Wild Yeast Project: Plating yeast from a Lambic

This experiment hopefully will finish Aim 1 of my Wild Yeast Project which is to isolate different strains of wild yeast. Aim 2 of the project would be to use these strains in forced fermentations tests to see which strains are ideal for fermenting homebrew. I have tried plating the dregs from other American Wild ales, most notably Russian River’s Temptation and Sanctification. Unfortunately, while these yeast grew fine if plated directly from the bottle, they did not survive the freezing process in glycerol at -80°C.

My attention then started to turn to other sources of wild yeast. Namely, Chad Yakobson of Crooked Stave and the Brettanomyces Project mentioned that Vinnie (brewmaster at Russian River) inoculates his barrels and brews with commercially available strains of Brett. To get something truly unique, he mentioned I should look to lambics brewed in Belgium.

Lambics are spontaneously fermented beers predominantly from the region of Belgium, though spontaneous fermentation could happen anywhere. These beers are made with a significant amount of unmalted wheat in a process known as a “turbid mash”.  This results in a wort with a high percentage of protein, allowing for the beer to have a foamy head and distinct wheat taste. The hops that are employed are also unique. Lambic brewers use aged hops that have lost their bitterness potential but not their antimicrobial properties. High IBUs will inhibit the growth of lactic acid producing bacteria which is a strongly desired quality in lambics. The wort is then transferred to a vented attic and placed in large open tanks called coolships (see picture above). This is the critical moment and birth of a lambic – once cooled, the wort is inoculated with local microflora found in the air. Once inoculated, the wort is racked to used wine barrels to finish fermentation (which could take 2-3 years). For more reading on lambics I suggest a great blog called Lambic and Wild Ale.

Fermentation of this style of beer occurs in stages depending on the growth of the fermenting organism. In the first stage, wild yeast (Kloeckera apiculata) enterobacteria contribute little to the beer’s final taste but are important in breaking down proteins in the beer. The second stage is where Saccharomyces cerevisae rapidly multiply and ferment most of the sugars into alcohol and some esters. In the third stage, lactobacillus becomes the focus and produces lactic acid giving the beer a twangy sour character. The fourth and final stage involves numerous strains of Brettanomyces which give lambics the characteristic funky character. It is the yeast from this final stage that I am interested in and can be found in commercial lambic bottles.

I utilized the beer trading forum over at Beer Advocate to hunt down a great lambic from Belgium. While I could get Belgian lambics from off the shelves, I wanted something unique and hard to find. I traded for a bottle from the famous Belgian lambic brewery, Brasserie Cantillon. While I originally traded for a bottle of Cantillon’s famed Bläbær (a lambic brewed with blueberries), the generous trader found out about my project and threw in a bottle of Lou Pepe Framboise from 2004 as well. This lambic was brewed with raspberries and is wonderfully tart with an aggressive sourness and classic barnyard notes. I have tried a bottle from 2006 which was less astringent, suggesting that the yeast present in the bottle are still active and will sour the beer for years to come.

I shared the bottle with some friends and isolated the yeast sediment at the bottom of the bottle. I used a sterile 10 ml pipette and transferred the dregs into a sanitized mason jar. As of now the dregs are still in my fridge but I will bring them in the lab for testing after the July 4th holiday. Here will be my protocol for plating:

  1. Transfer to a sterile 50 ml conical.
  2. Centrifuge the sample at 1000 rpm to clarify yeast from raspberry material. If there are multiple bands resulting from the centrifugation, I may need to isolate each of those separately.
  3. Count cells under a microscope
  4. Dilute cells enough to plate on selective media (might need to dilute to 100,000 fold).
  5. Plate on WLN/WLD and MYPG with and without Bromocresol green.
I will post the result in about a week and hopefully can start working on fermentation of different Brett isolates.

4 Comments

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4 responses to “Wild Yeast Project: Plating yeast from a Lambic

  1. Pingback: Wild Yeast Project: Lambic Plating Results | Brew Science – Homebrewing Blog

  2. Owen

    Hi,

    Again, I am reading your blog with interest. One thing that has popped into my mind is this…

    Why go through the trouble of centrifuging and using differential plates, when you could simply dilute the “detritus” from a commercial bottle, streak out a few plates using the diluted beer, and then cultivate the colonies that formed into separate containers. The assumption here is that each colony is formed by a single cell, and thus each colony would give you a pure (and potentially different) strain. Then, if you brewed small batches (ca. 1 Liter) of each under controlled conditions, it would then be up to your nose / taste buds to determine which strains you liked the best. Or am I missing your main goal?

    • You are absolutely correct on this thought and is what I have already done in my other posts. I suggests you check em out. Everything you mentioned in your comment is something I plan on doing once I isolate strains. You are not missing my main goal (you basically stated it) and you should check out Aim 2 and Aim 3 of the project.

      Centrifugation is a great tool actually. Yeast is several times larger than bacteria and thus will sediment at a faster rate. Depending on rotor speed (g forces) and diameter, one can isolate different organisms based on sized. Something that I’m thinking of playing around with is using a sucrose cushion to separate different centrifuged layers (each with different organisms). For my future posts I plate straight slurries.

  3. Pingback: Wild Yeast Project: Success! | Brew Science – Homebrewing Blog

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