When to aerate yeast starter?

[mashdar]

When I do a stir plate starter, I aerate the headspace to ensure there is ample O2 for prolonged sterol production, etc. I cut off the air at some point to allow the solution to go anaerobic for storage etc, but I always have an internal debate about when best to do this.

My schedule is typically ~48 hours on stir plate, with air cut off at 12-24 hours.

Does anyone have any thoughts/opinions/references?

I don't have a firm grasp of the "phases" of yeast growth, and whether they are rigid internal states of the yeast, or just general descriptions of yeast behavior under different conditions.

 

[DuncB]

Make the starter with the stir bar running. When you think it's done. Turn off stirrer. Should see the wort settling from the top. If it's bubbling or doesn't start clearing likely more stirring needed.
I don't send mine anaerobic just let it finish, cold crash pour off excess liquid and then store the rest in a sterile vessel that is sealed up with minimum headspace.

 

[day_trippr]

fwiw, I do the same, using pint jars.
If I keep the head space near zero the yeast stay "good looking" longer in the fridge

Cheers!

 

[IslandLizard]

On a stir plate (or shaker) I wait for the color of the starter to have turned significantly lighter. That could be after 1-2 days (fresh yeast) or as long as 4-7 days (old yeast). That's my sign of good growth taking place. Once there's the color change, I give it another 24 hours of stirring/shaking.
Then let it stand for 6 hours (no stirring/shaking) before it goes into the fridge for cold crashing.

 

[day_trippr]

Interesting. The longest I have ever kept a starter intended for a full batch of beer on a stir plate was 24 hours. When I check the gravity it's always terminal by then. But I'm working with highly viable over-builds in those cases - if I'm resurrecting something it could spin for (much) longer...

Cheers!

 

[DuncB]

I start off with lower gravity say 1.020 and lower volume if resurrecting an old yeast. So perhaps only 50ml in a small erlenmeyer and then go to a few hundred ml adding wort of strength that will be 1.035 with the dilution of the already present fermented wort.

 

[mashdar]

No thoughts specifically about aeration, though? (The stirplate headspace will blow off most O2 pretty soon after CO2 starts leaving solution, afaik ending cell membrane production. I'm hoping to farm some fat yeast by giving them O2 for longer.)

Is there a point where yeast physically will not divide/hoard membrane components? Or a point after which O2 availability harms vitality/longevity?

I feel like I read about continuous-feed bioreactors for commercial propagation, but it's a hazy memory.

Edit: I realize the best solution would be to steal my wife's microscope and start doing cell counts/stains to compare. I'll add that to the when-I-retire-and-have-the-time list.

edit2: I haven't finished reading, but this says stationary phase O2 gets used for other stuff. Oxygen Consumption by Anaerobic Saccharomyces cerevisiae under Enological Conditions: Effect on Fermentation Kinetics
It offhandedly mentions that stationary phase growth is YAN limited? So my understanding may be very flawed.

 

[VikeMan]

Here's a study that suggests that, at least under some condition(s), the addition of sufficient O2 can cause yeast to (partially) switch from fermentation to respiration, effectively (in my words, not theirs) partially suppressing the crabtree effect. At the risk of taking the study out of context, it's worth pondering if continuous aeration of a starter might increase biomass as compared with aeration only at the start. I think that has been posited before, but I don't recall seeing evidence for it.

Oxygen Response of the Wine Yeast Saccharomyces cerevisiae EC1118 Grown under Carbon-Sufficient, Nitrogen-Limited Enological Conditions

 

[mkopec1]

I have been out of the game for years but I remember doing starters back in the 90s with no aeration and no stirring in a 750ml Absolut bottle, and they all worked out fine. Nice cake at the bottom which was way more yeast than started with 3x 4x?. Sometimes I would get fermentation starting in 6 hours after pitching. First beer I brewed on the Anvil I just got I pitched dry us-05 and it took a good 12-14 hours to get that going. So there is definitely a difference in pitching a starter than just a dry packet.

 

[McMullan]

I wait for the color of the starter to have turned significantly lighter.

Do you mean opaque, “milky”? Lots of cells in suspension?

 

[tracer bullet]

Perusing the Wyeast site, assuming they know some things (but I'm not suggesting it's a final word in the slightest) they indicate:

"Agitation aids in removing inhibitive CO₂ from suspension as well as possibly adding small amounts of oxygen. Stirring or shaking the starter periodically or using a stir plate will improve cell growth. The use of stir plates has been shown to increase cell growth 25-50% over a non-stirred starter.

Small additions of oxygen periodically throughout the growth of a starter will replenish sterols and improve cell yield."

Also

"Because starters are inoculated at high cell densities, growth is usually maximized within 24-36 hours."

White Labs has:

"In this step, you want to ensure the yeast continues to get a healthy dose of oxygen and remain aerobic. This will allow the yeast to build their cell walls and prepare for the stressful environment of fermentation. Make sure the flask cover is loose to promote gas exchange. Allow your starter to stir for 24 hours..."

And elsewhere:

"When using a stir plate, 24-48 hours is generally enough time for a starter to be completed."

I too would be interested in a best-practice. I typically start my starters 2 nights before brewing. Stir plate with filtered air from a mini aquarium pump. About 48 hours later I turn it off and cold crash it in the beer frig overnight. Next morning it comes out and warms to room temp while I brew. When I'm ready for it later in the day I dump some off the top, swirl the rest, and pitch it in.

That may not be the best thing to do of course, I'm not advocating it. Seems to work but certainly curious if there are improvements to be had.

 

[McMullan]

Not much point trying to aerate the headspace. Aerate (oxygenate) the starter wort before adding the yeast. The best thing to do to maximise cell growth is to use pure O2 to oxygenate the starter wort. Then just leave it in a warm cupboard for a few days. It’ll stir itself, if you don’t stir it. I just swirl mine whenever I pass by, just to eyeball progress too. This assumes you are culturing up some harvested yeast or stepping up a starter, though. If you are making a starter with a liquid yeast pack, it might not be necessary at all, because the cells (membranes) should already be pretty packed full with lipids necessary for budding, as they’re grown under conditions that promote sterol production. Excess O2 might only serve as a stress factor here. In this case, it might be better to oxygenate towards the end of the starter, while the yeast are still active. Then not bother oxygenating the FV wort before pitching.

 

[IslandLizard]

Do you mean opaque, “milky”? Lots of cells in suspension?

Yes, of course, that's the cause.

 

[mashdar]

Not much point trying to aerate the headspace. Aerate (oxygenate) the starter wort before adding the yeast. The best thing to do to maximise cell growth is to use pure O2 to oxygenate the starter wort. Then just leave it in a warm cupboard for a few days. It’ll stir itself, if you don’t stir it. I just swirl mine whenever I pass by, just to eyeball progress too. This assumes you are culturing up some harvested yeast or stepping up a starter, though. If you are making a starter with a liquid yeast pack, it might not be necessary at all, because the cells (membranes) should already be pretty packed full with lipids necessary for budding, as they’re grown under conditions that promote sterol production. Excess O2 might only serve as a stress factor here. In this case, it might be better to oxygenate towards the end of the starter, while the yeast are still active. Then not bother oxygenating the FV wort before pitching.

Unless I have made an order-of-magnitude error (quite possible), the head space appears to contain many times the O2 of even a 12ppm O2 solution. Seems like 12mg/L vs 271mg/L.

The O2 advantages of a stir plate (by my reconning) are readily absorbing headspace O2 and mixing headspace gas. The former lets the yeast access the O2, and the latter delays blowoff by mixing instead of sweeping/blanketing.

Since mixed O2 levels in a 50/50 starter/headspace will be <1% by ~6 gravity point drops, adding O2 to the headspace during exponential phase should be beneficial, in theory?

Re lipids, the goal is to have sufficient stored sterols etc to allow ample cell division in the eventual beer. You want your starter yeast to be fat and happy at the end of the starter process, similar to the lab-culture yeast.

edit: I should say I used to aerate the starter directly, but foaming was an issue occasionally.

edit2: Also, I'm just talking optimal. I respect "good enough" as a process. To each their own!

 

[McMullan]

Perusing the Wyeast site, assuming they know some things (but I'm not suggesting it's a final word in the slightest) they indicate:

"Agitation aids in removing inhibitive CO₂ from suspension as well as possibly adding small amounts of oxygen. Stirring or shaking the starter periodically or using a stir plate will improve cell growth. The use of stir plates has been shown to increase cell growth 25-50% over a non-stirred starter.

Small additions of oxygen periodically throughout the growth of a starter will replenish sterols and improve cell yield."

Also

"Because starters are inoculated at high cell densities, growth is usually maximized within 24-36 hours."

White Labs has:

"In this step, you want to ensure the yeast continues to get a healthy dose of oxygen and remain aerobic. This will allow the yeast to build their cell walls and prepare for the stressful environment of fermentation. Make sure the flask cover is loose to promote gas exchange. Allow your starter to stir for 24 hours..."

And elsewhere:

"When using a stir plate, 24-48 hours is generally enough time for a starter to be completed."

I too would be interested in a best-practice. I typically start my starters 2 nights before brewing. Stir plate with filtered air from a mini aquarium pump. About 48 hours later I turn it off and cold crash it in the beer frig overnight. Next morning it comes out and warms to room temp while I brew. When I'm ready for it later in the day I dump some off the top, swirl the rest, and pitch it in.

That may not be the best thing to do of course, I'm not advocating it. Seems to work but certainly curious if there are improvements to be had.

A stir plate is optional. In most cases, it’s only going to promote a marginal increase (<<25%) in biomass, relative to non-stirred, therefore not worth bothering with, imho. Easy to test. Split batch: stirred vs non-stirred; compare fermentations and beers.

Assuming sufficient healthy yeast are used in starters (and FV worts), volume of the media/wort actually limits growth. Adding fewer cells promotes more growth and adding more cells promotes less growth. Cell density ends up approximately the same regardless. The cells communicate via chemical signals using growth-related compounds, released into media, as surrogate measures of population density and adapt their behaviour accordingly. Yeast manufacturers need to put in a lot more effort than brewers for obvious reasons. For brewer’s starters, oxygenating wort at the beginning then just leaving it works just fine. There’s really no need to complicate life.

It’s considered good practice to let the starter finish, which can take about 48-72 hours with healthy yeast. ‘Finished’ here means the cells have adapted physiology to a more stress-tolerant resting state therefore more suited for short-term storage and being pitched into FV worts, which are often stressful environments to be pitched in. The true lag phase only lasts several hours. As brewers, it’s best practice to get fermentation started and finished as soon as possible, to promote ethanol production therefore preservation of beer. Which is only beer at the end of the day. Liquid bread.

 

[tracer bullet]

A stir plate is optional. In most cases, it’s only going to promote a marginal increase (<<25%) in biomass, relative to non-stirred, therefore not worth bothering with, imho. Easy to test. Split batch: stirred vs non-stirred; compare fermentations and beers.

One of the "beauty is in the eye of the beholder" cases. I got the stir plate and bars for free, and it's ~ 60 seconds extra work to use them. I'll take up to 25% additional cell count.

I wouldn't knock anyone without a stir plate for deciding to not invest in one, however.

 

[mashdar]

A stir plate is optional. In most cases, it’s only going to promote a marginal increase (<<25%) in biomass, relative to non-stirred, therefore not worth bothering with, imho. Easy to test. Split batch: stirred vs non-stirred; compare fermentations and beers.

Assuming sufficient healthy yeast are used in starters (and FV worts), volume of the media/wort actually limits growth. Adding fewer cells promotes more growth and adding more cells promotes less growth. Cell density ends up approximately the same regardless. The cells communicate via chemical signals using growth-related compounds, released into media, as surrogate measures of population density and adapt their behaviour accordingly. Yeast manufacturers need to put in a lot more effort than brewers for obvious reasons. For brewer’s starters, oxygenating wort at the beginning then just leaving it works just fine. There’s really no need to complicate life.

It’s considered good practice to let the starter finish, which can take about 48-72 hours with healthy yeast. ‘Finished’ here means the cells have adapted physiology to a more stress-tolerant resting state therefore more suited for short-term storage and being pitched into FV worts, which are often stressful environments to be pitched in. The true lag phase only lasts several hours. As brewers, it’s best practice to get fermentation started and finished as soon as possible, to promote ethanol production therefore preservation of beer. Which is only beer at the end of the day. Liquid bread.

Kai's cell counts increased >25% even comparing slow to fast stir rates, so surely no-agitation to fast stir is quite a bit more still.
http://braukaiser.com/blog/blog/2013/03/25/stir-speed-and-yeast-growth/
I don't know of a direct comparison of SNS vs stir plate - if you have one I'd be interested.

Re shaking, I leave the starter in the basement (out of the way of small humans), so I prefer a set-and-forget approach. If shaking works for your situation, great!

I'm just optimizing, as a moderately obsessive person does with a hobby (and seems common to brewing in particular). If I can get by with 1 stage for my "big" starter instead of 2, that's a win IMO.

Speaking of Kai, here's an old blog entry where he aerated (stone in liquid) a stirred starter and got ~25% increased yield vs otherwise similar stirring. I hadn't read this one.
https://braukaiser.com/wiki/index.php/Yeast_Propagator
edit: Actually Kai did compare stir vs shake in this slide deck. He got ~250% cell count stirring, although IDK what the shake method was. Probably lurking on his blog somewhere. It's a good slide deck generally.
http://braukaiser.com/documents/Troester_NHC_2013_Step_Up_Your_Starter.pdf

 

[McMullan]

Kai's cell counts increased >25% even comparing slow to fast stir rates, so surely no-avitation to fast stir is quite a bit more still.
http://braukaiser.com/blog/blog/2013/03/25/stir-speed-and-yeast-growth/
I don't know of a direct comparison of SNS vs stir plate - if you have one I'd be interested.

Re shaking, I leave the starter in the basement (out of the way of small humans), so I prefer a set-and-forget approach. If shaking works for your situation, great!

I'm just optimizing, as a moderately obsessive person does with a hobby (and seems common to brewing in particular). If I can get by with 1 stage for my "big" starter instead of 2, that's a win IMO.

Speaking of Kai, here's an old blog entry where he aerated (stone in liquid) a stirred starter and got ~25% increased yield vs otherwise similar stirring. I hadn't read this one.
https://braukaiser.com/wiki/index.php/Yeast_Propagator
edit: Actually Kai did compare stir vs shake in this slide deck. He got ~250% cell count stirring, although IDK what the shake method was. Probably lurking on his blog somewhere. It's a good slide deck generally.
http://braukaiser.com/documents/Troester_NHC_2013_Step_Up_Your_Starter.pdf

Not very scientific, to be honest, with most likely very crude cell number estimates. It is what it is, I'm afraid. Doesn't match my observations based on more tightly controlled experiments. Otherwise I'd still be using and recommending a stir plate.

 

[day_trippr]

Does increasing dissolved CO2 levels inhibit growth?

 

[McMullan]

Does increasing dissolved CO2 levels inhibit growth?

It’s a recognised stress factor for yeast, but not a growth inhibitor. Certainly not in something like a small yeast starter under atmospheric pressure at room temperature or higher. I think it can slow fermentation, but it depends on the yeast. Recirculating wort in a Yorkshire square periodically knocks out dissolved CO2 and probably helps increase fermentation rate. Then again, my lagers fermented cool under pressure, if anything, seem to ferment at a slightly higher rate than a comparable recipe fermented at atmospheric pressure. I pitch at a high rate, though. I just assume it’s actually normal, because chemical reactions tend to usually occur at a higher rate proportional to pressure. Others have different experiences apparently, possibly because they are under pitching.

Edit: sorry, went off on one and forgot to finish by trying to address what I thought you were getting at. Yes, a stir plate will help knock out dissolved CO2, and that probably helps increase the rate of growth slightly. So, if in a hurry, a stir plate might be your best option.

 

[mashdar]

Not very scientific, to be honest, with most likely very crude cell number estimates. It is what it is, I'm afraid. Doesn't match my observations based on more tightly controlled experiments. Otherwise I'd still be using and recommending a stir plate.

Have you written up your results? I'd be happy to read that and be convinced otherwise.

Do you have access to a proper microbio lab?

Kai's counting methods are here. I feel they're about as sound as you can get without a commercial/institutional lab. https://braukaiser.com/wiki/index.php/Microscope_use_in_brewing
(edit: this is actually his guide for others, but I assume this is what he's doing. It's possible he has fancy equipment.)

 

[day_trippr]

Edit: sorry, went off on one and forgot to finish by trying to address what I thought you were getting at. Yes, a stir plate will help knock out dissolved CO2, and that probably helps increase the rate of growth slightly. So, if in a hurry, a stir plate might be your best option.

Bingo, exactly where I was heading. I once woke up to a thrown stir bar and unaware of the implications reset the stir plate which resulted in a CO2-driven volcanic eruption. While I was cleaning that up I was wondering if either the CO2 concentration outright or the lowered pH could have affected the starter viability. As it turned out it was a prototypical ferment for that recipe, so no evident effects, but as an engineering type it still left that question hanging out there...

Cheers!

 

[McMullan]

Have you written up your results?

To be published under copyright elsewhere.

Do you have access to a proper microbio lab?

Mostly decommissioned and sold off now. Just the basics left for rainy days.

Kai's counting methods are here. I feel they're about as sound as you can get without a commercial/institutional lab.

As suspected, not very scientific and most likely providing estimates of biased cell numbers. The most important (and most challenging) step of all - taking representative samples, part of the science vs how to use a microscope, etc. - gets brushed over with gapingly incomplete advice. Not really what I’d accept as being valid, tbh. Just another enthusiastic blogger. But, being ‘shackled’ by knowledge how to design scientific experiments, and why, I do find most home brew ‘experiments’ cringeworthy at best. No doubt my procedures are very different, being standardised and tightly controlled from start to finish. Because that’s just how I work, when running experiments.

I’ve managed to discover, unintentionally, how it’s totally unnecessary to complicate life as a brewer. Knowledge of why it works isn’t required, it doesn’t change the process. It can’t be improved beyond tossing in a cup of healthy yeast. No microscope or stirplate required. I’ve been banging on about this for a few years or so. A lot of the yeast related advice for home brewers is poor at best. It’s a bit ironic that some choose to complicate life by getting overly ‘technical’ merely to support a narrative that promotes one of the worst practices a brewer can follow. Underpitching. A stir plate doesn’t make much difference. A comparable non-stirred starter ferments a comparable FV wort just the same as a stirred (and ‘SNS’) starter. Home brewers have been following superstition not science. That includes me, btw. Until the science pulled me away from superstitions, that is. The science just recommends doing what skilled brewers have known (empirically) for generations. Bung in a cup of healthy yeast. No ‘rocket science’ required. It’s only beer.

 

[mashdar]

OK, well I respect that you have an opinion, but I can only work with the data at hand. You don't have to prove anything, obviously. If you do ever publish anything, let me know!

I'm not going to argue re cell counts, as I have no idea what kind of sampling/unfloccing Kai did. He wrote a ton on it, gathering from multiple sources, and his own data is accross multiple brews for each method, with surprisingly consistent results.

I've only ever seen hand wavy arguments that shaking is roughly equivalent, never with any attempt at quantification, so I don't have anything better to look at than Kai's results (and a bunch of marginally relevant journal articles). I'm perfectly willing to be convinced to throw out the stir plate some day.

I AM pretty convinced that cell count is not a huge deal in general, but again, it's a hobby : )

edit: Also I'm onboard with vitality/SNS - I use different starter methods depending on what I'm up to and how much time I have.

 

[McMullan]

It's very easy to simply run a side-by-side comparison, stirred vs non-stirred then ferment a split batch of kettle wort. Any increase in cell number in the stirred starter is unlikely to be sufficient to make any noticeable difference. There's going to be slightly less growth in the FV compared to the non-stirred starter pitched into a comparable wort. The perceived benefit is of no significance downstream. Nothing wrong with using a stirplate, though. It probably takes up to a day off the time taken. I sometimes use mine with an O2 stone to oxygenate starter wort, but I rarely make starters and prefer to repitch freshly harvested yeast, which don't even need oxygenated wort, if pitched at a high enough rate. An interesting one for LODO brewers, if they haven't realised already.