Orval dregs under a microscope

[CaptainK]

I am culturing up the dregs of a bottle of Orval and doing counts to check progress. I can see what appears to be two distinct yeast populations. A large "saccharomyces looking" population, and a smaller population. This would make sense since there should be primary fermentation yeast in there as well as 3 Brett strains?

I am counting them separately since I want to see the Brett multiply (assuming the small ones are Brett).

I started with 250ml total volume (with the dregs) of approx 1.025 wort on a stir plate.

2 days later I dumped that into a total volume of 1,000ml 1.040 wort and added constant air with a stone (I do this 4X step up with ale yeast and get a 10X increase in cell volume usually)

I got a 2X increase with the 250ml propagation.

Can anybody weigh in on this regiment? I assume Brett likes O2 for sterol synthesis just like Sacc?

Thanks

 

[CaptainK]

I didn't see any activity after 24 hours in the stepped up starter. This morning (40 hours) I had significant activity and dense creamy krausen with a super light bubble bath looknig krausen growing on top of that. It is starting to smell like fresh cheese in here. I read somewhere that Brett takes longer to propagate. Seems true. Maybe my first step just woke them up ( I did double the cell count and saw a drop to terminal gravity though). This was before I was counting the yeast as 2 separate populations though.

Brett can continue to ferment past what is considered normal for Sacc though. With the constant air I think I should get a bumper crop. I was going to pitch tomorrow, but I will wait until this is done. The good thing about having a microscope is that if it gets contaminated (bacteria) I will be able to see them (if they are in high enough numbers that is).

 

[CaptainK]

Still high white frothy krausen. Starting to smell like acetone. Wife is not happy.

Edit: Wife is really unhappy. We have a newborn in the house and she is losing tolerance for this project. It is in a pretty small room and it really stinks.

This is what OSHA has to say about ethyl acetate:

OSHA Permissible Exposure Limit (PEL) - General Industry
See 29 CFR 1910.1000 Table Z-1
400 ppm
(1,400 mg/m3) TWA
HE8
Mild narcosis at high concentrations

HE16
Mild eye, nose, and upper respiratory irritation

HE20
Strong odor

I have all 3 of these symptoms. The window is sealed with plastic for the winter. I guess I will have to take it off to ventilate this room.

This is what leads me to believe it is ethyl acetate:
"The volatile fatty acids produced by Brettanomyces that impact wine quality are quite diverse. The most well known to winemakers is acetic acid. This is produced during Brettanomyces growth by the oxidation of ethanol and can lead to the formation of ethyl acetate which can have an acetone type aroma".

Full article can be found here.
http://www.makewine.com/winemaking/materials/brettanomyces/

 

[CaptainK]

I decided to stop the experiment due to asphyxiation and unhappy wife. I also started to think that if ethanol was being oxidized to ethyl acetate then the yeast were past the reproduction stage (even in the presence of oxygen which is constantly being added) so I was done doing what I set out to do anyway. Also since I was adding O2 it makes sense that a lot of the ethanol would be converted to oxidized products just from that.

Too make a long story short I noticed a large population of bacilli under the scope. Looks like I prpagated bacteria. They could be the cause of the ethyl acetate as well. I don't have time to research this just yet I am getting behind in my real school work. I will be in the microbiology lab soon so I will be doing a lot of fun experimenting in the future.

 

[CaptainK]

One last post to wrap this up. I did a count and looks like I doubled my population. out of 335 cells counted only 22 were the large type I thought were "saccharomyces looking". My gravity did not drop however. Started out at 1.040 and ended up at 1.040. I don't know how accurate this is because I am using a refractometer with the correction tool in Beersmith for "fermenting wort".

 

[cfonnes]

3 brett strains? I thought Orval only used Brett C.

 

[smokinghole]

Only thing I ever got a strong acetone odor from was a fermenting acetobacter culture in some red wine. I was making red wine vinegar and it smells awful after a while but the acetone smell went away.

 

[TNGabe]

3 brett strains? I thought Orval only used Brett C.

Orval is finished with Bruxellensis

 

[Orval]

I am culturing up the dregs of a bottle of Orval and doing counts to check progress. I can see what appears to be two distinct yeast populations. A large "saccharomyces looking" population, and a smaller population. This would make sense since there should be primary fermentation yeast in there as well as 3 Brett strains?

At Orval, there's a first fermentation using saccharomyces type of yeast, after a two week stay in tanks, brett yeasts are added and yeasts are also added for carbonation in bottle, Brett as well but I'm not sure any more, so no wonder you find those Brett yeasts.