Tasting: Mikkeller’s Yeast Series 2.0

Eureka, I would like to share some of my tasting experiences of Mikkeller’s Yeast Series 2.0. The basic idea behind this series was to compare different yeast strains and their effects on the beer’s aroma and taste. I could get my hands on five of the six beers in the series (English Ale yeast is missing) and did a side-by-side tasting.

The base beer was all the same. In one case, the beer was fermented with a Lager strain, another one with an American Ale strain, yet another one with a Saison strain and two with Brettanomyces lambicus and Brettanomyces bruxellensis respectively. Lets see how they tasted and the individual strain’s impact on the flavor profile.

Lager yeast

Aroma: Very hoppy aroma (lots of grapes, fruits). The combination of all the hops used (Simcoe, Nugget, Warrior, Amarillo and Centennial) remind me of Nelson Sauvin hops. No yeast character.

Appearance: Orange, clear, 1 finger white head, nice bubbling.

Flavor: Fruity, nice bitterness level.

Mouthfeel: Medium body, average carbonation level, bitter/fruity aftertaste and a grassy finish

Overall Impression: Rather clean beer (in terms of yeast character). Very pronounced hop aroma and bitterness and a grassy finish

 

American Ale yeast

Aroma: Less hoppy than Lager example. Even a musty component in there. Doesn’t smell clean at all.

Appearance: Orange, clear, 1 finger white head, nice bubbling.

Flavor: Luckily nothing of the weird musty aroma is on the palate. Very fruity beer with a well-balanced bitterness. No typical yeast character.

Mouthfeel: Medium body, average carbonation level, bitter/fruity aftertaste. No grassy finish

Overall Impression: Compared to the Lager version, this beer is smoother in terms of bitterness. The bitterness is well incorporated and there is no grassy finish. However, the aroma in this beer is not as nice. We could not detect any yeast character in this example.

Saison yeast

Aroma: Pine, lots of tropical fruits and citrus and some spicy character (pepper).

Appearance: Orange, clear, 1 finger white head, nice bubbling.

Flavor: Again some fruits and some spiciness in addition.

Mouthfeel: Medium body, average carbonation level, slight bitter aftertaste and a grassy finish and even a bit astringent.

Overall Impression: Slightly different aroma compared to the previous two examples. This time, we could detect some yeast specific character (pepper). This yeast seems to accentuate the bitterness in the aftertaste including a grassy, astringent finish.

Brettanomyces lambicus yeast

Aroma: Subtle hop aroma, no funk…

Appearance: Orange, clear, 1 finger white head, nice bubbling.

Flavor: A bit of a disappointment. Subtile fruity beer with a well-balanced bitterness. No typical yeast character and no funk. Actually a rather clean beer.

Mouthfeel: Medium to low body, average carbonation level, slight bitter aftertaste.

Overall Impression: Not very funky nor very interesting. Average beer. We could not detect any yeast character.

Brettanomyces bruxellensis yeast

Aroma: Wow, now we are talking. There is some Brett funk going on: Wood notes, horse blanket, slight vinegar and the hop profile in the back. This beer reminds me of Cantillon’s Iris with Nelson Sauvin hops instead of the Saaz hops they use. Simply amazing smell!

Appearance: Orange, clear, 1 finger white head, nice bubbling.

Flavor: Unfortunately, not a lot of funk on the palate. Some leathery notes are present. Some fruity notes as well and a well incorporated bitterness. Rather clean beer.

Mouthfeel: Medium to low body, average carbonation level, no bitter nor grassy aftertaste. Hint of tartness reminds of the Brettanomyces in this beer.

Overall Impression: Judging from the smell, the most interesting one in the series for sure. B. bruxellensis really shows itself here. The aroma profile of this beer is surprisingly complex in my opinion. The flavor on the other side is not very yeast pronounced. But the finish is rather pleasant again.

What we learned from this tasting:

Lager strain: Gives a hop forward beer. Clean and very pronounced hop aroma. More pronounced bitterness and a grassy finish.

American Ale strain: Well incorporated bitterness and nice finish. This strain seems to work for more hop forward beers.

Saison strain: Some yeast specific character in the nose and palate. This strain accentuates the bitterness and leads to a grassy and astringent finish. Not really working for me. The spicy character, the grassy thing and the astringency makes it hard to enjoy this beer.

B. lambicus strain: Not a very funky Brett strain. Rather clean beer (compared to B. bruxellensis version). A side note. This doesn’t have to be true for every B. lambicus strain. There are so many B. lambicus strains with different flavor profiles.

B. bruxellensis strain: Lots of Brett character in the nose. But not so much on the palate. Rather clean and smooth beer with a nice bitterness level and no grassy finish.

I will put some efforts into brewing something like the B. bruxellensis beer myself. I am really fascinated about the complexity one can get with a single Brettanomyces fermented beer. Unfortunately, I tried to isolate some yeast from different Mikkeller beers before (brewed by DeProef) but never managed to recover any viable yeasts from the sediments in the bottles. I guess all the DeProef’s beers are pasteurized and therefore no (or a very small) chance to get any living yeasts out of bottles. That’s why I did not bother to isolate the B. bruxellensis strain at all. Thanks for reading, commenting and stay tuned!

Freezing Brettanomyces

Eureka, another Brettanomyces post. This time about a feasibility study if you can freeze Brettanomyces like any other Saccharomyces strain. I would hereby like to discuss my latest results.

All started by preparing some Brettanomyces strains I either bought or isolated for cryo storage like described in a previous post of mine concerning freezing yeasts. I put the following Brettanomyces strains in my -20°C (-4°F) freezer in August/September 2012:

- Brettanomyces isolated from WY3191 Berliner Weisse blend
- Brettanomyces isolated from Girardin Gueuze
- Brettanomyces isolated from 3 Fonteinen Gueuze
- Brettanomyces isolated from Cantillon Kriek (3 strains)
- Brettanomyces isolated from Cantillon 2007 Lou Pepe Gueuze (2 strains)
- Brettanomyces bruxellensis (WY5526)
- Brettanomyces lambicus (WY5112)

Some isolates consisted of more than one strain which were separated during trial runs with bromocresol green (not published). All these different strains were frozen separately.

In mid November 2012, the Brettanomyces were taken out of the freezer and transferred into fresh YPD media. After two weeks some of the yeasts showed signs of growth such as turbid media and gas production. In the end all media showed signs of activity and formed off-white coloured sediments. Both yeasts isolated from the Cantillon beers even showed signs of pellicle formation (not shown). Although activity could be observed it still has to be evaluated if the activity originates from the yeasts and not any contamination. Due to lack of time the yeasts remained in the YPD media for nearly two months until further experiments could be conducted.

Micrographs

Some micrographs showing the yeasts from YPD liquid cultures before freezing and afterwards.

Fig 1: Brettanomyces from Cantillon Kriek before freezing

Typical elongated cell shape of Brettanomyces visible (Fig 1 and 2). Even some hyphae formation (Fig 2). Somehow the colonies in Fig 1 look smaller than the ones in Fig 2 although both pictures were taken with the exact same setup.

Fig 2: Brettanomyces from Cantillon Kriek after freezing

Fig 3: Brettanomyces from Cantillon Lou Pepe after freezing

Again some hyphae formation (Fig 3).

Concluding from the micrographs shown (Fig 1-3), Brettanomyces yeasts could be found in the YPD media after reviving them. Although Brettanomyces yeasts could be observed in the microscope observations still does not prove that the yeasts are viable. Liquid cultures were first streaked on some Sabouraud agar plates and incubated at room temperature until colonies were visible. Colonies were then picked from the Sabouraud plates and streaked on Sabouraud agar with an addition of bromocresol green.

Some yeasts had different morphologies like WY5526 B. bruxellensis on bromocresol green containing agar media (Fig 4). Some colonies grew as green, others as white ones. For the next agar platings, each a white and green colony were picked.

Fig 4: WY5526 B. bruxellensis on bromocresol green agar

One strain of Cantillon’s Kriek and the strain isolated from a 3 Fonteinen Gueuze grew in different morphologies (white and green colored colonies) as well and were treated separately for the next agar platings. Please further notice that WY5112 Brettanomyces lambicus was not streaked on Sabouraud Bromocresol green due to a mold contamination on the first Sabouraud plate. However, typical colonies of B. lambicus could be observed (not shown).

Agar plate results

All the revived Brettanomyces strains formed colonies on Sabouraud Bromocresol green agar (Fig 5-7).

Fig 5: Brettanomyces on Sabouraud agar after six days of incubation. Left: Cantillon Kriek_green colony (B04_green); Top: Cantillon Gueuze 2007 (B05); Right: WY5526 B. bruxellensis colony 1; Bottom: WY5526 B. bruxellensis colony 2

All the yeasts grew as white colonies expect the one known to grow as green colonies (B04_green) (Fig 5). In the case of WY5526 B. bruxellensis, the two picked colonies from Fig 4 showed the same morphology again. Both grew as white colonies (Fig 5).

Fig 6: Brettanomyces on Sabouraud agar after six days of incubation. Left: Cantillon Kriek (B04_2); Top: Cantillon Kriek (B04_1); Right: Girardin (B01); Bottom: Cantillon Geuze 2007 (B05 dark_1)

The two colonies that grew differently on the first bromocresol green agar from the Cantillon’s Kriek isolate grew again as white colonies (Fig 6).

Fig 7: Brettanomyces on Sabouraud agar after six days of incubation. Left: Cantillon Geuze 2007 (B05 dark_2); Top: Newly isolated Brett (nothing to do with this experiment); Right: 3 Fonteinen (B02_2); Bottom: 3 Fonteinen (B02_1)

The same is true for the different morphologies from a 3 Fonteinen isolate (Fig 6, 7). The Brettanomyces strain(s) isolated from WY3191 Berliner Weisse blend formed colonies as well (not shown).

In all cases, the bromocresol agar media turned from a blue color to yellow indicating the secretion of acid. Some plates even had a strong acetic acid smell. An un-streaked bromocresol agar media was included as a control and the color remained blue throughout the whole experiments (not shown).

Summary/ Conclusion of agar platings

It could be shown that all the frozen Brettanomyces strains formed colonies on Sabouraud agar. Some of the isolated yeasts grew in different forms (white and green colonies) but such a differentiation could not be observed after a second streak on agar media. This is not the case for the yeast strain isolated from Cantillon’s Kriek (B04 green) which grew in green colonies on every plating.

The differentiation between Brettanomyces and Saccharomyces based on bromocresol green and its issues will be covered in a future post. One could already observe in these platings that some of the yeast colonies grew as green colonies in a first run but grew as white colonies in a second run again.

Micrographs of the colonies

At last some micrographs of the colonies.

Fig 8: WY5226 Brettanomyces bruxellensis (5526_1)

Fig 9: WY5226 Brettanomyces bruxellensis (5526_2)

The two different samples of WY5226 B. bruxellensis look very similar (Fig 8, 9). The differences in color appearance on the bromocresol green might be due to some issues of bromocresol green as an indicator for Brettanomyces and wild yeasts. As mentioned already, more about that in a future post.

Fig 10: Brettanomyces from Girardin Gueuze (B01)

Typical Brettanomyces cells visible in the Girardin isolate (Fig 10).

Fig 11: Brettanomyces from 3 Fonteinen’s OudeGueuze (B02_1)

Fig 12: Brettanomyces from 3 Fonteinen’s OudeGueuze (B02_2)

Both colonies from 3 Fonteinen isolate seem to be Brettanomyces (Fig 11, 12). Hard to tell based on the morphology of the cells if the two samples are the same or not.

Fig 13: Brettanomyces from Cantillon’s Kriek (B04_1)

Not a lot of elongated cells were visible in B04_1 (Fig 13) like in B04_2 (Fig 14). Maybe these two samples are not the same strain of Brettanomyces. Maybe B04_1 is not a Brettanomyces strain. Further studies are necessary.

Fig 14: Brettanomyces from Cantillon’s Kriek (B04_2)

Fig 15: Brettanomyces(?) from Cantillon’s Kriek (B04_green)

Some elongated cells visible in B04_green (Fig 15). Yet a lot of the cells looked like Saccharomyces cerevisiae. Might be a mixture of S. cerevisiae and Brettanomyces.

Fig 16: Brettanomyces from Cantillon’s Kriek (B04_white_1)

Lots of hyphae visible in one of the isolates from Cantillon’s Kriek (Fig 16). Very typical for Brettanomyces.

Fig 17: Brettanomyces from Cantillon’s Kriek (B04_white_2)

On the other hand, in the second sample of B04_white not that many Brettanomyces cells visible form hyphae as shown in Fig 16 (Fig 17). Yet again, maybe these two samples are not the same strain of Brettanomyces. Further studies necessary.

Fig 18: Brettanomyces from Cantillon’s Lou Pepe 07 Gueuze (B05)

Lots of elongated, boat-shaped cells in Cantillon’s Lou Pepe isolate B05 visible (Fig 18).

Fig 19: Brettanomyces from Cantillon’s Lou Pepe 07 Gueuze (B05_dark_1)

The second strain from Cantillon’s Lou Pepe looks different from the first one shown in Fig 18 (Fig 19). Hard to tell if the second sample B05_dark_2 is another strain than B05_dark_1 or not.

Fig 20: Brettanomyces from Cantillon’s Lou Pepe 07 Gueuze (B05_dark_2)

Summary/ Conclusion of micrographs

Brettanomyces were visible in most of the micrographs shown above. However the shape of Brettanomyces can differ significantly. The experiment strongly suggest that it is possible to freeze Brettanomyces and successfully revive them. In addition, it could be shown that some of the frozen samples might contain further strains of yeasts. Additional experiments are necessary to further look into this possibility.

Unfortunately, the yeast isolated as Brettanomyces from WY3191 Berliner Blend looked very similar as Saccharomyces cerevisiae cells (not shown). It might be possible that the yeast isolated from the blend wasn’t a Brettanomyces strain in the first place. But beside the S. cerevisiae colonies were some smaller colonies visible as well (Fig 21).

Fig 21: Isolated cells from WY3191 Berliner Weisse blend

The cells shown in Fig 21 are no yeast cells. Theses cells look like Lactobacillus. Interestingly, these bacteria cells were in the freezer as well (therefore possible to freeze Lactobacillus like yeast cells as well) and they grew on bromocresol green containing agar.

Enough with the experimental part. I am very happy about theses results. Took me some time to do all the platings, micrographs but I have the feeling it was all worth the efforts. At least now I know that I can easily freeze all my Brettanomyces strains I have (well over 20) without any worrying. The only thing to keep in mind here is that theses yeast might take some additional time for reviving than normal S. cerevisiae strains. Some preliminary results even suggest that it is possible to freeze Lactobacillus like you would any yeast cells.

The next post will be about another big yeast experiment. Stay tuned and thanks for commenting!

Insight into the Brettanomyces Mitochondrial genome

Eureka, I have to mention first that this is a very scientific biochemistry, bioinformatics post about Brettanomyces. However, I hope those of you with less biology experience can follow as well or at least get the idea of the main messages. This post is mainly about an insight into the mitochondrial genome of two Brettanomyces species and comparing those sequences with Saccharomyces.

I always wondered how similar the genome sequence of a Saccharomyces cerevisiae and Dekkera/Brettanomyces yeasts are. To do such comparisons the DNA sequences have to be published first or at least sequenced. I am not yet through all the publications about the Brettanomyces genome sequencing and therefore don’t know yet if any complete genome of Brettanomyces/Dekkera is available at the moment. More about the yeast genomes in future posts.

I would like to start with a quick introduction about the whole topic to give those without a biology background a chance to understand the following points. This post is about comparing different DNA sequences from different yeasts. You might know that every living organism contains DNA which can be seen as the manual of the cell. The DNA encodes nearly everything the cell needs to work such as proteins, different RNAs etc. The whole DNA within a cell is commonly called genome. The DNA consists of four bases: Adenine, guanine, cytosine and thymine. In the DNA every information is encrypted with these four bases. The code to decipher the information from the four bases code into a protein is called the genetic code. If the genetic code is known you can encode a DNA sequence into proteins. Or vice versa. Before one can encode a DNA sequence the sequence has to be known. And this is the process called DNA sequencing. By DNA sequencing you determine the sequence of the four bases in the DNA. Knowing DNA sequences is very important for modern biology to for example understand certain diseases. On the other hand, DNA sequences are in general very specific for every living organism. This can be used to detect certain organisms. I hope this is enough information for the beginning.

One disadvantage of sequencing genomes of yeasts is their size. Sequencing big genomes is always challenging. Before modern techniques such as shotgun sequencing came widely available the only way of sequencing large genomes was really time-consuming and thus really expensive. This is different today and a lot of people around the world are working on sequencing projects. Me included.

To compare any genomic sequences or get any information about the evolutionary relationship of Saccharomyces and Brettanomyces, one has to look at the DNA of the two yeasts and compare them. And since I don’t know yet if the full genome sequence of at least one Dekkera/Brettanomyces strain is sequenced one has to look at a different DNA. And that’s where the mitochondrial genome comes into play. Mitochondria are the organelles in the cell responsible for several pathways such as supplying the cell with energy. And mitochondria have their own DNA, called mitochondrial DNA (mtDNA) because mitochondria originate from bacteria cells (see endosymbiotic theory). To summarize this theory, some time ago a cell incorporated a bacteria cell and the incorporated bacteria cell lived on within the first one and became the mitochondria. And since the mitochondria was a bacteria cell with DNA, the DNA still exists in some part within the mitochondria. The whole amount of this mtDNA is then called the mitochondrial genome. Because mtDNA are relatively short compared with the genomic DNA of a yeast, sequencing mtDNA is relatively easy. The first sequencing of a mtDNA sequence of D. bruxellensis and B. custersianus has been completed by E. Procházka et al in 2010 [Procházka et al, 2010]. All the data below is from this publication.

Since Saccharomyces cerevisia is an eukaryotic model organism for scientists, the whole genome of this yeast is already sequenced. Including the mtDNA. The mitochondrial genome sequence has been published and can be found here under the accession number AJ011856. For D. bruxellensis (strain CBS 2499) the sequence is deposited with the accession number NC_013147. And for the B. custerianus strain (CBS 4805) with the accession number GQ354525. If you look at one of the deposited genomes you can see the sequence at the bottom of the entry.

Lets begin with a look at the mtDNA of Saccharomyces cerevisiae. One can see that the mtDNA is circular and 85779 bp long (Fig 1). All the red arrows represent a specific gene encoded on the DNA. I only included the genes because with all the other annotated stuff such as tRNA etc the picture would simply be unreadable. One can observe that it seems that all the genes are facing in the same direction. This simply means that only one strand of the DNA is used for coding. (DNA is double stranded).

Fig 1: mtDNA Saccharomyces cerevisiae

Moving on to D. bruxellensis. Again a circular mtDNA (Fig 2). By the way, the publication mentioned above was the first to demonstrate that the mtDNA in Brettanomyces is circular. This mtDNA is 76453 bp long and includes a lot of genes as well. However, some genes face a different direction than others. The mitochondrial genome in D. bruxellensis therefore uses both strands as coding strands. This is already different compared to S. cerevisiae in Fig 1.

Fig 2: mtDNA D. bruxellensis

And at last a quick look at the mtDNA in B. custersianus (Fig 3). Yet again a circular mtDNA with a length of 30058 bp. This is much shorter than in S. cerevisiae and D. bruxellensis. And yet again, the genes are read on only one strand.

Fig 3: mtDNA B. custersianus

Lets briefly summarize the first few observations and lets compare them. All the mtDNA genomes are circular. The sizes in S. cerevisiae and D. bruxellensis are more or less the same. The mtDNA in B. custersianus though is significantly shorter than the other two yeasts. On the other hand the genes in both S. cerevisiae and B. custersianus are one single strand and genes in D. bruxellensis on both.

All these observations already tell me that these three yeasts are really not the same based on their mitochondrial genomic setup. One might argue about the size differences but the different gene orientation is quite remarkable in my opinion.

Moving on with further comparisons. Below is a table with the number of genes encoded in the mitochondrial genomes in all three yeasts and the number of tRNAs (transfer RNA). If you don’t know what tRNAs are just don’t bother. I will not get into any details about these tRNAs in this post.

	S. cerevisiae	D. bruxellensis	B. custersianus
Genes (including tRNA)	42	46	47
tRNA	24	25	25
Genes (without tRNA)	18	21	22

By just looking at the number of genes one might already tell that there seems to be a difference in the number of genes without tRNAs between S. cerevisiae and Brettanomyces/Dekkera. All the mitochondria seem to have roughly the same number of tRNAs. I will not get into further detail about the tRNAs here. If you need further information please have a look at the original publication. I would like to talk about the differences in genes instead.

Lets have a closer look at some of the genes encoded in the mitochondrial DNA in the three yeasts. Please keep in mind that over 99% of the proteins present in the mitochondria originate from the cytosol: Newly synthesized cytosolic proteins are transported from the cytosol across the outer membrane by the TOM40:TOM70 complex. Thus the mitochondria DNA does not have to encode for a lot of proteins as it can easily be seen by looking at the table above.

The following genes are encoded within the mtDNA:

• Cytochrome oxidase subunits 1, 2, 3 (cox1, cox2, cox2)
• Apocytochrome b (cob)
• ATP-synthase subunits 6, 8, 9 (atp6, atp8, atp9)
• Mitochondrial small and large rRNAs (rns, rnl)
• RNAse P (rnpB)
• Mitochondrial subunit ribosomal protein 3 (rps3)

A lot of these proteins can be found in S. cerevisiae as well. But this does not mean the DNA sequences are the same though. More about that later on. Indeed there are some genes only encoded in the two Brettanomyces/Dekkera mtDNAs:

• NADH dehydrogenase subunits 1, 2, 3, 4, 4L, 5, 6 (nad1-4, nad4L, nad5-6)

This is an enzyme complex (also known as respiratory chain complex I) and catalyzes the reaction of NADH to NAD+.

The next question to answer is if the genes found in both yeast species have the same sequences or not. I would like to look at one set of genes.

Cytochrome c oxidase:

The cytochrome c oxidase complex consists of three different subunits, subunit 1 to 3. Cytochrome c oxidase catalyzes the reduction of oxygen to water and plays a very important part in the respiratory chain. Below is a table showing the length [bp] of the different genes.

	S. cerevisiae	D. bruxellensis	B. custersianus
COX1: cytochrome c oxidase subunit 1	12884*	5348*	5436*
COX2: cytochrome c oxidase subunit 2	756	738	744
COX3: cytochrome c oxidase subunit 3	810	810	810

Both yeasts have the three subunits encoded in their mitochondrial DNA. However, in D. bruxellensis subunit 1 has 3 exons, 4 exons in B. custersianus, and 8 exons in case of S. cerevisiae. The CDS (coding sequence) in D. bruxellensis and B. custersianus are 1629 bp and 1605 bp long in S. cerevisiae.

Comparing the two protein sequences of the Brettanomyces/Dekkera strains shows a pairwise identity of 85.1% (MAFFT alignment). For the other two subunits 2 and 3 in the two strains 88.8% for COX2, 81.9% for COX3.

Comparing COX1 from D. bruxellensis with S. cerevisiae shows a pairwise identity of 70.3%. COX1 from B. custersianus and S. cerevisiae are 70.5% identical.

Comparing COX2 from D. bruxellensis with S. cerevisiae shows a pairwise identity of 75.8% (Fig 4). COX2 from B. custersianus and S. cerevisiae are 73.9% identical.

Comparing COX3 from D. bruxellensis with S. cerevisiae shows a pairwise identity of 70.4%. COX3 from B. custersianus and S. cerevisiae are 70.1% identical.

Fig 4: Comparing COX2 from D. bruxellensis with S. cerevisiae shows a pairwise identity of 75.8%

This is a very nice example that all the three yeasts have the same protein function (cytochrome c oxidase) but the sequences are not the same and not even encoded the same way (different exons) or direction (as shown above). On the other hand not even the two Brettanomyces/Dekkera strains have the same sequences. Still the sequences are more identical in the two Brettanomyces/Dekkera strains than compared to S. cerevisiae.

Putting this all together, the three different yeast strains look very different at a molecular level (different size, using two strands as coding strands). The genes don’t even have the same exact sequences. In the end all the yeasts produce proteins with the same function. This is quite remarkable in my opinion. Another big difference between S. cerevisiae and Dekkera/Brettanomyces is the existence of the NADH dehydrogenase in the latter yeasts.

I hope this post was not too complicated and got you some ideas about the different genetic setup of Saccharomyces and Brettanomyces/Dekkera. In the end all the yeasts achieve the same but all with a different setup. This is simply remarkable and in my opinion a brilliant example how evolution impacts an organism. Not to say that even within the same species such as Brettanomyces/Dekkera two different strains (D. bruxellensis and B. custersianus) might have very different setups as well. This makes me wonder what a look at the genomic DNA might reveal…

I would like to end by mentioning that this is a nice example to show people what one can do by just looking at DNA sequences and why it is important in my opinion to sequence organisms in the first place. Such insights are not possible if no DNA sequences are available. Don’t expect the genomic Brettanomyces DNA insight soon. This will take me much longer to prepare because there is much more data to process…

Bibliography

• E. Procházka, S. Poláková, J. Piskur and P. Sulo, 2010. Mitochondrial genome from the facultative yeast Dekkera bruxellensis contains the NADH dehydrogenase subunit gene. FEMS Yeast Res, 10, 545-557 (Pubmed)

#57 Lambic 2012

Eureka, its time for another very cool project of mine. I have to apologize for the very few postings lately. I am very busy with my lab research lately. However, I am still homebrewing, drinking beers and yeast ranching. So no worries.

Fig 1: Girardin’s Faro

I visited my local last week after several months once again and had the opportunity to try a lot of great beers from other homebrewers and even some really amazing commercial examples. They even had Girardin’s Oude Lambik on tab. Wow! Even a Girardin Lambic tastes great. However, a Lambic is not as complex as you would expect from a Geuze because Geuzes are blends of different Lambics to improve the complexity. And this Lambic was no exception. The Oude Lambik has a very subtle sourness and even a malty aftertaste. By the way, the Oude Lambik was flat as expected. I then tried Girardin’s Faro. A Faro is basically a blend of Lambics and then bottled with sugar and sometimes spices or other sources of sugar such as molasses, caramel are added in addition. In the Faro from Girardin caramel is added. I really liked the Faro because it has some carbonation which makes it easier to appreciate in my opinion.The taste and aroma reminded my of a very young Lambic. Once again, the complexity, sourness and funkiness were rather subtle. It even had a bitter aftertaste. I then tried Girardin’s Framboise before heading home. I assume this Framboise was bottled just a few weeks before. The aroma was just incredible. I have never encountered such an intense raspberry aroma in a beer before. It smelled like a homemade raspberry jam. Amazing! And no sourness or funkiness at all. Just amazing! On the other hand, this particular Framboise was not sweet like other even pasteurized examples. The last Framboise beers I had (like 3 Fonteinen’s) were rather “old” and the fruity character was no longer detectable or lets say rather subtle. Sourness and funkiness were the main components there. However, I would prefer a Gueuze in this case instead. It is somewhat not worthy in my opinion to store a Framboise, Kriek etc. to lose the fruity character. Enough with commercial examples. The whole sour beer stuff reminded me of my latest Lambic attempt.

This post is all about my second attempt to brew a Lambic style beer. However not a traditional Lambic beer with the spontaneous fermentation method.

My first attempt to brew a Lambic ended in a very sour beer. Nevertheless, I still wanted to give this style another go. It took me nearly five years to brew my second attempt… The five years gave me a lot of opportunities to read books, blogs and other sources about sour brewing. And tasted a lot of great Lambics and Gueuzes (like described above). In Spring 2012, I decided to finally brew another Lambic. This time, I went with a traditional turbid mash instead of a single infusion step. Lets go through the recipe:

Recipe:	Lambic 2012
Numbers:	Volume [L]	45 (11.9 gal)
	Original gravity	13.5°P
	Terminal gravity	N/A
	Color	Around 5 EBC
	IBU	< 5 IBU
	ABV	N/A
Grains:	Pilsner malt (4 EBC)	6.3 kg
	Wheat flakes	3.5 kg
Hops:	Old hops (< 1% AA)	240 g and boil for 120 min
Yeast:	Wyeast’s	#3278 Lambic Blend and Milupa1 for primary (see description below)
Water:	Burgdorf	Mash: 41 L (10.8 gal), sparge: 34 L (9 gal) @78°C (172°F)
Rest:	Turbid mash (see description below)	Mash in @45°C (113°F), 20 min @45°C (113°F), 15 min @52°C (126°F), 45 min @65°C (149°F), 30 min @72°C (162°F), 5 min @78°C (172°F)
Boil:		Total 120 min
Fermentation:	Primary	> 365 days @20°C (68°F) in glass fermenters
	Secondary	N/A
Maturation:	Carbonation (CO2 vol)	N/A
	Maturation time	N/A

I would like to give you a short overview about the whole turbid mash schedule first. Have a quick look at the schedule below. The whole process sounds really difficult but it is not. You basically need three different kettles and that’s it. To get you an idea what a turbid mash schedule looks like have a look at this Youtube video.

Fig 2: Mash kettle preparation with perforated bottom

06/28/12: Lambic brew day. I chose to do a traditional turbid mash this time. All started with heating up 41 L of water to approximately 45°C in the water kettle. I added my false bottom in the mash kettle to drain off the turbid mash later on (Fig 2). I then transferred 8.2 L of water from the water kettle into the mash kettle and mashed in (Fig 3). The whole mash was very, very thick. I then left the mash rest for 20 min at 45°C. The remaining water in the water kettle was heated up to a boil.

After the first rest at 45°C, I added another 8.2 L of boiling water to the mash. The temperature now was 52°C. Another 15 min rest.

Fig 3: First rest at 45°C

Fig 4: First turbid wort

After the second rest, I removed 5.5 L of the liquid from the mash kettle. This wort is called turbid wort. You can easily see why in Fig 4. The turbid wort was heated up to 88°C.

After the second rest in the mash kettle (and after removing the turbid wort), I added 12.3 L of boiling water. The temperature now was at 65°C. Rest for 45 min at 65°C.

As the third rest passed, I removed another 5.5 L of turbid wort and added it to the preheated turbid wort from the previous removal and reheated to 88°C again.

I then added another 12.3 L of boiling wort to the mash kettle. The temperature now was at 72°C. Rest for 30 min at 72°C.

Now was the time to heat up the 34 L of sparging water to 88°C.

After the rest at 72°C, I added the turbid worts back to the mash and the temperature rose to 78°C. I then left the mash rest for 5 min and sparged to a gravity of 2°P. By the way, the wort was iodine positive. Indicating some starches were still left in the wort.

This was the whole turbid mash schedule already. Adding hot or boiling water to the mash to increase the temperature is basically a decoction mash. The special step in turbid mashing are the removal of turbid worts. The turbid worts contain enzymes, sugars and starches. By heating them up to 88°C, the enzymes get denatured (destroyed) and can’t work anymore. This leaves the sugars and starches in this worts. The resting time of approximately 5 min at 78°C after you add these turbid worts back to the mash are not enough to convert the remaining starches and sugars. Meaning, you add some unfermentable sugars and starches back to your wort. These unfermentable sugars are quite important for the Brettanomyces and maybe some bacteria later on during the fermentation. Brewer’s yeast (Saccharomyces cerevisiae) can’t ferment unfermentable sugars and starches. I guess this why they are called unfermentable sugars after all. These unfermentable sugars however can be metabolized by the Brettanomyces and gives these yeasts an opportunity to grow during the fermentation process as these yeasts are rather slow growers.

Back to the wort. I boiled the wort for two hours with the addition of 240 g of old hops I stored for at least two years. Can’t remember the variety. I guess it was a German variety like Tettnanger, Hallertauer or Saazer. Then cooled the wort down, added two packages of Wyeast’s Lambic Blend and filled two 10 L, one 20 L and half a 10 L glass carboy with the wort.

Fig 5: Fermentation in progress…

06/29/12: I already had to replace all the airlocks because the fermentation was already in progress (Fig 5).

07/07/12: Lambic already nine days in the carboys. The fermentation calmed down and it was time to add the real souring bugs. I first added medium toasted French oak chips to the carboys (25 g per 10 L). I boiled the chips in some water first (approximately 2 min) and discarded the dark brown water. Then added fresh water again and repeated the boiling process once again. Then discharged the water again and added the chips to the carboys. Then added my own souring mixture called Milupa1. This mixture consists of the following bugs: Wyeast’s Brettanomyces bruxellensis, Wyeast’s Brettanomyces lambicus, some Girardin Gueze dregs, some dregs from Les Trois Dames’s Oud Bruin and a Brettanomyces strain I previously isolated from a Cantillon Kriek. This mixture was quite aggressive in some trial fermentations and a pellicle formed just after a few days. Good enough for me to let this mixture eat through some Lambic wort. I added some of the mixture to each of the glass carboys (except one) and left the wort to the new introduced bugs. The one without the Milupa1 bugs is a control to test how the single Lambic Blend from Wyeast works.

10/13/12: Lambic now three months in the carboys (Fig 6). The carboys on the left side and right side are the ones with Milupa1. The carboy in the middle has no Milupa1 bugs. The 20 L carboy is not shown. The Lambic already smells amazing. However, I haven’t tasted any of the Lambics. Still a long time to go…

Fig 6: Lambic after three months in carboys

I hope I could give you some insight into the whole Lambic homebrewing process and maybe give you some new information as well. I will keep updating this post in the future. Stay tuned!

Isolating the bugs from Cantillon Gueuze 2007

Fig 1: Cantillon’s Lou Pepe 2007 Gueuze

Eureka, this is another post concerning wild yeast isolation from a commercial beer. Today’s beer is Cantillon’s Lou Pepe 2007 Gueuze. I got a bottle of this particular Gueuze a year ago and stored it for another year in my cellar. By the end of June 2012, I finally got the opportunity to open the bottle and taste it.

Before heading into the tasting notes, let me give you some background information about the beer. The label on the bottle says: “Our Lou Pepe beers are all exceptional products. We only use the finest lambic to make these beers. The Lou Pepe Gueuze is a blending of only 2 years old lambics. Beer with tasteevolution. Best before 12/2029″ (Fig 2). It comes in 0.75 L bottles and 5 ABV. Bottled on the 12th of October in 2009.

Smell: Very funky and a lot of horse blanket, leather and barnyard

Taste: Very light sourness, pretty dry on the palate, grainy. Some lemon and wood notes as well. Rather nice sourness (no vinegar). Subtle notes of funkiness.

Appearance: Pours in a golden-yellow color, clear and a pretty nice white head. Not very long lasting head though. Very fizzy. Looks like a champagne

Mouthfeel: Light body, average carbonation, dry and astringent aftertaste. Some bitterness is there as well

Overall: Not bad and very easy drinkable. Not a very sour and complex Gueuze compared to others. However, a good example for the style. My rating: 80/100. I expected this beer to be more complex and less astringent.

Fig 2: Bottle description

I then streaked some of the bottle’s sediment on some Sabouraud agar plates and left the plates at room temperature for approximately three weeks until colonies were visible. I could observe two different kinds of colonies (Fig 3).

Fig 3: Cantillon’s Lou Pepe 2007 sediments on Sabouraud agar

The most colonies were similar to the whitish colonies marked as 2 in Fig 3. And there were some darker colonies (light beige) marked as 1 in Fig 3. Nearly two years after bottling the Gueuze there are still some living organisms in the bottle. The morphology of these colonies is very similar to other Brettanomyces I isolated before. I expect these colonies (marked 2) to be Brettanomyces. On the other hand, I have no clue what the microorganisms in colony 1 could be since the color is very different from Brettanomyces or Saccharomyces colonies. Maybe the micrographs give me further information? Next step was to do some microscopy observations of the two different colonies. Lets begin with the colonies marked 2 in Fig 3.

Fig 4: Micrograph of colony 2 (see Fig 3)

Fig 5: Micrograph of colony 2 (see Fig 3)

To me, the colonies shown in Fig 4 and 5 look like a kind of wild yeast. At least no Saccharomyces cerevisiae for sure. Or any other kind of bacteria due to the size of these cells. I expect these cells to be Brettanomyces due to the elongated shape of the cells and other characteristics. The cells could be Kloeckera apiculata, Pichia membranaefaciens or Hansenula… The list is not complete here. Further investigations are necessary. What about the other colonies?

Fig 6: Micrograph of colony 1 (see Fig 3)

Fig 7: Micrograph of colony 1 (see Fig 3)

First of all, the cells shown in Fig 6 and 7 look very different from the ones shown in Fig 4 and 5. These cells here are mostly circular and look very similar to Saccharomyces cerevisiae cells. Aggregation of Saccharomyces cerevisiae as it can be seen on the upper left corner in Fig 7 can be observed in wheat yeast samples as well. For me theses cells look like typical Saccharomyces cerevisiae cells although some cells seem to have a more elongated form as well. I will have to do further investigations to get more information about the cells in colony 1. Wouldn’t it be cool to have isolated some Saccharomyces cerevisiae yeast cells from an old Gueuze from Cantillon?

To summarize, I could isolate two different kinds of yeasts from a Cantillon Gueuze bottled in 2009. I have a strong feeling the cells from colony 2 belong to the specie of Brettanomyces. This is just a feeling. The strains go into my library as B05 (colony 2) and Y03 (colony 1). Further investigations are necessary to differentiate the two different strains. Cool stuff. The only verified conclusion here is: It is possible to isolate some yeasts from a Gueuze that is nearly two years in the bottle. Stay tuned for further yeast related posts!

Isolating the bugs from Cantillon Kriek

Eureka, another post about a Brettanomyces isolation. Other posts about the same topic can be found here. The beer we are talking about today is the Kriek made by Cantillon. Further information about the Kriek can be found on the Cantillon webpage. Got myself a bottle (bottled on 23 December 2009) and decided to have a look at the sediment of the beer. Maybe some tasting notes first.

Fig 1: Cantillon Kriek

Aroma: Lots of horse blanket and very funky. Could not detect any cherry flavor.

Appearance: Red appearance, pink foam. Some particles from the bottle in the glass.

Flavor: Light fruitiness detectable, right amount of sourness and a bit tart. Pretty neat!

Mouthfeel: Light body, average carbonation level, pretty dry and sour finish.

Overall Impression: Very well made. Although the fruity character is gone. The beer was bottled 2.5 years ago. Maybe the fruitiness vanishes with time? No idea if this is true. All in all a very nice brew. My rating: 95/100. If you can get yourself a bottle. One of the best Krieks I had so far. Although no pronounced cherry aroma.

05/17/12: Streaked some of the bottle’s sediment on a Sabouraud agar plate and incubated it at room temperature.

Fig 2: Cantillon Kriek dregs on Sabouraud agar after nearly 14 days

06/02/12: Colonies were visible on the plate (Fig 2). Only one kind of colonies. Seem to me like very typical yeast colonies. Took a colony from the plate in Fig 2 and re-streaked it on another plate. The colonies now looked quite different as you would expect from normal brewer’s yeast.

Fig 3: Cantillon Kriek yeast on Sabouraud agar after 14 days

Next, look at the colonies under the microscope.

Fig 3: Cantillon yeast

These cells are yeast cells for sure (due to the size and appearance). In my opinion those cells belong to the Brettanomyces species. And they look very similar as the Brettanomyces in Wyeast’s Roeselare Blend (shown here in the pictures at the bottom). Well the cells could be something different than Brettanomyces for sure. However, from the smell of the plate, the look of the colonies on the agar plate and micrograph and the time it took for colonies to appear, I would assign these cells to Brettanomyces.

To summarize, I could isolate Brettanomyces strain(s) from a Cantillon Kriek nearly 2.5 years after it was bottled. The strain goes into my library as B04 (Brettanomyces 04).

The next post concerning a Brettanomyces isolation will be about another Cantillon beer. Stay tuned!