Launching Eureka Brewing’s Yeast lab

Eureka, I am really pleased to announce that I finally have all the equipment ready to send out yeasts from my yeast library to other (home)brewers. My yeast lab will mainly focus on different Brettanomyces strains, other souring bugs, blends and any kind of not commercially available yeast strains. To have a look at my preliminary yeast program please visit the respective site.

Sources for further Brettanomyces strains

Please further notice that I don’t accept any orders. Currently, the strains are only available to collaborators for testing purposes. I plan to release strains on an irregular basis. All information concerning releases, costs, cell counts etc. are and/or will be available on the respective site.

Cheers, Sam

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A glimpse into copper sulfate agar

Eureka, I would like to publish some preliminary results from my latest plating experiments. I am still interested in isolating Brettanomyces from different sources and still play around with different agar media to see what their impact is on the entire isolation process. The latest experiment I performed was a large scale bromocresol screening on different Saccharomyces yeasts to see whether bromocresol can be used to differentiate between Saccharomyces and Brettanoymces. My insight from this experiment: bromocresol green as a tool to differentiate between Brettanomyces (known to grow as white colonies) and Saccharomyces might only work within a small time frame. In addition, some Saccharomyces strains grew as white colonies in presence of bromocresol green (possible false positive strains).

Yet another approach is to add copper sulfate to the agar media to inhibit the growth of domesticated yeasts [Yakobson, 2010, Taylor et al, 1984]. Wild yeasts therefore should be able to grow in presence of copper sulfate. I wanted to give this agar a go to see if it can be used to differentiate between domesticated Saccharomyces strains and wild yeasts (Brettanoymces in my case). I started by adding 0.6 g copper sulfate to 1 L of Sabouraud agar and streaked some strains on the plates. As controls, plain Sabouraud agar plates were used to test the viability of the strains (not all plates shown).

Fig 1: Saccharomyces yeasts on Sabouraud agar (1056 = Wyeast American Ale, 1084 = Wyeast Irish Ale, PtPtince = EBY049, Y05 = EBY050)

The four domesticated Saccharomyces strains plated on plain Sabouraud agar showed a nice growth phenotype (Fig 1). Streaking the same strains on copper sulfate containing Sabouraud agar revealed that only one strain (WY1084 Irish Ale) was impaired in its growth (Fig 2). All the remaining Saccharomyces strains grew as normal. From this observation one can already conclude that the addition of copper sulfate to the agar media impaired only 25% of the domesticated Saccharomyces strains tested.

Fig 2: Saccharomyces yeasts on CuSo4-Sabouraud agar (1056 = Wyeast American Ale, 1084 = Wyeast Irish Ale, PtPtince = EBY049, Y05 = EBY050)

Plating Brettanomyces and isolated Saccharomyces strains on copper agar media revealed a growth phenotype for all tested Brettanomyces strains (Fig 3, 4). Only the Saccharomyces isolate (B04 green in Fig 3) and the bacteria strain (I10 in Fig 4) did not grow on copper sulfate agar. Since B04 green was isolated from a Gueuze, it can be argued that this particular strain might be a non-domesticated Saccharomyces strain. On the other hand, it might be a domesticated yeast strain concluding from the lacking growth on copper sulfate. Including the previous observation that only a small part of domesticated Saccharomyces strains were impaired in their growth makes it even harder to allocate the isolated yeast strain to domesticated or non-domesticated Saccharomyces.

Fig 3: Different yeasts on CuSO4-Sabouraud agar (B04 = EBY004 Brettanomyces, B04green = EBY041 Saccharomyces, B05 = EBY005 Brettanomyces, B02 = EBY002 Brettanomyces)

Fig 4: Different yeasts/bacteria on CuSO4-Sabouraud agar (B01 = EBY001 Brettanomyces, I10 = EBY024 Bacteria, I05 = EBY009 Brettanomyces, I11 = EBY013 Brettanomyces)

This small-scale experiment revealed that a copper sulfate addition to Sabouraud agar media does not impair most of the domesticated Saccharomyces strains tested. All the Brettanomyces strains tested in this experiment grew in presence of copper sulfate.

It seems to me that copper sulfate used at a concentration of 0.6 g per liter of Sabouraud agar media was not useful to differentiate non-domesticated from domesticated Saccharomyces yeasts. Simply because it could not inhibit the growth of most of the domesticated yeasts tested. As an outlook, one might increase the concentration of copper sulfate to levels where it impairs most of the domesticated Saccharomyces strains. Then test the Brettanomyces under the same conditions and see if they still grow or not. Maybe even change the Sabouraud agar to MYGP like published by Taylor et al. It is not clear to me yet if I even further investigate the use of copper sulfate.

References:

• Yakobson C. (2010) Brettanomyces project, http://www.brettanomycesproject.com/dissertation/analysis-of-culturability-on-various-media-agar/brettanomyces-identification-methods/
• Taylor GT, Marsh AS (1984) MYGP+Copper, A medium that detects both Saccharomyces and non-Saccharomyces wild yeasts in the presence of culture yeast, J. Inst. Brew., Vol 90, p 134-145

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!

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!

Isolating the bugs from Trois Dames Oud Bruin

Eureka, it has been a while since the last post about isolating some wild bugs from a commercial beer. Luckily, there are still some beers in my cellar with bugs I would like to isolate (Orval, Lost Abbey, BFM, Cantillon, Jolly Pumpkin…). The beer I am talking about today is from a Swiss brewery called Les Trois Dames (The three women). This brewery produces an Oud Bruin (Flanders Brown) made with apricot wine. The beer comes with an ABV of 7.2%. Maybe some of my tasting notes first.

Aroma: Sour and funky smell. Pretty awesome! Could detect some cherry notes. In my opinion one of the best smelling beers in Switzerland…

Appearance: Red-brown color, clear appearance, 1 finger tan head with some carbonation. Nothing special here.

Flavor: Now begins the fun part… Unfortunately, the complexity of this beer is very limited. I could detect some sour cherry notes. The sourness is just right. Not too overpowering. However, nothing else. Not even a hint of apricots…

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

Overall Impression: There is the right amount sourness in this beer and the aroma of this beer promises a lot. Unfortunately, the flavors speak another language. The beer has a very limited complexity in my opinion. Maybe the beer was too young? Only a second tasting could tell. To summarize, this is a pretty nice beer and because this is a beer made from a Swiss brewery, it is a winner for me for sure. I would give this beer a rating of 85 out of 100. I am a bit disappointed about the lacking apricot notes. I should get myself a bottle of apricot wine one day and compare the apricot flavors there. Maybe the apricot flavors are already very subtle in the apricot wine?

Anyway, this post is about the bugs in the beer. What I did is already a standardized technique for me to isolate some bugs from such a beer. I made myself a small DME (dry malt extract) starter and dumped the dregs of the beer in there and left it for some days. Approximately a month later, I plated some of the starter liquid on some Sabouraud agar plates and waited… Eleven days later, there were some colonies visible on the agar plate (Fig 2).

Fig 2: Sabouraud agar with colonies of Les Trois Dames Oud Bruin bugs after eleven days

The colonies morphology: White, not glossy, wavy margin, convex elevation, circular and the plate had a hint of an acetic acid smell. The acetic acid smell already made me wonder what I got myself here. I then picked a single colony and had a look at it with my microscope.

Fig 3: Les Trois Dames Oud Bruin bugs

Fig 4: Les Trois Dames Oud Bruin bugs

First of all, due to the size of the cells, I assume that all the cells visible in the following pictures are yeasts. I could observe some oval formed cells and some with the mysterious vacuole (Fig 3). And yet again, some cells which adhere to others (Fig 3). For me some of the cells in Fig 3 look like normal brewers yeast (Saccharomyces cerevisiae) and others don’t. Then again, I could observe some elongated cells (Fig 4). I already have an idea about these cells but will have to do further research first. Lets move on to the picture at a higher resolution.

Fig 5: Les Trois Dames Oud Bruin bugs

There are these dark spots again which I could observe in Brettanomyces bruxellensis before (Fig 5). My conclusion from the observations. I assume that I got myself some Brettanomyces bruxellensis and maybe some other Brettanomyces species. Some Saccharomyces cerevisiae could be in there as well. I guess this is all I can tell you about the bugs right now without doing any sophisticated lab tests. Nevertheless, I once again could show that it is possible to harvest some wild yeasts from a bottle. My next steps concerning wild yeasts will be further investigations about the elongated cells as it can be seen in Fig 4. There is a hypothesis in my head what these cells might be and I already found some promising evidence for my hypothesis. Another running project is to isolate the bugs from a Cantillon Kriek. Will post about the results for sure. By the way, the starter I made with the dregs from the Les Trois Dames bugs had no pellicle. Stay tuned for further yeast ranching experiments. Cheers!