Make Yeast Collection Wort Vials
For collecting the yeast I made a fresh wort and put 10 mL in 15 mL tubes using a sterilized burette pipette. I wanted the 5 mL of head space to allow space for any krausen formation and so that when I mixed the wort I could move a fair amount of oxygen into the liquid to get the yeast going.
Collection Wort Recipe (~1.04 gravity):
20 g DME (Dried Malt Extract)
180 mL Distilled water
boil for 1 hour and then transfer to tubes and allow them to cool before using. Label each 15 mL tube with the date of creation.
In this mock up I made vials that I added 100 ul of Vodka (40% by volume ethanol) to 10 mL of wort to see if a very low amount of alcohol (0.4%) could help select for yeast over other organisms and denoted these vials with a B.
Collection Tool Kit
1) 70% rubbing alcohol in a spray bottle - used for cleaning hands and tools. I would use 70% ethanol but its harder for me to get.
2) Knife with scissors - used for cutting sections of fruit etc to get in vial. Sterilized every time before use
3) Ziplock bag with napkins - used for cleaning tools with alcohol
4) Yeast Collection Wort Vials - always visually check them before heading out, just in case of contamination. The fresher the batch you can make the better, but vials should be able to be kept for 1-2 months at 4C.
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| Field collection kit |
Field Collection of Yeast
I went to Lake Merritt's gardens in downtown Oakland to hunt for wild yeast. I looked for fruit and flowers since they would be potential sources of sugars and complex carbohydrates that might attract yeast. Before each sampling I would spray my hands, the top of the vials and the knife with alcohol. For fruit and flowers that were small enough I would use the alcohol treated knife/scissors to deposit fruit into the Yeast Collection Wort Vials. I would then screw on the top and then shake vigorously for 30 seconds in order to try and get any yeast present into solution and also aerate the wort. I immediately labeled the vial with the date of collection, a unique identifying number, and a comment about the collection.
Controls - upon getting home I used 1 ul of German Hefeweizen yeast from White labs as a control to make sure that the system was a viable condition for yeast. Since I wanted to see if the addition of alcohol (vials labeled B) had any selectivity I added 1ul of the White Labs yeast and 1ul of Lactobacillus from yogurt to a few vials.
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| Vials ready to start fermentation |
Fermentation
Ideally I would have an incubator that I could control the temperature of the collected materials, but my apartment does not afford me this luxury, so I simply placed the vials in a jar in relatively dark space and let them incubate. I checked them about every other day to see if there were obvious signs of fermentation (krausen, bubbles, etc). It took almost three days before I saw bubbles coming from the bottom of the tubes and krausen formation in the controls. The photos below are all from three days after sampling. |
| Controls made with Hefeweizen Yeast. Notice the krausen and bubbles |
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| a) Hefe yeast - krausen present b) Grape - krausen present c) Catctus fruit - mold present, no krausen |
I was expecting the fermentation to take about 2 weeks, but I was surprised to see some still going at that time so let them go a full 3 weeks before moving onto the next step - sampling and plating.
Plating and Sampling
Part of the reason for choosing a 10 mL ferment for the collection vials is that there is enough liquid to taste multiple times and still plenty to either start a new fermentation with or streak out plates. Notes on each sample are in the summary table at the end of this blog and I will just highlight the few that I think are noteworthy here. The grape was just as expected. It was boozy in flavor and had a grappa like nose to it. Microscopy showed plenty of health budding yeast cells. I really was hoping the burgmansia would come through, but it was lack luster, not much taste compared to the wort control and the scent was reminiscent of the flower but not as intoxicating. The orange flower was the one that caught me off guard. Upon opening the vial it had much more pressure then the hefeweizen yeast controls and the scent, oh the scent! its smelled just like the flower. The flavor was like a warm fermented saison and microscopy showed a healthy number of budding yeasts. After getting a feel for each ferment I then decided to streak out the material on DME Agar plates.DME Agar Plates
30g DME
5 g Agar
1g Fermax Yeast Nutrient
264 mL distilled water
boil for 10 minutes in an attempt to completely dissolve the agar. Pour into 5.5 cm diameter petri dishes. This made approximately 15 plates. I tried to make a relatively sterile place to pour by cleaning my bathroom floor with 70% isopropanol alcohol and then putting down a small sheet of aluminum foil to work on and cleaning my hands and all tools with isopropanol. I let the plates set up for 1 hour and then place in the oven at the lowest setting to help the plates dry out. All plates were labeled with a pour date. These plates should be able to keep 1-2 months at 4C. It might be helpful to add ampicillin to these plates if you are just looking for yeast, but I was curious to see everything that grew from these collections. I believe it would also be helpful to boil the material for ~30 minutes to one hour before pouring as I think some contamination I saw on control plates was due to spores in the DME, agar, or yeast nutrient.
To some of the plates I added 50ul of the fermentation culture and then used 6mm sterilized (boiling water follow by isopropanol) to spread the plates. 50ul turned out to be way to much and 1 ul would probably be a better spreading concentration.
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| Orange flower yeast plated and under the microscope |
Juniper Berry yeast - looks like a Brettanomyces due to the pseudohypha
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| Left to right: Budding yeast from Orange flower, Bacteria clusters from Burgmansia, Fungal hyphae from mold in cactus fruit ferment. All 400X with inverted scope. |
Next step is to get single colonies from these cultures and then to scale up fermentation from these single colonies by transferring krausen to select for faster fermentinng yeast...or at leas that is the theory! I will start with 500 mL or 1 L ferementers so that they are small
A = regular wort
B = wort + 0.4% ethanol
Isolation of single yeast cell (colony)
From the streaked plate a loop was used to get just a small amount of yeast and then used quadrant streaking technique (use the loop first in one quadrant, turn it or clean it, run it through first quadrant into next, repeat until 4 quadrants touched) in the hope of getting a single colony that could be picked and propagated. |
| single colonies of Orange Flower Yeast |
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| 1.5 mL tubes with DME Agar. Orange Yeast inoculated "mini stab" |
Currently working on making stabs of the other yeast isolates from grape and juniper and then the big task: figuring out how to domesticate these yeast. Here is the best paper I've read on domestication of yeasts and what happens to their genome over the process of domestication.
http://www.cell.com/cell/
The biggest take homes are domestication involves increasing the ability of yeast to metabolize maltose (Mal1 locus) and for most yeast strains, excluding German Hefeweizen and Saison, it also means losing the pathways that cause phenolic flavor (production of 4-vinyl guaiacol ). Yeasts at the far end of domestication also show a decrease in sexual reproduction.
