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General Category => Yeast and Fermentation => Topic started by: James K on July 23, 2018, 10:25:45 PM

Title: Dregs/stir plate
Post by: James K on July 23, 2018, 10:25:45 PM
I am working on building up some dregs and was curious about my process.
I made some 1.040 wort and cooled it down in my sink. Then I drank the first bottle of a beer called “future knowledge” and left a small amount of beer in the bottle before I swirled it around to get the remaining yeast off of the bottom. I poured those dregs into my flask and set it on the stirplate, turned it on high for about 30 minutes while I drank the bottle. Before I poured the dregs I spayed a paper towel with isopropyl and cleaned the top of the bottle.
I have about 3/4 inch or head on the starter so I poured the 2nd bottle of future knowledge and and left the dregs, swirled it around, wiped the top, and poured them into the flask on the stir plate.
I’m trying the build the yeast and Brett up by Friday.
At this point I’m at a wait and see moment.
Am I ok? Not okay? I’ve only built up dregs once before but used 3 bottles from beer I made myself. Should I keep my stir plate on? Continuously shake my starter?
Looking for advice and feedback.

Future knowledge - farmhouse golden ale, farmhouse microbes with lab-isolated brettanomyces, and House-mixed culture yeast.
Title: Re: Dregs/stir plate
Post by: Robert on July 23, 2018, 11:02:58 PM
You didn't say how much wort.  There's a limit to how much growth you can get from a given number of cells in a given volume -- there's an optimal inoculation rate.  I've generally been successful when I've started the dregs of 3 or 4 (12 oz) bottles in 500mL and left it 24 hours on the stir plate, crashed, and then built through a couple of 2L shaken or O2 starters.   Not a magic formula, just an example.   Some people swear by tenfold increases, etc. Whatever your volume, especially if it's a liter or two (kinda big for 2 bottles' dregs,) maybe leave it on the plate 24 hours keeping it in the growth phase, and it will have done all it can.  (I'm not really Brett-savvy, but it's not going to grow much at this stage, is it?)
Title: Re: Dregs/stir plate
Post by: James K on July 23, 2018, 11:12:58 PM
I’m not really worried about the Brett because if it’s there eventually it should just eat away at the sugars. Right? It was a 500ml starter. I plan to decant and make another one in a few days. Should I leave it stirring on the plate? And at what speed? Usually I just crank it up for a while and then turn it off, let the yeast reproduce, that kinda thing.

Normally when I make a starter I have like 200billion cells.  This is only my second run with dregs.
Title: Re: Dregs/stir plate
Post by: Robert on July 23, 2018, 11:32:03 PM
I figure if you leave it cranking for 24 hours, you'll maximize growth, it will stay in aerobic growth rather than fermentation.   In 500mL, I believe you'll max out with about 100B cells, like starting with a regular pack of yeast,maybe less than that, I can't find my references right now.  So then do your regular starter.  That's how I've done dregs, again with good results.  The complaint you hear about leaving a starter on the plate for the duration, especially from advocates of SNS, is that the starter wort tastes funny.  Fair enough. At this stage, I wouldn't care about that, you just want as much yeast as you can get, to then do a starter by your preferred method.

As for the Brett, I'm thinking the same as you.  Don't really want to grow it in proportion now, do you?

EDIT Sorry, way off.  500mL will probably max out around 15B cells.  I was thinking what I have after my second stage.  Going by data in the White and Jamil book.
Title: Re: Dregs/stir plate
Post by: narcout on July 24, 2018, 12:04:17 AM
I figure if you leave it cranking for 24 hours, you'll maximize growth, it will stay in aerobic growth rather than fermentation.

Doesn't the Crabtree Effect state otherwise?
Title: Re: Dregs/stir plate
Post by: Robert on July 24, 2018, 12:09:44 AM
I figure if you leave it cranking for 24 hours, you'll maximize growth, it will stay in aerobic growth rather than fermentation.

Doesn't the Crabtree Effect state otherwise?
Good catch.  To clarify, it will stay in the growth phase as long as possible.   So a good tactic at this stage of propagation.
Title: Re: Dregs/stir plate
Post by: James K on July 24, 2018, 01:45:03 AM
So it sounds like I need to decant tomorrow and then do another starter, leave it cranked tonight and tomorrow, cranked after tomorrow’s starter, and then do one more growth before the final pitch.

Correct?

Doesn't the Crabtree Effect state otherwise?
Can you explain this? I’ll look it up, I just have never heard of it before.

The Brett is there, I’m more concerned about growing the farmhouse and house mixed culture, although I think what I am doing will grow everything.
Title: Re: Dregs/stir plate
Post by: Robert on July 24, 2018, 02:02:20 AM
Crabtree TLDR: at some point it will start fermenting even when fed oxygen.

Yeah, I find the 500mL from dregs, decanted to a 1-2L starter (stir, shake, O2, what have you) yields the functional equivalent of a vial or smack pack, which you can make a final starter from.  And you have plenty of time to do it by Friday.   Dude, BTW, you do some crazy experimental brews.  Kudos.
Title: Re: Dregs/stir plate
Post by: James K on July 24, 2018, 02:20:40 AM
Crabtree TLDR: at some point it will start fermenting even when fed oxygen.

Yeah, I find the 500mL from dregs, decanted to a 1-2L starter (stir, shake, O2, what have you) yields the functional equivalent of a vial or smack pack, which you can make a final starter from.  And you have plenty of time to do it by Friday.   Dude, BTW, you do some crazy experimental brews.  Kudos.

Thanks man,
I’m all about trying different and non conventional. I’m still in early stages of learning and the more I push myself the more I learn.

I was going to try that coolship but I’m gonna have to wait till it’s not raining every other day (and when it’s cooler at night), plus I was reading a book about sours “American Sour Beers” and it recommend lactic acid, and I didn’t feel like dying of ecoli.

This beer, used from dregs is going to be 4# rye, 4# pils, 4# flaked wheat.
Usually when I go out of town I look for unique, and I build on that.

I’m probably going to cut that stir plate when I go to sleep, and start over rebuilding when I wake up, do that for a few days. Hope it doesn’t rain this Friday.
Title: Re: Dregs/stir plate
Post by: narcout on July 24, 2018, 04:18:38 PM
Can you explain this? I’ll look it up, I just have never heard of it before.

I'll let S. Cerevisiae do it, since he explains it way better than I can.

The thing that confuses most people is that yeast cells have two separate metabolic pathways. The first metabolic pathway is called the respirative (aerobic) metabolic pathway.  The respirative metabolic pathway is extremely efficient. It basically converts sugar to water and carbon dioxide gas.  The second metabolic pathway is called the fermentative (anaerobic) metabolic pathway. It is the pathway through which sugar is converted primarily to ethanol (which is a carbon-based compound) and carbon dioxide gas.  The fermentative metabolic pathway is not anywhere near as efficient as the respirative metabolic pathway.   

Many older home brewing texts erroneously refer to the growth phase as respiration.   Brewing yeast cells do not respire in beer wort due to something known as the Crabtree Effect.  The Crabtree Effect states that yeast cells will favor fermentation over respiration when subjected to dissolved glucose levels above the Crabtree threshold, and they will do so even in the presence of dissolved oxygen (O2).  The Crabtree threshold is around 0.3% weight/volume (w/v). 

To put things into context, the extract from the average mash contains roughly 14% glucose, which means that wort with a specific gravity above 1.008 contains a glucose level above the Crabtree threshold.

Here's the math:

A 1.008 solution is a 2% sugar weight/weight (w/w) solution, which is the same thing as w/v when dealing with a solute dissolved into water because 1ml of water weighs one gram.  Of that 2%, only 14% is glucose; hence, 0.02 x 0.14 = 0.0028, or 0.28% glucose w/v, which is below the Crabtree threshold.

As all beer and batch yeast propagation worts have specific gravities above 1.008, yeast biomass growth in brewing is fermentative.  Now, the metabolic pathways in yeast cells are a little on the leaky side.  What yeast cells do while there is still O2 in solution is shunt a small percentage of the glucose being consumed to the respirative metabolic pathway for the synthesization of ergosterol and unsaturated fatty acids (UFA).   These compounds make cell membranes more pliable, which, in turn, make passage of nutrients and waste products in and out of the cells easier.
Title: Re: Dregs/stir plate
Post by: James K on July 24, 2018, 08:44:17 PM
(http://i86.photobucket.com/albums/k97/Jrk264/561FBFD6-F7E0-4A2F-8990-67CF76C4967D_zpskqodqkmq.jpeg) (http://s86.photobucket.com/user/Jrk264/media/561FBFD6-F7E0-4A2F-8990-67CF76C4967D_zpskqodqkmq.jpeg.html)


Here’s what I have so far, a considerable amount of yeast, much more than what I started with.

What what I gather about this crab tree effect, is that it’s best to have yeast in an aerobic state when you are trying to grow the cell count, because in an aerobic state the yeast will eventually start to ferment. Correct? Whereas if I just turned the starter plate on for a short period I would retain a smaller yield of reproduction from the yeast because it’s in a more anaerobic state and the yeast doesn’t reproduce as fast. 

Does that sound right or am I confusing myself?
Thank you for the help. I’m about to decant and pitch fresh wort, this time with some yeast nutrient, I forgot about using some last time.
Title: Re: Dregs/stir plate
Post by: denny on July 24, 2018, 08:57:27 PM
AFAIK, Crabtree is only about the presence of glucose, not whether it's aerobic.
Title: Re: Dregs/stir plate
Post by: Robert on July 24, 2018, 09:49:08 PM
I figure the first stage you've just done is where you're most concerned with maximizing and hastening growth, as you start with so little.  Your flask looks like it's got about as much as I'd expect IME.  You can probably just go with whatever your SOP is through a couple of starter steps now and have a good pitch.  And a fresh, healthy pitch which is most important.

And yes, Crabtree effect is about glucose.  As long as yeast have a critical amount of carbon available they will at some point stop respiring and go anaerobic.   Producers of dry yeast prevent Crabtree and keep yeast in aerobic growth by using a continuous or fed-batch process, constantly adding tiny amounts of glucose, keeping the level sufficient for growth but below that threshold.  For us, it's inevitable, but a stir plate will ensure as much oxygen as we can supply at the start and will keep yeast in suspension and in contact with the medium for rapid growth and fermentation.  Your call.
Title: Re: Dregs/stir plate
Post by: narcout on July 24, 2018, 09:53:29 PM
What what I gather about this crab tree effect, is that it’s best to have yeast in an aerobic state when you are trying to grow the cell count, because in an aerobic state the yeast will eventually start to ferment. Correct? Whereas if I just turned the starter plate on for a short period I would retain a smaller yield of reproduction from the yeast because it’s in a more anaerobic state and the yeast doesn’t reproduce as fast. 

Does that sound right or am I confusing myself?

Sorry, I kind of dragged us into the weeds.

The Crabtree Effect just tells us that certain types of yeast will (mostly) ferment rather than respire when sufficient glucose is present, regardless of the presence of oxygen.  This is what is going to happen in your starter; reducing or adding more oxygen isn't going to change that.

Yeast still use oxygen to build up reserves of compounds that are shared with daughter cells during reproduction (and which could eventually limit the number of growth cycles), so providing sufficient O2 is still important.   
Title: Re: Dregs/stir plate
Post by: James K on July 25, 2018, 09:02:11 PM
(http://i86.photobucket.com/albums/k97/Jrk264/66D6BDF9-A741-4003-A2B2-5CEF0EF5E543_zps2gbgsyxo.jpeg) (http://s86.photobucket.com/user/Jrk264/media/66D6BDF9-A741-4003-A2B2-5CEF0EF5E543_zps2gbgsyxo.jpeg.html)

Looks like we’ve added some family members to the starter. I’m building it up again today and then I’ll probably do one more build up before the final pitch.

I tasted some of the decanted wort and it seemed fine. No noticeable off flavor.



The Crabtree Effect just tells us that certain types of yeast will (mostly) ferment rather than respire when sufficient glucose is present, regardless of the presence of oxygen.  This is what is going to happen in your starter; reducing or adding more oxygen isn't going to change that.

That helps. The last time I took a biology class was high school and I dropped out of high school so. Not really bio savvy. I read all this technical jargon and was like “still don’t get it”.

On the last starter I do I think I’m going to let the starter ferment a bit and use the fermenting starter as my pitch. When I’ve been aerating and turning the starter off at night there has been a slightly larger yeast cake, but no signs of fermentation.

Usually my starters grow a layer of head/foam. Why might that be? Is it the large presence of oxygen inhibiting fermentation in the starter?
Title: Re: Dregs/stir plate
Post by: narcout on July 26, 2018, 10:18:35 PM
On the last starter I do I think I’m going to let the starter ferment a bit and use the fermenting starter as my pitch.

A lot of people do that, and it's fine.  The only thing I would say is that sometimes starter wort that's been on a stirplate can develop some undesirable flavors.  I think most people that pitch the full volume of an actively fermenting starter will skip the stir plate and just aerate/oxygenate the starter right after pitching the yeast into it. 

When I’ve been aerating and turning the starter off at night there has been a slightly larger yeast cake, but no signs of fermentation.

That's not unusual, starters tend to ferment out pretty quickly.

Usually my starters grow a layer of head/foam. Why might that be? Is it the large presence of oxygen inhibiting fermentation in the starter?

That sounds like krausen, and is a sign of active fermentation.  Unless you've overoxgenated to the point where it's toxic to the yeast (not possible if you are only using air and not O2 from a tank), oxygen isn't inhibiting the fermentation of the starter.
Title: Re: Dregs/stir plate
Post by: Saccharomyces on July 27, 2018, 12:15:20 AM
AFAIK, Crabtree is only about the presence of glucose, not whether it's aerobic.

The Crabtree effect is based on percentage of glucose in the medium.  Below the Crabtree threshold and in the presence of O2, reproduction is respirative. Above the Crabtree threshold, all reproduction is fermentative, regardless of O2 level.  The difference is lies ergosterol and unsaturated fatty acid (UFA) reserves when maximum cell density is reached as well as the usage of adenosine triphosphate (ATP).  Cells produced under the Crabtree threshold in the presence of O2 have fully-charged ergosterol and UFA reserves.  Cells produced above the Crabtree threshold share the ergosterol reserves of their mother cells; therefore, a mother cell shares a portion of its ergosterol and UFA reserves every time it buds a daughter cell.  Dry yeast is produced using a continuous process that holds the medium in a steady state below the Crabtree threshold, which means that dry yeast cells have fully-charged ergosterol and UFA reserves.  That is why dry yeast can be pitched into poorly aerated wort.

As I have mentioned many times, a stir plate does not oxygenate wort after it is offgasing. All it does is keep cells in suspension.  One should be pitching or stepping a culture at high krausen, which means that a stir plate does not buy one much when propagating yeast cells.

When propagating a culture from dregs, one should start with about 25 to 50mls of 1.020 wort.  That culture can be stepped by a factor of 10, but a factor of 4 to 5 is more foolproof (50 -> 250ml -> 1L).  The culture should be stepped as soon as it reaches high krausen.  High krausen signals the transition from exponential growth to the stationary phase where reproduction is for replacement only.  I cover this area of yeast management in the following blog entry: https://www.experimentalbrew.com/blogs/saccharomyces/yeast-cultures-are-nuclear-weapons



Saccharomyces (a.k.a. S. cerevisiae)
Title: Re: Dregs/stir plate
Post by: Robert on July 27, 2018, 12:47:54 AM
Saccharomyces, I arrived while you were away, but given your reputation, I'm glad you're back, and of course well,  and I have the opportunity to avail myself of your knowledge.    I understand why the method of propagation from dregs you've just detailed is ideal.  Nonetheless, I have always had success with the simple (crude? ) method I recommended above:  a few steps, decanting in between (use of stir plate for initial oxygenation and maintaining yeast in suspension purely optional.)  It is convenient for those limited in equipment or the practical ability to precisely time steps to high kräusen.  So, my question:  does it actually do any harm to the resultant pitch and/or first generation fermentation, or is it simply inefficient in terms of yield over medium supplied?
Title: Re: Dregs/stir plate
Post by: James K on August 07, 2018, 08:41:45 AM
Ok so, this beer has been in the carboy for 10 solid days, it is still fairly active. After I switched from a blow off valve to airlock, I still had to replace the airlock twice because the beer was still blowing off.
The airlock is burping once every second-three seconds and the inside of the carboy still has movement.

Do you think this is the Brett working away? Or the farmhouse strain? I raised the temp from high 70s into the 80s yesterday but I do not see any signs of the beer slowing down.

I made another farmhouse beer 3 days after this experiment and it finished out completely and is already cleared up.  Also. Usually when I have made a beer with Brett I transferred into secondary and then pitched the Brett. Am I ok with leaving this beer on the trub for a long duration of time? I could transfer to secondary but don’t really care how clear the beer gets, I just don’t want to impart off flavors like autolyzing from leaving the beer on the yeast too long.

Thanks for the input. For now I just plan on letting this beer sit in the carboy another week-2weeks.

Update: current gravity is 1.002, so I think the Brett is working away, I taste the Brett.