILV6 gene, encoding for a regulatory subunit of the acetohydroxy acid synthase enzyme (responsible for converting pyruvate into the diacetyl precursor α-acetolactate), showed greater expression early during fermentation in a strain producing more diacetyl. To confirm the role of this ILV6 gene in controlling α-acetolactate/diacetyl formation, we overexpressed both the S. cerevisiae- and S. eubayanus-form of ILV6 in our standard production lager strain A15, and found that overexpression of either form of ILV6 resulted in an identical two-fold increase in wort total diacetyl concentration relative to a control. These results suggest that both forms of the gene influence diacetyl formation, and different levels of transcription (this could be a result of a gene-dosage effect, since different lager yeast hybrids have inherited different amounts of the parental genomes; for a recent review on the subject click here) may contribute to differences in diacetyl production in various lager yeast strains.

Link to the publication:

E-mail me if you are interested in the article, but don’t have access to the full-text.


A screen of 14 S. pastorianus lager-brewing strains showed as much as a nine-fold difference in wort total diacetyl concentration at equivalent stages of fermentation of 15°Plato brewer’s wort. Two strains (A153 and W34), with relatively low and high diacetyl production, respectively, but which did not otherwise differ in fermentation performance, growth or flavour production, were selected for further investigation. Transcriptional analysis of key genes involved in valine biosynthesis showed differences between the two strains that were consistent with the differences in wort diacetyl concentration. In particular, the ILV6 gene, encoding a regulatory subunit of acetohydroxy acid synthase, showed early transcription (only 6 h after inoculation) and up to five-fold greater expression in W34 compared to A153. This earlier transcription was observed for both orthologues of ILV6 in the S. pastorianus hybrid (S. cerevisiae × S. eubayanus), although the S. cerevisiae form of ILV6 in W34 also showed a consistently higher transcript level throughout fermentation relative to the same gene in A153. Overexpression of either form of ILV6 (by placing it under the control of the PGK1 promoter) resulted in an identical two-fold increase in wort total diacetyl concentration relative to a control. The results confirm the role of the Ilv6 subunit in controlling α-acetolactate/diacetyl concentration and indicate no functional divergence between the two forms of Ilv6. The greater contribution of the S. cerevisiae ILV6 to acetolactate production in natural brewing yeast hybrids appears rather to be due to higher levels of transcription relative to the S. eubayanus form.

Hops Growing Update

I planted some hop rhizomes earlier this year, and they’ve been growing extremely well overall. Shortly after I moved the plants from the indoor pots to the garden in April, we had a spell of some really cold nights, which unfortunately killed both Centennial plants. The other 4 (Cascade, Chinook, Columbus and Nugget) survived and have been growing well (the bines are at least 3 meters long and they’ve reached the roof). Not getting my hopes up for a first-year harvest, but hopefully some of the plants will at least produce something!