Cascade Kriek Bug Isolation Part I

Greetings, fellow yeast mates!

First of all I’d like to wish you all happy holidays and I hope the New Year will bring you all only joy.

As the last post of this year I thought I’d do a little progress report on bug isolation from dregs and even beers that some of you were nice enough to send me.

I think of most interest to you will be the progress of Cascade Brewing Barrel House dregs because of the Lactobacillus brevis strain that they inoculate their beers with. Even though L. brevis is commonly thought of as a hop resistant species that produces aggressive acidity in beers it isn’t exactly right and some strains of L. brevis are killed by hop acids. Genetics of lactic acid bacteria hop resistance are interesting and are not bound to a particular species, but that will be discussed in a future post, which I’m already working on. In any case let us see what’s been happening at the lab these last few weeks.

Cascade Kriek

From their website: Our Kriek spends over six months in lactic fermentation and aging in oak barrels. This NW style sour red ale is fermented for eight months with fresh whole Bing and sour pie cherries.

Those who have had it will know that it’s very sour and aggressive. Actually when I had it before I became interested in reculturing and thus just poured away the dregs, I thought my tongue and tooth enamel were dissolving with every sip. Sounds like a very good source for that particular Lactobacillus strain, doesn’t it? Luckily, ‪Ryan Wagner from KROC (Keg Ran Out Club) had a culture of it growing in his yeast ranch and was kind enough to send some during the Cantillon Brettanomyces sharing event. Per his description, the culture was maintained at practically ideal conditions for Lactobacillus maintenance and produces a clean, strong and bright lactic character in wort within just a few weeks after inoculation with flavors and smells reminiscent of apricots. It was really exciting to get my hands on this culture because it sounded very promising. Shortly after receiving the culture I streaked it on some plates and took a peak at it under microscope. It was immediately apparent that the sample is practically loaded with Brettanomyces. This was a little strange since, as far as common knowledge goes, they do not use Brett in most of their beers. I contacted Ryan the same day and he confirmed that the culture contains only whatever was raised from the Kriek dregs. After that I contacted Brandon at Embrace the Funk since he asked to keep an eye out for Brett in Cascade beers because their flavors seem too “bretty” to be just regular Saccharomyces. He was not very surprised and said that Chad Yakobson also saw Brettanomyces in Cascade beers and this confirms it. The only logical conclusion as to the origin of this yeast in the beer then would be that it’s wild and got there from the barrels or the cherries used during fermentation and aging.

Micrograph of the Cascade Kriek culture. This definitely looks like Brettanomyces to me.

Micrograph of the Cascade Kriek culture. This definitely looks like Brettanomyces to me.

After a few weeks on different agar plates I ended up with three types of colonies that you can see below in the 12-3 o’clock portion of the MYPG+BG+p-C plate. These colonies look much better on other agars, but I’ll refrain from telling you about those for now for the sake of staying on topic. One seems to be Brettanomyces – medium to large, round, glossy, convex colonies. Another looks like Saccharomyces – round, flat, and matte. Perhaps it’s just some contaminant because those are very rare. The third looks like Lactobacillus – small, irregular/filamentous, colorless/off-white/yellowish. Each organism was inoculated into a small primary culture and will be used later to raise bigger ones.

MYPGBGp-C plate with various cultures streaked in quadrants.

MYPGBGp-C plate with various cultures streaked in quadrants.

Saccharomyces?

Saccharomyces?

While looking at yeast is fairly straightforward, looking at bacteria is a lot more difficult. These organisms are a lot smaller and my usual 600x magnification doesn’t suffice at all. They are so small and there is so little to them that the light just goes straight through and around the cells, making them difficult to see and requiring some playing around with the light, the condenser and the light filters on occasion. Here you can see micrographs taken at 1500x magnification, which is 2.5 times greater than that at which I usually take yeast pictures. They still look smaller than yeast at 600x so imagine just how tiny these guys are. Staining also helps a great deal. Compare the images of unstained and methylene blue stained cells and what difference in viewing it makes.

Unstained Lactobacilli

Unstained Lactobacilli

Methylene Blue Stained Lactobacilli

Methylene Blue Stained Lactobacilli

While I was at it, I also took some high power images of the Brettanomyces from this culture and even captured a short real-time video where you can see what I think are vacuolar granules or fat globules moving inside the cells and me playing with the focal planes a little bit.

Cascade Kriek Brettanomyces

Cascade Kriek Brettanomyces

Cascade Kriek Brettanomyces

Cascade Kriek Brettanomyces

Cascade Kriek Brettanomyces

Cascade Kriek Brettanomyces

Cascade Kriek Brettanomyces

Cascade Kriek Brettanomyces

Cascade Kriek Brettanomyces

Cascade Kriek Brettanomyces

These organisms don’t seem to metabolize bromocresol green and stay as green colonies on BG-containing plates. That is not really a big deal since not all Brettanomyces species possess that ability anyway.

What is curious, though, is that the bromocresol green containing colonies did not remain blue, but turned green-yellow. There are about 4 other Brettanomyces strains on that same plate and they depleted the dye from the entire plate, but some of it stayed in the colonies. You can even see some blue colonies that did not turn green or white. Other than being a dye BG is also a pH indicator changing color from blue to yellow as it becomes more acidic, and as I discussed in the “Size Matters?” post, in aerobic conditions with available glucose Brettanomyces may produce acetic acid. The pKa of acetic acid is 4.76, which is exactly enough to turn BG green-yellow. This could also explain the yellow-green color of Lactobacillus colonies. The pKa of Lactic acid is 3.86, which is exactly at the border of where BG spectrum turns completely yellow. Of course right now it’s all just speculation.

Findings so far:

–       Cascade Kriek contains a lot of what seems to be Brettanomyces, which may account for the fruity character that results from fermenting with it. Not only does Brettanomyces produce fruity esters during fermentation, but it also produces fruity compounds during its acid breakdown as a survival action (see my “Size Matters?” post) and Lactobacillus provides plenty of acid.

–       This strain of Brettanomyces does not seem to metabolize bromocresol green.

–       The presumptive Lactobacillus cells are short and thick, which is a typical look of L. brevis. For example L. delbrueckii cells are longer. Sorry I don’t have my own images to show you for comparison because my entire L. delbrueckii stock died. Slightly off topic, but don’t keep your Lactobacillus and Brettanomyces cultures in the fridge. They seem to die surprisingly quickly in the cold and it’s better to keep them warmer and passage once in a while. But that’s for another post. Here is a nice collection of pseudocolored SEMs that you could look at for comparison, just keep in mind the scale differences (L. delbrueckii is a subspecies of L. bulgaricus.)

–       These bacteria are aerobic (another L. brevis trait) and grow much better in aerobic conditions.

–       Both organisms seem to produce acid in aerobic conditions with glucose as the carbon source.

Future directions

–       First and foremost I’ll try to test the hop resistance of these bacilli and, hopefully, confirm their beer souring ability. There seemed to be a chance to do genetic testing of that trait for a while, but I don’t think it’ll play out in the end and it’ll have to be done by a stepwise increase of IBUs in the growing medium. Luckily I saved a few hundred mL of a 120 IBU IIPA back when I brewed it and it’s been frozen for a few weeks just for that purpose.

–       Irregular shape of the presumptive Lactobacillus colonies suggests motility (ability to move), which is a trait that L. brevis is supposed to possess. For example Lactobacillus delbrueckii that is readily available from WYeast or White Labs is non-motile and forms round colonies. Just to be sure I’ll make some motility agar tubes next time I pour plates and stab them.

–       Gram stains to confirm that they are gram-positive.

–       What’s a little troubling is that these bacteria do not seem to produce acid when given lactose as the sole carbon source. Both presumptive Brettanomyces and Lactobacillus grow very happily on lactose, though not as quickly as on glucose, but do not produce any acid. Perhaps anaerobic conditions are required and I’ve already put them into an anaerobic chamber to see what happens.

–       The seeming acid production from glucose seems intriguing and will have to be done in liquid cultures with proper pH measurements and indicators.

Sounds exciting, doesn’t it? I’m curious to see what results will turn up from this little study.

The Kriek culture is just one of the few that you guys sent me to analyze and each one deserves attention and work. Sounds like a busy year already! Thanks again to all my readers for your support, collaborations and yeast trades and I wish you well. Some of you, however, didn’t send the cultures that you promised or stiffed me on the shipment costs, and for that may your beers be infected and not in a good way!

Happy Holidays!

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13 thoughts on “Cascade Kriek Bug Isolation Part I

  1. Great post! The detail you put in is wonderful. The first image looks more like hyphae. Does Brett chain instead of bud later in it’s growth cycle? .

    • Some strains make pseudohyphae as far as I know. I notice they do that when they’re maintained in liquid for a time with lots of nutrients and simple sugars. You can see that in some of my previous posts when I tried growing some Brett in Luria Broth.

  2. Hi Dmitri, really nice post. I agree with all your micro-organism genus assignments based on your micrographs. I only have one question. Do you incubate your agar plates containing bromocresol green at a dark place? If you don’t maybe the color change from green to yellow might be due to photobleaching? A easy way to test for acid production is to add calcium carbonate to your agar media. If acid is formed, the agar around the colonies turns clear.

    By the way, I successfully revived all my nine frozen Brettanomyces strains after spending nearly 4 months in the refrigerator. Will publish a post about that in the future. However, I transferred the yeast pellets from the cryo stocks into WLP media and it took the yeast nearly three to four weeks until they showed signs of fermentation. I even took micrographs from all the WLP media to be sure that only Brettanomyces are in there. The colonies in the WLP media look like the colonies before freezing. I just streaked all the different strains on agar plates to test if the yeasts are indeed viable and now wait for colonies to form. I haven’t revived the frozen Lactobacillus strains so far…
    Keep up the great work!
    cheers, Sam

  3. Yeah I just kept picking up these different Brett type characteristics in the beers I tasted from them. I ‘m sure its just in their oak now and got there as a truly wild occurance, It’s nice to see my suspicions confirmed in the lab though!

  4. Great work on breaking down the culture. I think the Brett must come in with the cherries as none of our other beers produce such acid levels. Long term aging on the fruit gives it the opportunity.
    Servus! Ron

    • Actually Brett doesn’t produce much acid. It’s mainly contributed by the lactic acid bacteria. Check out my previous post “Size Matters?” On a little more info about it.

      • BKY, The base beer for the Kriek has already been soured by our house strain of Lactobacillus prior to being put on the fruit. What is the likely pathway to this higher level of acidity than is in our other beers?
        Servus! Ron

      • Hmmm… Good question, Ron.
        Is that Lacto strain used in other beers as well, but doesn’t produce such acidity?
        Knowing what I know about your beers (and it isn’t much at all since I’ve only seen them twice in my life – the Kriek and the Apricot) I’d guess souring it first with Bacteria makes it sour to begin with, and by the time you add the cherries there are still a lot of Lactobacillus cells left in suspension. Since they are small, simple, and multiply at a greater rate than yeast, they overtake at that point as well and convert the sugars from cherries to lactic acid. As far as I know L. brevis (that’s what you use, right?) is one of the slower growing members of the family, but it should still be able to overtake the yeast anyway. I really can’t tell because I don’t know your process. What yeast do you use and how is it pitched relative to Lacto both time-wise and density-wise?
        Brettanomyces probably comes from barrels and fruit and for sure adds its beautiful touch, but acid production is a common misconception with brewers. As you probably know, all-Brett beers are quite mild and fruity without the sharp acidity. In conditions of oxygen and glucose availability some Brett strains produce acetic acid (vinegar), but not high levels of it and from what I remember of the Kriek there was barely any hint of vinegar. In beer and wine it’s usually Acetobacter that converts ethanol into acetate (alcohol to vinegar) and thus ruins the product. During conditioning the environment of the beer, especially a sour, is very unwelcoming to most organisms due to low nutrient availability, high waste product content, and high acidity. This suits Brettanomyces because they are naturally slow growers and don’t meet much competition at this point. In order to survive in the acidic environment, Brett actually starts breaking down the acids and other byproducts and turning them into various funky phenolic compounds and fruity esters.
        What it comes down to, essentially, is the natural aggressiveness of your Lacto strain that is fed more with abundant sugars from the cherries plus the acidity of the cherries themselves. Brettanomyces most likely plays an auxiliary role in the flavor and aroma profiles, but not additional acidity production. I could be wrong, of course, but that’s just what I think.
        Have you ever tried keeping the base beer in same conditions and keeping for the same time, but without adding cherries and then tasting the cherried and uncherried versions side by side? It’s really hard for me to guess what’s going on without knowing how its process differs from other beers. If it’s a trade secret, that’s fine and I understand. Or you can email me if you’d like to continue this discussion in private.
        Also if you happen to have some extra slurry from your barrels or fermentation vessels from various beers that you’d like to share and/or have me look at, I’d be more than happy 🙂
        Cheers!
        Dmitri

  5. Hi,

    I also have cultured the dregs from a Cascade sour beer (2009 Sang Royal).

    Here are my findings:
    I used plates containing cycloheximide as I was only interested in isolating the bacteria. I did not observe any brettanomyces growth. This is interesting, but I suppose different beers could be inoculated with different microbes.

    I observed 2 distinct bacterial colonies that I subsequently streaked to obtain pure cultures. Both were Gram positive, catalase negative rods. At first look, I thought I had just ended up with 2 isolates of the same bacteria, but upon closer evaluation, I realized that the rods were distinctly different lengths. The short rods I am assuming are L. brevis. I am not sure what the others are. I expanded each on a plate and the inoculated some apple juice (10P). After a few weeks, the juice with the shorter rods was quite sour.

    I am currently trying both isolates in a malt-based solution. It would be interesting to confirm the use of L. brevis as it is not the most often used in sour beers. However, it would make sense as it can be resistant to hops and can be “trained” to live in high alcohol environments.

  6. Does anyone know what type of saccharomyces strain was found in the dregs? I stepped up the dregs from a Cascade Strawberry and pitched the starter into my wort as the sole/primary yeast/bacteria for the fermentation. The 5 gallon batch took off within 5 hours. I just hope this batch wont be a waste because Cascade bottles with wine/champagne yeast. I’ve heard on random forums that cascade bottles live and someone even said an employee told them to go ahead and make a starter. If anyone has any input on this please let me know. I love Cascade’s sours and want to try to get a similar profile using their strains. I called the barrel house but they weren’t willing to give up any info. The ferment seems very healthy and smells nice but it’s only been a week. No way of knowing unless Ron or someone else chimes in on this one. Happy brewing!

    • I know a little of their process, but Ron asked me to not disclose it. I’m going to honor this request, and even though I don’t know the exact strain they use, I know what general ale category it’s in. I’d say go ahead and see if you can propagate the culture. I’m sure you’ll like it.

  7. I am currently disecting Cascade Vine to test the hop resistance of their Lacto strain. I pitched the dregs into 3 jars, one with lactose, one with maltose and one with dextrose. All were incubated in a water bath around 110F. Within 24hrs dextrose had hit pH 3.8, while maltose and lactose were around 4.3. Within 48hrs matlose was at pH 3.6, dextrose at 3.8 and lactose 4.3. I then made up a mix of maltose, dextrose, yeast nutrient, dead yeast cells and loaded with citra hops. I inoculated a jar with the cascade dextrose culture and another jar with a culture propagated from malt. After 24hrs of incubation both were at pH 4.3. I will continue to monitor them for the next few weeks/months.

    I too observed a brett strain in Vine under the scope. I will post some pics when I put up a post on my blog.

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