Wild Yeast Strains from Natural Wine Lees

In my last post, I plated the gross lees from naturally fermented wine on agar without and with drugs that will kill non-Brettanomyces species. In one plate in particular, I got several different types of colonies that metabolized the bromocresol dye at different rates, suggesting different species of wild yeast. After picking some colonies to grow in culture, I will link some photos of their morphology in this post.

To refresh everyone’s memory, Garrett Oliver, brewmaster of Brooklyn Brewery, has used the lees from naturally fermented wine for either a primary or secondary fermentation of wort. Some of these beers are described here. His overall goal is to isolate wild yeast from spontaneously fermented wine to eventually brew wild beers. Interestingly, spontaneously fermented wine can have any number of yeast species depending on the grapes used in the must and where they were grown. This includes species such as Saccharomyces, Pichia, Candida, and Brettanomyces.

For these photos, I picked one colony from the TO sample and five colonies from the GL sample. GL stands for Gross Lees and is taken directly from the fermented must. I’m only showing one colony of TO since all of the colonies that I picked from this sample looked exactly the same:

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The cells are very different from I expected compared to Brettanomyces bruxellensis. These cells are not long and sausage-shaped, but more ovoid. They do not appear as round as Saccharomyces cerevisae.

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GL-1. Although the yeast in the sample was diluted, cells that are clumped together appear slightly elongated while cells alone in suspension are more ovoid. This is very distinct from TO-1

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GL-2. This was the only sample that have very long shaped cells, but in very low abundance. Could this be the teleomorph (sexual reproduction) stage of the yeast.

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GL-3. Similar to TO-1.

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GL-4. A bit more elongated and thicker (see cell in above right corner).

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GL-5. Very similar to GL-3.

Conclusions

Hard to say what strains I have. All of these strains have different abilities to metabolize the bromocresol dye suggesting different strains. Some of the colonies look different, but some are the same, as in GL-3 and GL-5. I cannot rule out that I’ve isolated the same strain either. These cells look very different from the Brettanomyces that I have isolated from Lambics. More importantly, I was hoping to get some wild Saccharomyces in these samples but I found none, since all the colonies that I grew all had the classic funky Brett character. I refuse to believe that the gross lees has no Saccharomyces since it must appear during spontaneous fermentation of wine. My growth media might be missing them somehow and I may have to make media selective for only wild Saccharomyces. Importantly, I’m coming to realize that just looking at cell and colony morphology is not enough to ascertain what these strains are. To truly identify these strains from different classes of fungi, PCR genotyping and growth on limited carbon sources might be the way to go.

Future

I will next take these six strains (same or different) and run some basic fermentation trails to test their ability to ferment wort. I may also include coumaric acid as a test for Brett character since it worked well on my previous plates. As an alternate approach, I am going to inoculate the gross lees with sterile wort, stimulating whatever is growing there. Hopefully, any Saccharomyces that is there will grow faster than Brettanomyces.

6 Comments

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6 responses to “Wild Yeast Strains from Natural Wine Lees

  1. Keep it up! Looking forward to further progress.

  2. Congrats on the realization of how inconclusive a microscope is, I;m not joking.. Pure cultures are impossible to decipher with a microscope as your cell morphology will change daily. So if you took photos of the same strains the following days you’ll find different cell shapes and it will look like a different yeast even though it’s cells from the same single organism. This is why I choose media and used giant colony morphology. You will be able to tell from that if it is Sacch/Wild Sacch, Brett, Pichia, Candida as they all form quite different morphologies. Then you can better choose which mediums to use to test for a more strain specific differentiation. At the end of the day PCR will tell the most.. Remember as well wild Sacch will have POF+ genes and could produce phenolics so the p-coumeric acid test is not always a sure fire way either.

    A few of your photos if not most look like Brettanomyces. B. brux can have small lemon shaped cells. Most of the Brett strains I use to brew with are the smaller lemon shaped type cells.

    Compare your photos.

    http://www.brettanomycesproject.com/gallery/brettanomyces-yeast-cell-images/

    Sacch is also a pretty weak organism so some cells might have died already, I know it seems un-likely but cell death can happen quickly especially if you have K1 strains which are killing off a lot of the native yeast first present. Can you get fresh lees from the winery?

    Chad

    • Hi Chad,

      Nice to hear form you again, thanks for commenting. Yes, I agree giant colony morphology may be the safest bet. However, after growing my colonies all seem to have the same shape just differences in color due to Bromocresol dye metabolization.

      Are there clear differences between giant colonies of wild sacch and Brett? All of my giant colonies are smooth and round in shape and differ only by their ability to metabolize Bromocresol.

      Going back to fresh wine lees may be the best chance of getting wild Sacch.

      Jason

    • Nice comment, Chad. I think everyone who uses microscope would agree with you, even if they look at tissues. Because cells just look how they want to look and there is nothing you can do about it :)
      Metabolic assays and PCR are indeed the best way to go.
      I recently read a paper where this group analyzed the microflora of American Coolship Ales (same thing as Lambic, made the same way, just made in the States) and they found that Brett. b pretty much dominates above all after about a year or so, with occasional Candidas making an appearance. I would imagine it’s probably very similar situation with wild wines. Then again Brett. b is a very large family with dozens of strains with different characteristics (also got a paper somewhere with metabolic assays of 47 Brett. b strains).
      Fun fun fun!

  3. Pingback: Packin’ Porter | Brew Science – Homebrewing Blog

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