Wild Saccharomyces?

[trentm]

I am afraid to walk on the "Wild Side" but love the beer. Being a plant pathologist, I realize the diversity of microorganisms present in the environment and being a long time "clean" home brewer, sanitation in ingrained in my process. I cannot imagine pitching a wild caught mixed culture into my hard work. Therefore, I have decided to take a walk on the "Semi-Domesticated Side" by isolating pure cultures of the wild things.

Wanting to capture a local wild Brettanomyces I added 5 strawberries (I live on the central coast of CA) to a flask of low gravity wort and encouraged fermentation. Dilution streaking from the resulting culture on to wort agar plates relieved 2 types yeast colonies (and a lot of fungi). One type grew very slowly (similar to Brettanomyces) the other grew very fast (similar to Saccharomyces). After re-streaking the yeasts onto clean plates, I have single colonies of each. Last night, I added 3 of the fast growing colonies to a 15 ml centrifuge tube containing 5 ml of wort. This morning activity was already apparent. The slow growing colonies are not yet big enough to increase.

All this said, I have 2 question: 1) Do you think the fast growing yeast is wild Saccharomyces? and 2) What would you expect of the fermentation performance if it is a wild Sacc?

 

[Warthaug]

If you follow the link in my sig, I have a whole section on my blog about capturing/characterizing wild yeasts. I've not done the best job keeping it upto date, but there is some stuff there that may help you out.

As for your yeasts, growth rates are not sufficient for species identification. There are a lot of yeast species that will grow on plates (and in media). But a few questions/points first:

1) How long did you you let the strawberries/wort mix ferment? This can be quite important as different yeast species dominate at different times. Some of my blog posts go into this in great detail (e.g. 1, 2), but the long and short is oxidative yeasts (picha, Kloeckera) dominate the first week, saccharomyces become dominant after a week or so, and bretts begin to emerge around the 1 month mark (but don't become dominant until many months after fermentation begins).

2) What precautions, if any, did you take against bacteria? Many can out-grow yeasts on plates, and appear early in the fermentation process.

2.5) Was your wort hopped? The plates? More than ~20IBU's worth? Over 20IBUs of alpha acids will inhibit the growth of some gram positive bacteria.

3) What did the two different yeast colonies look like? E.G. smooth or rough edges? Raised centre or flat? Shiny, mucoidy or powdery? What colour? Some limited ID can be made by looking at the colonies. A picture would be worth a thousand words, assuming you can get a decent macro of the plate.

Bryan

 

[trentm]

If you follow the link in my sig, I have a whole section on my blog about capturing/characterizing wild yeasts. I've not done the best job keeping it upto date, but there is some stuff there that may help you out.

As for your yeasts, growth rates are not sufficient for species identification. There are a lot of yeast species that will grow on plates (and in media). But a few questions/points first:

1) How long did you you let the strawberries/wort mix ferment? This can be quite important as different yeast species dominate at different times. Some of my blog posts go into this in great detail (e.g. 1, 2), but the long and short is oxidative yeasts (picha, Kloeckera) dominate the first week, saccharomyces become dominant after a week or so, and bretts begin to emerge around the 1 month mark (but don't become dominant until many months after fermentation begins).

2) What precautions, if any, did you take against bacteria? Many can out-grow yeasts on plates, and appear early in the fermentation process.

2.5) Was your wort hopped? The plates? More than ~20IBU's worth? Over 20IBUs of alpha acids will inhibit the growth of some gram positive bacteria.

3) What did the two different yeast colonies look like? E.G. smooth or rough edges? Raised centre or flat? Shiny, mucoidy or powdery? What colour? Some limited ID can be made by looking at the colonies. A picture would be worth a thousand words, assuming you can get a decent macro of the plate.

Bryan

Thanks for the reply. I found the initial look at your website to be a wealth of information. So I have a lot of reading to do when time permits. Regarding your questions:

1) Less than a week. It was fully contaminated with mold and other fungi. So as soon as yeast began to flocculate, I felt must move forward. The large colonies were definitely dominate with only a few of the smaller colonies on 1 of the three plate use for the initial isolation.

2) None. However, microscopic examination of the large colonies confirmed yeast. Actually they look like Brett but I don't have any experience with wild Sacc. or any of the other wild yeasts. I have not observed the small colonies.

2.5) No hops in the media

3) I'll look closer at the colonies tonight and see if I can get some good photos.

It looks like I have a lot to learn about spontaneous fermentation and isolating the type of yeasts I'm looking for. It seems likely these isolates are may not be what I'm after. If not, I will keep trying.

 

[Warthaug]

The presence of mold is generally a bad sign, often a sign of preparing the sample in a dirty/dusty place &/or in too moist an environment. Most wild ferments should appear clean, with no filamentous molds/etc (other than normal bacterial pellicles/etc). Molds typically won't grow in liquid media, so that's a sign something went wrong.

For media I'd actually recommend wort with a SG of 1.040 or so - that's high enough to keep a lot of things from growing, but is a perfectly happy place for yeast.

Don't give up!

Bryan

 

[Microscopist]

Nice to see someone else 'borrowing' centrifuge tubes from work there's no telling what you have growing without a fair bit of work with selective media, I picked a few good looking colonies, grew them up in 10ml malt media and tasted them to find promising strains.

I didn't use hops until after the initial selection process.

Got my yeast out of flowers by the way and all the reject strains got pitched into a Flour, water, sugar mix and now make fabulous bread

 

[Warthaug]

Its not always hard to tell what you have growing - depending on your resources. I've developed (well, copied) a DNA-sequencing based approach for species ID. It works pretty well - assuming you have access to a PCR machine & DNA sequencing lab...

Bryan

 

[trentm]

Thanks for the comments. Both of you seem to have a great deal of knowledge and I greatly appreciate your willingness to share.

Update: I increased the putative Saccharomyces to 250 ml. After fermentation was complete and the yeast had settled (only 48 hrs) I was excited to evaluate the resulting beer. First the aroma was pleasant and curious but also smelled of wort. Checking the gravity, to my grief, it was only at 8 degrees P from the original 10. After reading through Bryan's blog, it is likely an oxidative yeast. Although it is not what I'm looking for, I saved a sample in just in case.

Microscopist: My lab days are over as I have taken a new job in food safety management. I now rely on cynmar.com for lab supplies. It's a great place for homebrewers to get hard to find materials for a reasonable price. For example, 50 sterile 15ml centrifuge tubes for <$8. Unfortunately, sales of ingredients for selective media are restricted (at least in CA). While agar and yeast extract are no problem, they would not sale peptone to an individual. Do they make drugs with peptone?

 

[Microscopist]

While agar and yeast extract are no problem, they would not sale peptone to an individual. Do they make drugs with peptone?

You need it to grow anthrax, but then you need it for almost any bacteria so it's an odd one to block. If you're sufficiently determined you could make it from powdered skimmed milk by digesting it with a bit of rennet, fresh papaya or pineapple juice ( using two out of the three might give better solubility )

I'm trying to find a route at work to get a yeast sample into the genomics lab - trouble is the Id method uses primers noone uses here and I never order primers.

 

[trentm]

You need it to grow anthrax, but then you need it for almost any bacteria so it's an odd one to block. If you're sufficiently determined you could make it from powdered skimmed milk by digesting it with a bit of rennet, fresh papaya or pineapple juice ( using two out of the three might give better solubility )

I'm trying to find a route at work to get a yeast sample into the genomics lab - trouble is the Id method uses primers noone uses here and I never order primers.

Oh! Anthrax jeez. I was actually able to cop some peptone from a friend but thanks for the work around. Be careful with that knowledge you'll have Big Brother watching you.

My science background is as an applied plant pathologist so as a "Gene Jockey" I am very limited. Warthuag (Bryan) seems to have a strong biotech background. Perhaps he has some insight.

 

[Warthaug]

Update: I increased the putative Saccharomyces to 250 ml. After fermentation was complete and the yeast had settled (only 48 hrs) I was excited to evaluate the resulting beer. First the aroma was pleasant and curious but also smelled of wort. Checking the gravity, to my grief, it was only at 8 degrees P from the original 10. After reading through Bryan's blog, it is likely an oxidative yeast. Although it is not what I'm looking for, I saved a sample in just in case.

Keep the ferment running - oxidative yeasts really kick ass the first few days and tend to overwhelm everything else, but given time the sach & bret will take over. In my current run I'm planning on my first "harvest" around day 30, and every 20 or so days after that.

While agar and yeast extract are no problem, they would not sale peptone to an individual. Do they make drugs with peptone?

I'm a bit surprized by that - peptone is simply hydorlysed protein; there isn't really anything special about it. Like microscopist said, you need it to grow almost any bacteria, so that may be why.

Making peptone is dirt-easy, get some papain or bromelain (available at health food stores), or get an unseasoned papain or bremelain-based meat tenderizer and some skim-milk powder. Make a 5X solution of skim milk and add a crushed pill of the papain (or a pill-sized amount of tenderizor). Let sit at room temp for 18-24 hours. Bring to a boil briefly to kill the enzymes and then use as a 4x-8x concentrate of papain.
EDIT: Sorry, you need to acidify the milk solution before adding the enzyme; 4.5 is ideal.

Another option is to go to a health food store and get the body builder vitamins - many of them come complete with all the amino acids.

Although I have to ask why do you need peptone? There are a number of selective media you can make at home, using commonly available ingredients (plus a few easy-to-get lab chemicals like cyclohexamide & bromocresol green). Eureka brewing (a blogger) has some great posts on this.

Bryan

 

[trentm]

Well, I guess that was just an example of how some scientific supplies are not available for individuals. My need for peptone was to make YPD which is certainly not selective. While CA is known for liberal thinking our laws are often very conservative. We can't buy alcohol above 75%, syringes (without a prescription), peptone, etc.

 

[Warthaug]

You should be able to just buy YPD pre-mixed. But there are a number of cheaper/easier options. I'm a fan of potato-starch agar myself:
http://wiki.bugwood.org/Potato_dextrose_agar

Grows most organisms we'd be interested in, can incorporate selective agents like specific sugars in place of dextrose (e.g. lactose), added nutrients (yeast extract/nutrient), antibiotics, dyes (bromocresol green), etc.

Beer-wort agar is my go-to for 99% of my culture experiments:

1. Prepare a 1.002 to 1.004 wort solution. I make this by a 1:10 dilution of 1.040 starter wort that I pressure-can or freeze for later use. I'd recommend making sure the 1.040 wort has a double-dose of proper yeast nutrient (not DAP) for best growth.
2. Add in 1.5% w/v agar
3. Pressure cook/autoclave to sterilize
4. If adding dyes or antibiotics, cool media to ~55C and add these at this point
5. Pour plates/slants

http://bkyeast.wordpress.com/ played with a few others, but like me appears to have settled on wort-agar and potato-agar.

Eureka brewing also has done some culturing stuff, but he appears to prefer commercial agar preps over homebrewed ones:
http://eurekabrewing.wordpress.com/yeast-cultivation/

Bryan

 

[trentm]

Thanks for info Bryan. Wort agar works great for me as well. I use it for culturing as well as storage on slants. However, for me, Brettanomyces has not stored well on wort agar. I have heard it does better on YPD and thus my desire for peptone and other ingredients. I've only had my Brett on YPD for a couple of months but it looks good compared to the wort agar slants. I also tried the isotonic NaCl method (Eureka Brewing blog). Results to be determined. I am also trying the cowboy method of keeping a culture going in a flask. I really don't want to lose my isolate from Orval. I assume you are using cryostorage, but do you have any recommendations for storing Brettanomyces?

 

[Warthaug]

I freeze my cultures, Brett & sacc both. In a conventional freezer they appear to last for 3 years before needing to be recultured. If you have a frost-free you need to keep the tubes in a cooler with some ice packs to protect from the defrost cycle. In either case, store the yeast in a mix of 1.020 wort + 15% - 20% glycerol. I keep backups in my -80c at work; stored like that they are good for decades.

There are other options that work well. Slants can be stored under boiled/cooled water for 1-2 years. Historically, before lab-grade freezers existed, slants would be stored for 10+ years under steralized mineral (paraffin) oil. In either case, fill the slant, cap it, seal with electrical tape, and keep in the fridge. There are reports in the microbiology field of 50-year old wine yeasts being recovered from mineral oil stocks!

Bryan