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.
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.
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.
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.
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.
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.
– 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!