Various yeast biomass volume in starter

[Drunk Monk]

Hi,


I am experiencing significantly different behaviour of WLP802 and WLP860 lager yeasts while preparing starters.
Namely I did two starters recently with absolutely similar conditions:
1. Starting from single colony
2. 3 steps starter: 20mil/100mil/1lit
3. Same highly fermentable starter wort with added nutrients (pils malt, mashed at 64C for 1,5 hours)
4. Same aeration – 2 minutes with 50micron stone

But results for both yeasts are miles away from each other. While WLP860 had vigorous starter with a lot of yeast slurry after 24hours of last step, WLP802 had quite slow progress and after 72 hours had 4 times smaller yeast amount by volume.

What can be a reason of such behavior of WLP802?
BTW same picture I hade when I made WLP802 starter from fresh pack I purchased, so my yeast storage and growing on slants procedure is not the case.

 

[madscientist451]

I searched “WLP 802 yeast slow” and there are many reports of that strain being slow to get started, including a thread here on HBT: https://www.homebrewtalk.com/threads/slow-start-for-yeast-starter.694092/

 

[Drunk Monk]

I searched “WLP 802 yeast slow” and there are many reports of that strain being slow to get started, including s thread here on HBT: https://www.homebrewtalk.com/threads/slow-start-for-yeast-starter.694092/

That is about how slow it is. I know that wlp802 is really slow to start that is why I am giving it extra days on stir plate. But my issue is resulted yeast mass which is way too small.

 

[madscientist451]

I make lagers every year this time and then save the kegs in a very cold basement until I have room in the keezer. This year I switched to NovaLager and its been working pretty good for me so far.

 

[mashdar]

Take gravity readings?

72h is not very long for a slow 3 stage starter.

Why are you starting at 20ml? Slants or something?

edit: fwiw when resurecting 1yr+ yeast I sometimes need 4+ days for first stage.

 

[Drunk Monk]

Take gravity readings?

72h is not very long for a slow 3 stage starter.

Why are you starting at 20ml? Slants or something?

edit: fwiw when resurecting 1yr+ yeast I sometimes need 4+ days for first stage.

Gravity readings are more or less identical.
72 hours is for last stage. First stage 2 days, second stage 2 days, third stage 3+ days.
And yes, I start from 1 colony from petri dish.

 

[Drunk Monk]

So from 1L starter after cold crash I get 20ml of yeast slurry which result in 80bil of cells if we take maximum of 4bil/ml. According to calculator should be around 130-150bil of cells.

 

[IslandLizard]

Some yeasts are just slower growers. At what temperature are the starters being propagated at? The small volume steps (20 and 100 ml), are they agitated periodically, and if so, how?

I'd try longer grow times.
Instead of 2 days for each of the first 2 steps, give them 4 days each and a week for the last step, and see if that yields more biomass in each of the steps, especially the final one.

It's possible that WLP802 is just more finicky.
I haven't used 802 in ages. But I still have a culture left. I should start it up and see what it does.

 

[Drunk Monk]

Let me clarify this case a bit more—the issue is not the time but the fact that, although both strains consumed the same amount of sugars, the biomass (slurry) produced is different.

Specific Gravity (SG) of the starter: 1.036
Final Gravity (FG) of the starter: 1.009
Slurry amount for WLP802: 20 ml
Slurry amount for WLP860: 50 ml

 

[IslandLizard]

Let me clarify this case a bit more—the issue is not the time but the fact that, although both strains consumed the same amount of sugars, the biomass (slurry) produced is different.

Specific Gravity (SG) of the starter: 1.036
Final Gravity (FG) of the starter: 1.009
Slurry amount for WLP802: 20 ml
Slurry amount for WLP860: 50 ml

My emphasis (in bold/red).^
Thank you for reiterating this, that's the whole point you're making, yes!

When making yeast starters we want to promote growth (aerobic) over fermentation (anaerobic). They both occur at the same time but we can shift the mechanism of which one takes prevalence. By supplying oxygen the yeast will remain (mostly) in her aerobic reproduction mode, and will grow.

From what you observed, 802 doesn't reproduce as well as 860 under similar circumstances.
Maybe 802 needs more oxygen and or certain nutrients during her growth phase? Or she's just a slower grower, and needs more cycles to create the same biomass. A 4th step-up (in 1.6-2.0 liters) should bring her closer to 150 billion cells.

 

[Broothru]

My emphasis (in bold/red).^
Thank you for reiterating this, that's the whole point you're making, yes!

When making yeast starters we want to promote growth (aerobic) over fermentation (anaerobic). They both occur at the same time but we can shift the mechanism of which one takes prevalence. By supplying oxygen the yeast will remain (mostly) in her aerobic reproduction mode, and will grow.

From what you observed, 802 doesn't reproduce as well as 860 under similar circumstances.
Maybe 802 needs more oxygen and or certain nutrients during her growth phase? Or she's just a slower grower, and needs more cycles to create the same biomass. A 4th step-up (in 1.6-2.0 liters) should bring her closer to 150 billion cells.

So she's a "grower", not a "shower?" Usually associated with a different gender identity, isn't it?

On the other hand, isn't it possible that 802 is a 'fluffier' yeast that is just taking longer to settle? In my current project I'm comparing 5 different commercial Kolsch yeasts in side by side propagations. A test wort of a Kolsch recipe was split five ways, approximately 3 liters each of oxygenated wort. All were fresh 1st generation/1st step yeasts pitched into identical one gallon glass jugs at the same time. Each was fermented in the same location under identical ambient conditions and allowed to reach Final Gravity, with occasional agitation and stir plate used intermittently.

There was significant deviation in both the volume of biomass produced as well as the behavior of the fermentations themselves. Final gravities ranged from 1.006 to 1.008. All were then placed in a 38F refrigerator to settle and condition/'lager'. Two were mostly cleared shortly after FG was reached. Two were showing signs of partial clearing after two days' refrigeration. The fifth one is barely showing any clarification. It stands to reason that the slowest yeast to clear has the most microscopic suspended yeast cells that eventually will settle out, and consequently will appear to have the least amount of biomass until clearing has occurred.

The biggest unanticipated thing I've observed is that the fastest clearing, most attenuative yeasts are not the most prodigious biomass producers. Their yeasts fell most quickly and then became tightly compacted on the bottom of the jugs, being more difficult to dislodge and re-suspend in the wort. The yeast that has shown the least proclivity to clearing initially showed the greatest amount of biomass collecting at the bottom, but was very easy to re-suspend with agitation. The other two samples were somewhere in between.

My prediction (based on an educated guess rather than empirical data) is that after 3~4 weeks of lagering/conditioning at 38F that all the test samples will be mostly-to-spectacularly clear, within 1 point of the same specific gravity and equal %ABV, with near identical biomass deposits on the bottom of each jug, I'll share my final observations in a few weeks' time.

 

[mac_1103]

One could get a definitive answer in about 15 minutes with a centrifuge and/or a microscope. Or one could simply accept that it's difficult to do laboratory work without a laboratory.

 

[Drunk Monk]

So she's a "grower", not a "shower?" Usually associated with a different gender identity, isn't it?

On the other hand, isn't it possible that 802 is a 'fluffier' yeast that is just taking longer to settle?

It's possible, but I don't think that giving the starter additional time to settle will increase biomass by 2.5 times. Also, when preparing a starter, you're not going to wait a week for it to fully settle — the viability would suffer significantly. Pitching the whole starter with the liquid isn't a good option either — there's too much unwanted stuff in there.

Maybe 802 needs more oxygen and or certain nutrients during her growth phase?

That’s my guess as well. I suppose that at some point, WLP802 consumes all the available oxygen and switches over to anaerobic alcoholic fermentation, while WLP860 doesn’t require as much oxygen and has enough of it to fully convert all the sugars into new cells.

One could get a definitive answer in about 15 minutes with a centrifuge and/or a microscope. Or one could simply accept that it's difficult to do laboratory work without a laboratory.

If I had a counting chamber and a microscope, I wouldn’t be here guessing — this thread wouldn’t even exist!

 

[mac_1103]

Also, when preparing a starter, you're not going to wait a week for it to fully settle — the viability would suffer significantly

But you could take a small sample of each starter and let that fully settle to see how the fully compacted yeast cakes compare.

 

[Broothru]

That’s true. With my 5 yeast samples, approximately 100 ml each thick slurry, into equal portions of Kolsch wort, produced wide variations of deposited yeast cakes, at least initially. One sample showed about ¾” yeast in the bottom of the 1 gallon jug after reaching final gravity.

After a few days of refrigeration it had compacted to ¼”, which was about the same visual volume appearance of a different yeast both before and after refrigeration and settling. The first yeast in this example was the least clear (actually quite cloudy) at final gravity, and the other was the most clear and compacted at the end of fermentation before refrigeration.

Based on these casual observations, it only underscores that different yeasts show different characteristics, even though they are typical yeast strains used specifically for a single type of beer. Just like what the O.P. saw between WLP-802 and WLP-860. That should come as no surprise.

What matters in the end is how these different characteristics affect the final beer, and how more or less favorably we perceive those characteristics relative to other beers utilizing different yet similar strains.

 

[Broothru]

It's possible, but I don't think that giving the starter additional time to settle will increase biomass by 2.5 times. Also, when preparing a starter, you're not going to wait a week for it to fully settle — the viability would suffer significantly. Pitching the whole starter with the liquid isn't a good option either — there's too much unwanted stuff in there.

I don’t disagree that supernatant in large volumes is something you don’t necessarily want to pitch into your fresh wort. But yeast viability isn’t going to suffer that much in a five day refrigeration. You will, however, collect more than a few million extra yeast cells that are still suspended in the supernatant. They are also the most active and viable cells.

Normal viability decreases somewhere between 3-5% per month, IIFC. If your propagation is assumed to be 100% viable, then your pitch viability would be at least 95-97%, which is much more viable than that 6 month-old yeast packet you bought at your LHBS.

It only takes a single yeast cell to (in theory) start a fermentation, though it probably wouldn’t make a very good beer. That’s the whole reason to make a starter, because 2 billion active cells is always better than 250 million.

 

[mashdar]

I brew a lot of beer that needs <200M cells, and I'd be a bit worried to pitch 2B cells (>10x over pitch).

But you'd need a 10L stir plate starter or something to hit 2B, so I assume that was hyperbole.

 

[IslandLizard]

I brew a lot of beer that needs <200M cells

Did you mean to write <200 billion cells?
https://www.brewunited.com/yeast_calculator.php

 

[mashdar]

Did you mean to write <200 billion cells?
https://www.brewunited.com/yeast_calculator.php

Oh, yeah. What's a factor of 1000x between friends?

Why are we even talking about 2 billion? lol

 

[mac_1103]

A billion here, a billion there...

 

[Broothru]

I brew a lot of beer that needs <200M cells, and I'd be a bit worried to pitch 2B cells (>10x over pitch).

But you'd need a 10L stir plate starter or something to hit 2B, so I assume that was hyperbole.

Yeah, that's what I get for doing "math in public" again. In fairness, I'm an old guy and haven't studied Organic Chemistry or Math in over 55 years, so what's the big deal with a few orders of magnitude, anyway?

Actually I'm guilty of conflating volumes as much as being guilty of bad math. At least that's the fig leaf I'm trying to hide behind. Let me try to dig my hole a little deeper:

According to an AI script reply to a query I made, "A typical pitching rate for beer brewing yeast is around 10 million cells per milliliter (10 x 10^6 cells/mL), which translates to 10 billion cells per liter (10 x 10^9 cells/L)."

So, 200 ml of slurry (what I usually pitch) would be 2 billion cells. That explains the origin of that number. From the same query:

"Yeast Cell Count in a 1-Liter Batch:
Since a standard package contains 100 billion cells and is designed for a 5-gallon batch, that translates to approximately 5 billion cells per liter (100 billion cells / 20 liters).

And that's what I get for not questioning the accuracy and math of the source. The LLM generated answer even contradicted its own 'self'. But I was the one who ultimately failed both my scientific training and my inherent journalistic doubt.

Mea maxima culpa!