Wild Yeast Project: Success!

It has been a while since I last posted about my Wild Yeast Project and for good reason. To refresh everyone’s memory, I embarked on a project to isolate either wild yeast or bacteria from a commercial bottle of wild ale, culture it in my lab, and characterize it for a batch of homebrew. My previous efforts involved culturing microorganisms from an old bottle of Cantillon Lou Pepe from 2004.  Unfortunately, this experiment did not go well. All of the colonies that I did isolate were coliform bacteria and not what I was particularly looking for – wild yeast. It turns out that the bacteria grew much faster and outcompeted any wild yeast on the plate.

After many months, and finally having the time to come back to the project, I tried again from another commercial bottle of beer. Again I went to a potentially good source of wild yeast and obtained a bottle of Cantillon Blåbaer (2010 vintage). This is a lambic beer and one of their more famous beers. Aged on blueberries from Denmark, Blabåer is a collaboration between a Danish bottle shop, Ølbutikken, and Cantillon. It’s sold only once a year at this shop in Copenhagen and the limit is usually two per person. It is not distributed here in the states and I had the fortunate opportunity to trade for the beer on Beer Advocate.

I opened the bottle almost two months ago, but saved the dregs (about 50 mls) in a sterile conical tube and stored it at 4°C. The first couple of experiments that I did were complete failures:

  • 20 μls of lambic slurry into 180 μls of sterile double distlled water (1:10 dilution).
  • Made a serial dilution all the way out to 1:1,000,000.
  • Plated 50 μls each onto a MYPG, MYPG + bromocresol green, and a WLD plate.
  • Incubated at 30°C for two weeks.
  • No growth.

OK, not a problem. I must have plated a sample that was too diluted. The next time I plated straight slurry to see if anything would grow. Nothing. For two weeks. At this point I was pretty concerned but confused. I could clearly see wild yeast and bacteria in the dregs with the lab microscope and they looked similar to the Lou Pepe samples. However, there was no growth on agar plates. As a last ditch effort I decided to inoculate 3 mls of wort that was filter sterilized (completely removes any bacteria) with 20 μls of straight lambic slurry. After four days of shaking at 37°C I got this:

In the 15 ml conical is what grew from the slurry after four days. I will post photos soon, but examining the yeast under a microscope revealed a wild strain that looked alot like Brett (small elongated cells). Also, the small fermentation in the tube was not from Saccharomyces cerevisiae as it smelled like sweaty gym socks dipped in vinegar! Another interesting thing to note was the yeast looked homogenous, as in they all looked the same. I wonder if I have one dominant strain?

Future:

I already have this experiment going to but I will mention it here. I made a serial dilution of the whatever I grew and plated it to get single colonies onto different media to better characterize what I might have. Colony morphology will tell me whether I have one strain or many. Also, I will post microscope photos of culture shown above. More importantly, one question remains to be answered and that is why did the strain grow on liquid media but not solid agar media?

9 Comments

Filed under Uncategorized

9 responses to “Wild Yeast Project: Success!

  1. Thanks for the comment over on my blog. I’ve responded there, but will also mention here to let me know if you want me to get details about the photomicroscopy set-up I used to take my photos – it was a set-up that a colleague let me use in his lab, so I don’t know the particulars off hand.

    Interesting experiments you have going on here with the wild yeast culturing. It is weird that you have had such poor success getting the cultures to grow, but I like your new tactic of growing up a “micro-starter”. Maybe the yeast from the dregs just needed a kick start to get going. Have you tried plating up the resulting culture? That would be a sure way to tell if there’s an issue with the solid media.

    • Hi Jim,

      I have plated it out and the sample appears to be mostly one strain of wild yeast though I can’t tell for sure. I dilution streaked that culture to isolate single cells. I’ll post about it pretty soon however.

      J

  2. Jason,

    In the 15 mL conical, I wouldn’t be surprised at the smell after only a few days. In the lambic fermentation cycle, enteric bacteria grows in the first few days and then yields to sacc-type yeasts. Its not uncommon to have some funky smells! Fermentation kinetics should be faster in a lab-scale inoculation, but your yeast is probably tired after several fermentations in the brewery, bottle, and storage in your fridge! I’d say hold onto it for another week and see what happens.

    When you got what you described as coliform bacteria on your plates, did you ever step them up and test them in wort? These could be pedio or acetobater (or perhaps lacto?), especially considering the pH change.

    These experiments are great to read about! Good luck!

    • Hi Kyle,

      Thanks for the comment. Normally I would agree with you, enteric bacteria present in the dregs should come up first and yield some funky smells. However, after looking in the scope I could not find any motile bacteria, not even relatively small non-motile species like pedio. It was ALL wild yeast and predominantly one strain after looking in the scope. I did not try to grow the coliform bacteria since I just didn’t have enough time and I wanted to focus on wild yeast. This is something for the future though.

      Keep an eye out for another post about what these cells actually are. Not my next post, but the one after.

      Cheers,

      J

  3. Pingback: Wild Yeast Isolated From Cantillon Balbaer | Brew Science – Homebrewing Blog

  4. First time here at these blogs-came here off of an image search for Brett, Dekkera, Schizosacharomyces since I’ve been privileged to grow up large amounts of pellicle forming rod shaped yeasts in a 3000 gallon tank of waste beer in the ’80s -Great stuff here!

    I wonder if diluting into sterile Distilled water wasn’t a thorough osmotic shock to the the yeast dregs (bacteria can recover if they are biofilmed or have protective exo-polysaccharides). I used dilution into water to swell and lyse yeasts so we could free up the liposaccharides for biosurfactants.

    Your move to a weak wort undoubtedly helped get you away from making little balloons out of your Bretts ;-)

  5. JohnE

    I’m interested in this experiment because I was planning on trying to recover some brett (probably a brett/S. Cerevisiae mix?) from a bottle of New Belgium Lips of Faith Brett. My first thought was to add wort (well, a DME soultion as I would for a starter), as I’m not set up for plating onto agar right now. My theory on why this would be successful is basic natural selection. These organisms have multiplied many times in fermentations, so they are adapted to that environment, whereas on the agar they are not as well adapted and are easily out competed by other latent bacteria.
    So, this gives me confidence that I might be able to grow up a decent amount from the dregs without agar. Now I just have to decide what to use it for!

    • John,

      So if you inoculate a DME starter with the dregs of a bottle, you are definitely going to grow the dominant yeast that is in that bottle. This is the million dollar question though – what’s in that bottle. For example, if one were to culture a sour ale from a bottle fermented sour ale from Brooklyn brewery, you might just get Champagne yeast since this is what they use to bottle condition their beers. So if that lips of Faith bottle has a priming yeast just to get CO2, that’s all you will grow.

      Also keep in mind that if there is Brett in the bottle (we assume there is) then these will probably outcompeted by regular sacch. if there is a higher cell count.

      When you do your starter, if it smells funky then you most likely have Brett. However, you still have a mixed culture.

  6. Pingback: Bringing The Funk | Modern Times Beer

Leave a Reply

Fill in your details below or click an icon to log in:

WordPress.com Logo

You are commenting using your WordPress.com account. Log Out / Change )

Twitter picture

You are commenting using your Twitter account. Log Out / Change )

Facebook photo

You are commenting using your Facebook account. Log Out / Change )

Google+ photo

You are commenting using your Google+ account. Log Out / Change )

Connecting to %s