Yeast Sterol Production & Starter Growth

Here is a topic I have been pondering...

I have read that (i) membrane sterols are a rate limiting factor for yeast growth/division/budding, (ii) the production of sterols by the yeast require oxygen, and thus sterols are produced prior to fermentation. The rationale for doing step up starter cultures is to maximize sterol production (or minimize the limitation of insufficient sterols), and thus generate healthier yeast. At the same time, final cell number has a peculiar relationship to inoculation rate: when fewer yeast are pitched, the final number is greater (cf growth charts).

I know there are lengthy threads on aeration of starters on stir plates, etc. It seems to me that sterols should only be limiting if an airlock is used for a starter culture (i.e. sterols will be made from the dissolved oxygen & the yeast will begin to ferment in an anaerobic environment). In a non-airlocked starter, the yeast will be growing under aerobic conditions (not to re-open the previous debate, but there should be adequate oxygen diffusion from the atmosphere to provide oxygen for sterol production with a simple foil covered or foam stoppered Erlenmeyer...).

So taking all of this together, are step up starters a relic of using airlocks, where oxygen would be limiting? Given that smaller pitching rates yield greater final cell counts, it seems to me that if you are not using an airlock, it would be more advantageous to start with a small starter pitch to get a better yield. If this is the case, why bother with the step ups?

well, this is something I’m looking into and where I already have some data.

Yeast growth on a stir plate is in general limited the nutrients available in that wort and not by the available oxygen. I haven’t done any experiments that would show that, but that’s what I conclude from the results that I have so far.

As for the relationship between yeast growth and inoculation rate, very little work has been published when it comes to using a stir plate. Results have been published by Jamil, Chris White and Wyeast but none of them fully disclosed how they arrived at their growth curves. I have done some reverse engineering of Jamil’s calculator and the growth curve for a stirred starter is simply the growth curve for a still starter multiplied by 2.7 on both the x and y axis. I may follow the scaling of the y axis (growth) but stretching the x axis (initial cell density) doesn’t make sense since that means that the optimal inoculation rate for yeast growth in stirred starters is 2.7x the optimum in unstirred starters.

In my research thus far I have found that each gram of brewers extract (stuff dissolved in all malt wort) growth about 1.4 Billion new cells even if the inoculation rate is very low (less than 1 M/ml). Once the inoculation rate surpasses 1.4 Billion cells per gram of extract, the amount of new cells that can be grown in that wort, the growth rate drops. I think that this is simply because not every cell will be able to consume enough nutrients to grow a new cell.

Stepped starters still make sense, to some extent. If your starting cell density is too low it will take longer for the yeast to create a favorable pH environment for fermentation and this makes the starter more susceptible to infection. But when starting with a vial or pack of yeast, I don’t think that there is any need for stepping up a stirred starter unless your flask is not large enough to hold the volume of wort necessary for propagation.

Here are pointers to the data I have published so far:

http://braukaiser.com/blog/blog/2012/10/08/yeast-growth-experiments-some-early-results/
http://braukaiser.com/blog/blog/2012/11/03/estimating-yeast-growth/


Kai

Thanks for your reply, Kaiser. Interesting data you linked there. Nice to see someone approach hobby brewing in this way. Often things are done for no particular reason (because it works, right or wrong), while we should be trying to optimize everything!

I come from a lab background, so I've cultured a lot of bacteria and mammalian cells, but only starting to get into yeast for brewing. In the lab, we have the advantage of engineering antibiotic resistance so contamination isn't a problem. Picking a single colony off a plate with a sterile tip to transfer 100-200 bugs to a 1L broth is common, though doing a 3mL culture first just to ensure antibiotic resistance, and then inoculating 100-200 cells from that into 1L.

With brewing, we rely on the yeast being better at consuming the nutrients than other bugs, but there is the risk of other bugs getting in or tainting the batch at every handling step. With step up cultures for yeast, in my mind, you will have created a better pH environment for yeast growth and dominance of the culture, as you say. Adding media volume after the 1st step has completed (2X-10X), there will be so many yeast that they will still dominate the culture. However, it seems to me that the step cultures also add a risk with handling -- each time the new media might not be fully sterilized and there is a risk of airborne contaminants.

Having -80°C freezers at work, I started making yeast banks for the strains I have bought. Thusfar, my starter strategy has been to take 1mL of frozen yeast slurry (50% yeast cake from 1° fermenter:50% USP glycerol) and inoculating directly into cooled 1.5L of 1.040 DME media (Erlenmeyer with foil, sanitized 10 min boil on stovetop, stirbar boiled too). I inoculate with a flame going and sanitized work surface, but by no means aseptic technique! Using this method, I see krausen at about 48h-72h. So there is a good long lag phase where other bugs could reproduce; I don't know exactly how many yeast I am inoculating either -- several thousand or a million max. Apart from a systemic bacteria introduction from incorrect technique (and I am pretty good about gloves, sanitizer, timing, etc), as I only have the culture open once, the risk of airborne contaminants is limited.

So far, my method has been yielding good results, though I was concerned about reading that maximizing sterols was a reason for doing step cultures. I don't want to be inadvertently weakening my yeast strains... yeast abuse & whatnot I might switch to doing an 100mL initial step and then use that to inoculate the 1.5L starter to cover my bases, but under aerobic conditions, I am hoping that my yeasties are happy.

rockjetty said:

At the same time, final cell number has a peculiar relationship to inoculation rate: when fewer yeast are pitched, the final number is greater (cf growth charts).

Click to expand...

I think you're misreading the growth chart there, or maybe I am. All pitches were done with 100B cells. Likewise, the x-axis doesn't chart the number of cells pitched but just inoculation rate, which in this experiment is a simple proportion of starter size.

In any case, pitching few cells gets you fewer final cells on the yeast calc calculator itself.

Kai, that's some great work! I always am fasinated by what you are doing.

I'm actualy running a similar type of experiment with five different yeast strains and looking at a matrix of wort gravity and incoculation rate. Like Kia, I expect that the cell growth is limited by availbe sugar not cell density as some of the starter calculators seem to sugest.

I'm not nearly the scientist that Kai is, but here is the experiment setup:
http://woodlandbrew.blogspot.com/2012/12/how-many-cells-are-produced-by-starter.html

WoodlandBrew said:

Kai, that's some great work! I always am fasinated by what you are doing.

Click to expand...

Thanks

I'm actualy running a similar type of experiment with five different yeast strains and looking at a matrix of wort gravity and incoculation rate. Like Kai, I expect that the cell growth is limited by availbe sugar not cell density as some of the starter calculators seem to sugest.

I'm not nearly the scientist that Kai is, but here is the experiment setup:
http://woodlandbrew.blogspot.com/2012/12/how-many-cells-are-produced-by-starter.html

Click to expand...

I like that others are checking the assumptions that the currently available yeast growth calculators are making.

I assume that you don't have results yet? Is the table on top of the post the expected growth based on the common yeast calculators?

Kai

Kaiser said:

I assume that you don't have results yet? Is the table on top of the post the expected growth based on the common yeast calculators?

Click to expand...

Yes, I don't have results yet, and yes, the table at the top is based on the common yeast calculators as well as limited observations I have made, and reading. (Or when compared to a well run scientific experiment, it's just a guess based on a gut feeling.)

I will add a caption to the table so that it is not misleading.

I'm looking forward to seeing your results. You are always very thorough and disciplined with the data that you collect.

MalFet said:

I think you're misreading the growth chart there, or maybe I am. All pitches were done with 100B cells. Likewise, the x-axis doesn't chart the number of cells pitched but just inoculation rate, which in this experiment is a simple proportion of starter size.

In any case, pitching few cells gets you fewer final cells on the yeast calc calculator itself.

Click to expand...

Ha! You could be right... I thought they were using a 100B smack pack to inoculate their starter broths (0.5L-8L). If I am misreading the graphs, it seems like a strange experimental design to inoculate with different volumes of starters, despite the constant cell number. They say that the FG was the same in all starters ranging from 0.5L to 8L all with 100B cells, but how long was it at that gravity? The lag, exponential, and plateau phases of growth are going to last for different periods depending on the volume/availability of nutrients. Could be that the 8L culture has just finished the exponential phase, and is thus very healthy, and just starting into the plateau phase, whereas the 0.5L culture will plateau quite quickly, and may have been sitting in stasis for some time before pitching... Pitching different volumes would also require adjusting the calculation for final volume, depending on the size of the batch.

On a side note, now that I have brought up growth phases of yeast -- I am interested in peoples opinion of what is happening during active krausen. My thoughts are that cell division/budding is coming to a close, cell density is limiting oxygen availability (for aerobic culturing), so most of the yeast go into active fermentation. So inoculating your wort with starters at active krausen will be the healthiest and most dense your starter will ever be. Thoughts?

Recently I recorded observations of a fermentation. I was looking at specific gravity and cell counts recording both viable and non-viable. (the wort was pitched with 10% viable yeast). The amount of krausen roughly followed the number of viable cells in suspension, so it was when the krausen first peaked that there were the most new cells. If you looked at the data it would probably make more sense to you than to me. It does seem to follow what you are suggesting. That post is going up on December 23rd.

The axis on these charts are a bit odd and as a result it's not easy to see the actual information. You also need to know the volume to make sense of the y-axis, which is the actual number of cells and not a cells per volume number.

A more valuable chart is one that plots cell growth over initial density. Or even final cell density over initial density.

rockjetty, I don't think the starters were pitched into any beer. Their cell's were counted which it doesn't matter how long they sat. cells that grew won't disappear withing a few days (or even weeks).

As for high Kraeusen, that should be the end of the exponential phase. Fermentation starts right away. Even in the presence of some oxygen, the high sugar concentration forces the yeast to use fermentation (see Crabtree effect).

Pitching a starter at high kaeusen is best beause that's when the yeast is the most healthiest and can get started fermenting the new wort pretty much right away. Yeast that sat dormant for even a few days will have to replenish lost resources (the longer it sat dormant the more it will have to replenish) before it can start fermentation and cell growth.

WoodlandBrew, that's some mighty low yeast viability. If the 10% was determined with Methylene Blue, the number is pretty much useless since Methylene blue tends to ovestimate cell viability, especially in old cultures.

Kai

Kaiser said:

well, this is something I’m looking into and where I already have some data.

Yeast growth on a stir plate is in general limited the nutrients available in that wort and not by the available oxygen. I haven’t done any experiments that would show that, but that’s what I conclude from the results that I have so far.

Click to expand...

I've not done the experiments, but I am a biologist whose work frequently deals with lipids & sterols; sometimes even in yeast...

Yeast do indeed need oxygen to produce sterols, which is yet one more reason to use a stirred starter. However, sterols are readily synthesized from simple compounds, and as such are not a limiting factor in aerobic growth. Hence why you see growth in a starter being limited by nutrient availability; to make sterols a yeast's minimum requirement is a carbon source (i.e. sugar) and O2.

Where sterols become important is in the ferment itself. In theory, sterol-rich yeast can undergo more in-fermenter cell divisions. More importantly though, these yeast should be more stable as sterols help to maintain proper membrane fluidity and make membranes more resistant to things like temperature changes and alcohol.

In short, sterol production is more important in ensuring the maximum quality of yeast than quantity. Yeasts with lots of sterols (and unsaturated fatty acids, also produced during aerobic metabolism) have more stable membranes, making them more resistant to temperature changes & alcohol, and making them more resistant to autolysis. Ideal features if you want to make a high gravity beer, or re-use a yeast cake for multiple batches.

Bryan

Kaiser said:

WoodlandBrew, that's some mighty low yeast viability. If the 10% was determined with Methylene Blue, the number is pretty much useless since Methylene blue tends to ovestimate cell viability, especially in old cultures.

Click to expand...

Thanks Kai,
Yes, absolutly. That was low viability. The reason I was recording the data was to see if the dead cells would sink to the bottom or be suspended by the living cells durring fermentation.

I'm glad you mentioned the discrepancies in cell count with methylene blue. That is not something that I have seen before. I would certainly not want to post false findings. I have done counts with both trypan blue and methylene blue and seen similar numbers. I have also read articles that indicate they both work equally well. White Labs also uses methylene blue as does Wyeast.

But I will have to revisit that, especialy with older slurries.

WoodlandBrew said:

Thanks Kai,
Yes, absolutly. That was low viability. The real reason for recording the data was to see if the dead cells would sink to the bottom or be suspended by the living cells durring fermentation.

Click to expand...

Interesting experiment. I haven't thought of that.
If you want to be sure about healthy vs dead, you could take a healthy culture, heat 90% of it to 70 C to kill them and then mix with the rest and pitch. That way you know 90% are dead.

I'm glad you mentioned the discrepancies in cell count with methylene blue. That is not something that I have seen before. I would certainly not want to post false findings I have done counts with both trypan blue and methylene blue and seen similar numbers. I have also read articles that indicate they both work equally well. White Labs also uses methylene blue. And also by Wyeast.

But I will have to revisit that.

Click to expand...

Methylene blue's tendency to overestimate viability is actually well known. But because of its simplicity this stain has become the standard viability test in brewing. Here is some of the info I have on that: http://braukaiser.com/wiki/index.php?title=Microscope_use_in_brewing#Methylene_blue_staining

Typran blue may have the same issues and may be equally bad as MB.

Kai

Sorry to get distracted from the original topic of sterols, RockJetty. I commented on your thread because sterol production and use is something that I have been interested in. My understanding from Fix "Principals of Brewing Science" is that there are two metabolic pathways that produce sterols. To summaries my simple understanding: the primary pathway uses oxygen, but there is a secondary pathway that does not use oxygen but uses more energy.

Warthaug mentioned several reasons for the importance of sterols, and that matches what I have read:

Warthaug said:

Where sterols become important is in the ferment itself. In theory, sterol-rich yeast can undergo more in-fermenter cell divisions. More importantly though, these yeast should be more stable as sterols help to maintain proper membrane fluidity and make membranes more resistant to things like temperature changes and alcohol.

In short, sterol production is more important in ensuring the maximum quality of yeast than quantity. Yeasts with lots of sterols (and unsaturated fatty acids, also produced during aerobic metabolism) have more stable membranes, making them more resistant to temperature changes & alcohol, and making them more resistant to autolysis. Ideal features if you want to make a high gravity beer, or re-use a yeast cake for multiple batches.

Bryan

Click to expand...

My real curiosity is how this applies to saved slurries, and what the best way is to revive them. Would it be best to use a high pitch rate and a lower gravity with plenty of oxygen to boost sterol content while minimizing the number of new generations of cells to prevent mutation? Or perhaps mutation and cell selection is not a concern?

WoodlandBrew said:

To summaries my simple understanding: the primary pathway uses oxygen, but there is a secondary pathway that does not use oxygen but uses more energy.

Click to expand...

I haven't read Fix in a while, but it is my understanding that sterol production takes oxygen or unsaturated fatty acids (UFA). There is no way to make sterols by simply spending more energy.

My real curiosity is how this applies to saved slurries, and what the best way is to revive them. Would it be best to use a high pitch rate and a lower gravity with plenty of oxygen to boost sterol content while minimizing the number of new generations of cells to prevent mutation?

Click to expand...

I think the best way to revive an old slurry is to take a small amount of it and grew a new population of cells from it.

Kai

WoodlandBrew said:

My real curiosity is how this applies to saved slurries, and what the best way is to revive them. Would it be best to use a high pitch rate and a lower gravity with plenty of oxygen to boost sterol content while minimizing the number of new generations of cells to prevent mutation? Or perhaps mutation and cell selection is not a concern?

Click to expand...

Kaiser already hit the nail on the head - the best thing is to grab some yeast from the slurry, re-grow it aerobically, and use that. The yeast in a yeast cake will be somewhat sterol/unstaurated lipid-depleted, is typically glycogen depleted, is stressed, and thus is less than ideal for brewing. That said, many brewers will re-pitch the same yeast cake 4 or 5 times without an aerobic growth phase (aside from oxygenating the wort pre-pitch), and seem to not suffer overly from it.

In terms of mutation, no matter what you use for a yeast source there will be mutations (in lab strains, you get 1 mutation for every 3 or so cell divisions; presumable brewing strains will be similar). Re-pitching (vs going back to a low-generation stock) will lead to more mutations, and thus be more likely to lead to changes in the yeast characteristics. That said, the effect over a small number of re-pitchings (3-4) should be minimal. And more than that means you now have a house strain!

Bryan

Thank you both!