After characterizing some new wild yeast differential media with Brettanmonyces lambicus and Brettanomyces bruxellensis, I decided to plate all of the samples that Garrett Oliver gave me. Just to recap, I’ve been collaborating with the Brooklyn Brewery on isolating wild yeast from lees of naturally fermented wine. Garrett has done a novel approach to beer secondary fermentation with wild yeast by using microflora found in spontaneously fermented wine. The samples he gave me:
- CR: Crochet Rouge Rose
- GL: Gross Lees
- TO: Weisse wort inoculated with gross lees. Currently a nameless and experimental brew.
TO on Brett Agar: Sample was diluted 1:1000 and 50 uls was plated. This sample had the highest amount of wild yeast. If you click on the image, notice the colonies that are greenish. These yeast metabolized the bromocresol green dye to a lesser extent and suggests these are unique strains. Since Brett agar contains chloramphenicol and cycloheximide, brewers yeast and bacteria should be inhibited.
TO on CuSO4 Agar: This was diluted as the sample before. There were fewer number of colonies on this plate, indicating that this media is far more stringent in isolating wild yeast. As before some colonies are different in color. Click on the image for a full size view.
Brettanomyces may not be the only type of yeast that can be found on the grape skins. For example, this article found species of Candida, Hanseniaspora, and Issatchenkia on the skins of grapes. Even regular Saccharomyces cerevisae is found on grapes and often becomes dominant in the later half of wine fermentation. This article in Wine Make Magazine describes differences between spontaneous wine fermentation versus inoculated and is a good read. In relation to my project with Brooklyn Brewery, any indigenous yeast would be of interest to Garrett, including Saccharomyces cerevisae. To this end, I made Brett Agar plates as in my last post but without the drugs chloramphenicol and cycloheximide. This should allow everything to grow and any potential indigenous Saccharomyces cerevisae should not be able to metabolize the dye. These should be greener in color compared to the other colonies (Brett).
The above photo is the TO sample with the Brett Agar (-drugs) on the left and Brett Agar (+drugs) on the right. It is pretty apparent that the dominant microflora is bacteria (shallow glossy colonies). This plate also smelled normal – no Brett funkiness. The plate on the right is basically a replicate of photos at the beginning of the post. It seems that bacteria, most likely lactobacillus, is present at high levels in Weisse wort barrels. Garrett expressed an interest in characterizing Brett samples (plate on the right) for a future brew.
Now here is where things get interesting. Make sure to click on the photo for a closer look. This is the gross lees (GL) plated on Brett Agar without drugs (left) and with drugs (right). Instead of bacteria, colonies are predominantly wild yeast. However, the left plate there are numerous colonies of different shades and colors suggesting different species of indigenous yeast. This diverse microflora reflects the final stages of the fermentation as the wine was racked off the lees. The plate on the right indicates only Brettanomyces growth.
I’m basically in the final stages of the project. have already picked a bunch colonies from both GL and TO samples to grow in small fermentations. Next I will post microscopy pictures on the cell morphology of the colonies I picked and run mini-fermentations (3 ml volumes).