Insight into the genome of Saccharomyces carlsbergensis

Eureka, another yeast genome got recently published (May 2014) by scientists at the Carlsberg Laboratory in Denmark: Saccharomyces carlsbergensis, the world’s first pure lager yeast used in production since 1883. I would like to review the published article and point out some interesting results. Below the full reference of the paper I am talking about.

Walther A, Hesselbart A, Wendland J (2014) Genome Sequence of Saccharomyces carlsbergensis, the World’s First Pure Culture Lager Yeast, G3, 4:783-793; doi:10.1534/g3.113.010090, http://g3journal.org/content/4/5/783.full

The scientists sequenced the genome using next generation sequencing techniques and compared the genome with Saccharomyces cerevisiae (top-fermenting yeast) and Saccharomyces eubayanus (likely to be a parent of the lager yeasts). Lager yeasts are hybrids and resulted from an interbreeding event between a top fermenting S. cerevisiae yeasts parent as well as a non-cerevisiae parent (likely to be S. eubayanus). This means, the genome of lager yeasts consist of parts of the S. cerevisiae genome as well as parts of a non-cerevisiae parental genome.

Beside S. carlsbergensis, the authors re-sequenced another lager yeast (Weihenstephan WS34/70) for comparative reasons. Lager yeasts can be grouped into group I (Saaz-type lager yeasts) and group II (Frohberg-type lager yeasts). Members of the two groups reflect geographic associations with breweries like group I (Czech and Carlsberg) and group II (Weihenstephan and Heineken). The sequenced S. carlsbergensis strain (CBS1513) belongs to group I whereas WS34/70 belongs to group II. Comparing the two genomes therefore might give some insight into genetical differences between the two lager yeast groups.

Loss of parental S. cerevisiae DNA in S. carlsbergensis

The authors found substantial genome size differences between the two lager yeasts (about 3.5 Mb). A previous investigation showed, the Weihenstephan lager yeast contains two complete parental genomes (S. cerevisiae and S. eubayanus) with some losses at chromosome ends [Nakao et al, 2009]. To address how much of the S. carlsbergensis genome is from S. cerevisiae and S. eubayanus, the authors mapped the obtained S. carlsbergensis genome to the two parent yeast genomes. The comparison revealed, the genome of S. carlsbergensis does not contain information of the S. cerevisiae chromosomes VI, XI and XII (Fig 1, left), harbours some translocated S. eubayanus chromosomes (II, IV and VIII and XV) (Fig 1, right) as well as loss of heterozygosity in some S. cerevisiae chromosomes (Fig 1, right, chromosomes IV, XIII, XV and XVI). This lack of chromosomal information of S. cerevisiae chromosomes VI, XI and XII as well as some loss of heterozygosity is sufficient to explain the smaller genome size of S. carlsbergensis in comparison with the Weihenstephan lager yeast.

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Fig 1: Pairwise comparison of S. carlsbergensis genome with sub-genome of S. cerevisiae (left) and S. eubayanus (non-cerevisiae) sub-genome (right). Taken from Walther et al, 2014

Summarized, S. carlsbergensis (group I) lost some S. cerevisiae DNA which is still present in the Weihenstephan lager yeast (group II).

Chromosomal map of S. carlsbergensis

The authors generated a chromosomal map for S. carlsbergensis strain CBS 1513 which consists of 29 different chromosomes (Fig 2). Whereas the Weihenstephan lager yeasts harbours 36 different chromosomes (not shown). The individual chromosomes either contain only chromosomal information from the parental S. cerevisiae (parts in blue) or S. eubayanus (orange parts) yeasts or contain information from both yeasts (translocated chromosomes).

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Fig 2: Chromosomal map of S. carlsbergensis strain CBS 1513. Blue parts represent S. cerevisiae sub-genome, orange parts the S. eubayanus sub-genome. Taken from Walther et al, 2014

To investigate if group I lager yeasts resulted from a hybridization event of two haploid (one copy of each chromosome) S. cerevisiae and S. eubayanus cells, the authors determined the copy numbers of each chromosome present in the S. carlsbergensis genome. If this would be the case, one would expect to find a 1:1 ratio of S. cerevisiae and S. eubayanus chromosomes in the S. carlsbergensis genome.

Surprisingly, the S. carlsbergensis genome seems to be triploid (three copies) with one copy of S. cerevisiae and two copies of S. eubayanus genome (1:2 ratio). The complete S. carlsbergensis genome therefore consists of a total of 47 chromosomes (Fig 2). In comparison, the Weihenstephan lager yeasts is tetraploid (4 copies) with two S. cerevisiae and two S. eubayanus genomes (1:1 ratio).

The comparison showed a clear distinction between lager yeast group I and II with loss of S. cerevisiae DNA in group I. In terms of origin, one may suggest that group I lager yeasts were generated by a fusion event of a haploid S. cerevisiae with a diploid S. eubayanus yeast cell whereas group II lager yeasts originated from a diploid-diploid fusion generating tetraploid group II lager yeasts. Three conserved translocation events in both sequenced lager yeasts may however suggest a common ancestor of both lager yeast groups. And a DNA elimination event may have created group I lager yeasts afterwards.

There you have it. A pretty cool research project. I would like to finish with yet another astonishing result. The authors addressed the level of diversity of possibly one of the original S. carlsbergensis yeast strain isolated by Emil Chr. Hansen in the late 19th century (obtained from Carlsberg bottles of the late 19th century) with the strain deposited at CBS in 1947. The yeasts present in the bottles were identical with the CBS deposited yeast strain. “This suggests very limited evolution of pure cultured yeast strains under industrial fermentation conditions” [cited from Walther et al, 2014]. Pretty cool, right?

I hope you enjoyed reading my short review. Please have a look at the original genome paper as well. I think it is very well written publication. Hope to see some new lager yeast genomes coming out soon.

Bibliography

Nakao Y, Kanamori T, Itoh T, Kodama Y, Rainieri S et al (2009) Genome sequence of the lager brewing yeast, an interspecies hybrid. DNA Res. 16:115-129

#13 Pivo Pilsner

Eureka, its time for yet another recipe from the past. This time a Pilsner style beer.

Fig 1: Charles bridge in Prague very early in the morning before other tourists crowded the bridge

All started with a study trip to Prague and České Budějovice (aka Budweis) in 2008. I don’t want to write a trip journal here but if you have the opportunity to visit Prague, just go for it. It is such a nice place to visit.

České Budějovice, in the south of the Czech Republic, is very well-known for the Budweiser Budvar Czech Premium Lager. The trip was not about beer though but a friend of mine and me visited a local pub in our descover-the-city-time and enjoyed a freshly poured Czech Budweiser instead. What a treat. I can’t tell you much about the city though…

Fig 2: Drinking Czech beer in an Australian bar in Prague

After our one day stay in the south of the Czech Republic, we traveled north and stayed in Prague for several days. We both attended the city tours organized by our school and spent our spare time in different bars again… The beer was very cheap and really good. I am not into Pilsner style beers anymore but I would still order a Pilsner Urquell directly poured from a cask. Just amazing!

I went home with a lot of beer, a headache and decided to give this particular style a go to replicate a Pilsner Urquell. The recipe is a slightly changed version from SIOS (recipe in German) and brewed it in 2010.

Recipe: Pivo Pilsner
Numbers: Volume [L] 22 (5.8 gal)
Original gravity 12.7°P
Terminal gravity 5.5°P
Color Around 4.1 EBC (measured 3.3 EBC)
IBU 44 IBU
ABV 3.6 %
Grains: Pilsner malt (4 EBC) 4.3 kg
Carapils (5 EBC) 0.3 kg
Acidified malt (4.5 EBC) 0.1 kg
Hops: Hallertauer Nugget (12% AA) 23 g and boil for 75 min
Saazer (5% AA) 19 g and boil for 15 min
Saazer (5% AA) 19 g and boil for 5 min
Yeast: #2278 Czech Pils
Water: Burgdorf Mash: 12 L (3.2 gal), sparge: 20 L (5.3 gal) @78°C (172°F)
Rest: Mash in @48°C (118°F), 5 min @48°C (118°F), 10 min @53°C (127°F), 45 min @63°C (145°F), 10 min @73°C (163°F), 10 min @ 78°C (172°F)
Boil: Total 75 min
Fermentation: Primary 24 days @15°C (59°F) in plastic fermenter
Secondary None
Maturation: Carbonation (CO2 vol) 2.5
Maturation time 3 weeks

Fig 3: PLC and stirrer are preparing the water for the mash in

05/13/10: Brew day number 13 begins. It is so cold outside that I did this batch in my garage. PLC and stirrer did their job wonderfully, iodine test was negative after the last rest at 73°C (163°F). Then transferred the mash into my lautering bucket and begun to sparge. Then boiled the wort for 75 min with the additions of the hops according to the protocol above.

Fig 4: Surface of the boiling wort (not yet boiling)

Then cooled the wort down to 18°C (64°F) and added the yeast. Due to the very cold weather, I hoped the temperatures outside would stay low to cool down my cellar. Unfortunately for the beer, the weather changed and the temperatures in the cellar rose to 15°C (59°F). I therefore had to ferment the beer at a higher temperature than originally planned. The fermentation went not that well. My original gravity was 12.7°P and the gravity was still at 12.4°P after seven days… The gravity dropped to a final 5.5°P. A pretty slow fermentation although fermented at relatively high temperatures for a lager fermentation.

06/06/10: Bottled the beer after a primary fermentation of 24 days to a level of 2.5 vol. I then left the beer carbonate for nearly two weeks and left it mature at 15°C. Luckily, I bought myself a refrigerator in the summer of 2010 and so the maturation of the beer could take place at lower temperatures.

10/05/10: Tasting

Fig 5: Pivo Pilsner meets glass

Aroma: Very hoppy aroma. Bready, sweet and honey aroma. DMS!!!

Appearance: Yellow, clear with a white foamy head on top.

Flavor: Hint of hops, some sweetness and nice bitterness. Again, DMS detectable.

Mouthfeel: Light body, lively carbonation level, short bitter and malty aftertaste.

Overall Impression: Despite the DMS, the beer was not that bad. Nevertheless, the DMS made it pretty hard to enjoy and I dumped the last remaining bottles in 2012.

Changes and things I would consider important for a next batch: First, get enough viable yeast and ferment it at the appropriate temperature. I now own a temperature controlled fermentation chamber for such reasons. Second, increase the boil to a full length of at least 90 min to thrive of any DMS precursors. And chill the wort immediately after the whirlpool to minimize the DMS formation. Third, adapt the technique. Maybe go for a decoction mash, use an appropriate water profile, Premium Pilsner malt or even floor malted barley instead of normal Pilsner malt and use very fresh whole hops. Maybe even mature the beer or some of it in wooden vessels (as done by Urquell).

To summarize, brewing lager beers is not that easy. There are a lot of things to consider and taken care of. If you have a flaw (such as DMS) it will be easily detectable since theses beers are rather subtle in the aroma and flavor. On the other hand, it is very rewarding if you can brew such a beer (been there). Once again, I am not very into lager beers and such anymore but tasting a freshly well made Pilsner beer is very different to the ones you buy in bottles. Stay tuned!

#8 Bavarian Pilsner

Eureka, todays post is about my first attempt to brew a Pilsner style beer. Unfortunately, another recipe from the dark ages without useful tasting notes… Anyway, the following recipe is a straight forward approach for a Pilsner recipe. I went with Wyeast’s #2206 Bavarian Lager yeast and did the fermentation a bit higher than usual bottom fermenting temperatures. I brewed this particular batch in winter of 2007 when my cellar was at around 14°C (57°F). Today I know that the yeast I chose can be indeed used at higher temperatures without any inappropriate esters and such. But back then, just a lucky guess. Lets go through the recipe.

Recipe: Bavarian Pilsner
Numbers: Volume [L] 27 (7.1 gal)
Original gravity 12°P
Terminal gravity 3.3°P
Color Around 8 EBC
IBU 39 IBU
ABV 4.7 %
Grains: Pilsner malt (4 EBC) 4.65 kg
Carapils (4 EBC) 0.2 kg
Acidified malt (5 EBC) 0.14 kg
Hops: Tettnanger (5.2% AA) 17 g and boil for 70 min
Magnum (14.8% AA) 17 g and boil for 70 min
Tettnanger (4.1% AA) 17 g and boil for 3 min
Yeast: #2006 Bavarian Lager
Water: Burgdorf Mash: 18 L (4.8 gal), sparge: 18 L (4.8 gal) @78°C (172°F)
Rest: Mash in @45°C (113°F), 20 min @ 43°C (109°F), 30 min @ 63°C (145°F), 30 min @ 71°C (160°F), 10 min @ 78°C (172°F)
Boil: Total 70 min
Fermentation: Primary 7 days @ 14°C (57°F) in plastic fermenter
Secondary None
Maturation: Carbonation (CO2 vol) 2.5 (bottled at 4.5°P)
Maturation time > 3 weeks

02/17/07: Brew day number eight begins. All went according to the protocol above. I had nearly 27 L at the end of the brew day and pitched a package of Wyeast’s #2006 Bavarian Lager in there.

Sparging process

Looking back, this was probably a underpitched wort for sure. Anyway, I then left the fermenter at 14°C (57°C) and then bottled the beer with a gravity of 4.5°P to have just enough sugars for the carbonation. The forced fermentation test finished at a gravity of 3.3°P. This technique is called “Grünschlauchen” in German. It means that you bottle the beer at the exact moment where there is just enough sugars left for the carbonation process. In my case, bottled the beer with a gravity of 4.5°P. The remaining 1.2°P (4.5°P minus the gravity of the forced fermentation test) would be enough for the carbonation. There are several reasons why I do not use this Grünschlauchen method anymore. One difficulty here is to catch the right moment to bottle the beer. Another one is the lacking secondary fermentation or maturation process. Nowadays, I alway go through a secondary fermentation and bottle with either an addition of sugar or use wort. Way easier!

After bottling the beer, I stored it for several weeks before I tasted it for the first time. This time, I have some tasting notes. What a treat! Not the bests, but at least some notes. The beer had a nice white head and a yellow color. Light haziness. The aroma was very hoppy and nice. The flavor was again hoppy and some bitterness detectable. All in all a very easy drinkable beer.

Unfortunately, I have no idea how old the beer was as I did the tasting. As I read through the tasting notes now, the descriptions I made are very typical for a Pilsner beer. I therefore assume that this beer tasted like a typical Pilsner beer indeed. Cheers!