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Old 02-10-2013, 12:46 AM   #51
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In this case, 100-300 millions of cells/mL of culture (instead of billions). Four billions of cells/mL seems to be very high in a conventional starter, even for those yeast strains that have high growth rates, like belgian strains. How did you estimate this number of cells? Just curiosity...

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Old 02-10-2013, 01:25 PM   #52
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Originally Posted by diegobonatto View Post
In this case, 100-300 millions of cells/mL of culture (instead of billions). Four billions of cells/mL seems to be very high in a conventional starter, even for those yeast strains that have high growth rates, like belgian strains. How did you estimate this number of cells? Just curiosity...
Right 1-3E8 is 100-300 million. I shouldn't try to do even simple math in my head after a few beers. :-/

What you are saying makes much more sense now. It's good to know that we are all seeing about the same thing as someone with as much experience as you.

Cell counts were done with MB stained on a hemocytometer.
This is my procedure: http://woodlandbrew.blogspot.com/201...viability.html

4 billion per ml was the highest, but I have seen most of WLP566 and S-04 come in about there for the thick cell density. The actual culture density is about 100-200 million per ml. I've done a few hundred cell counts, and have pretty consistent results.
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Old 02-10-2013, 02:54 PM   #53
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Glad to know that I could help. In fact, neubauer counting + MB staining is a standard method to evaluate cell viability and number. Scientific literature reports some drawbacks by using this technique, seems that quiescent (non-dividing but viable cells) can be stained with MB and, thus, leading to an underestimation of the number of viable cells. The use of fluorescent dyes is more advisable to evaluate viability, but this is unpractical for homebrewers because epifluorescent microscopes ($$$) are needed.

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Old 02-11-2013, 01:48 PM   #54
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Now I know why d_striker posted in the science forum. I neglected this thread. So here is my answer to d_striker’s question:

The lower yeast growth per sugar is something I observed in my experiments while I don't have an exact justification for the actual numbers yet I have a theory of what's going on.

Let's assume two things:
(1) - 1 gram of extract gows X Billion new cells
(2) - yeast only bud once they consumed enough resources to grow a new cell

This means that if there are more yeast cells per extract than can be grown from that extract not every cell will be able to grow a daughter cell. In an idealized culture (all cells consume nutrients at the same rate) no new cells should be able to grow since none of the cells will be able to consume enough nutrients to grow a daughter cell. But the culture is not ideal which means some cells will be able to consume enough nutrients to grow a daughter cell while others won't. The ones who don't grow buds will consume extract but don't actually contribute to cell growth (though it makes them healthier and better prepared for fermentation). This mechanism also means: the more initial cells are trying to consume the existing extract the fewer will be reach nutrient levels sufficient for budding. That's why I expect cell growth to drop with increased initial cell density.

I'm still working on solidifying or disproving this theory with additional experiments. If the theory is correct, the drop in yeast growth should be earlier and more pronounced with old cultures compared to fresh cultures since old cultures have depleted their reserves further. I have experimental data on this, but the results are not as clear as I hoped them to be. I think I have to control a few more parameters.

But I do want to hear more from diegobonatto and have a few questions. I changes the exponential notation to Billions to make it more readable. At least for me

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Originally Posted by diegobonatto View Post
However, if we consider the stationary phase of growth for the major yeast strains, all yeast strains reach the maximum concentration of 100-300 B cellls/L of culture medium (wort, YEPD, or other rich medium that are plenty of complex nutrients). It is possible to achieve high cell numbers (above 300 B cells/L) by using special equipments (e.g., bioreactors), where the physico-chemical parameters are better controlled (pH, O2 concentration, nutrient levels, etc...).
What is causing this limit? is it simply the available amount of nutrients or are there other mechanisms at work? I don’t have my data here at work but tonight I want to check the max culture densities that I observed.

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In our case (homebrewer's case), we always perform what is called a "batch culture", that is the use of erlenmeyers, growlers, etc. with shaking to grow yeast cells. Thus, the maximum concentrations of cells always will be in the range of 100-300 B/L, even if more sugar or DME was added in the culture.
I’m wondering about that. One limiter might be the amount of alcohol that is produced when the initial sugar concentration is high.

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… It should be noted that these numbers are theoretical, and many factors can influence the final number of cells in the wort. Viability is an important factor, as well as the initial sugar concentration in wort, dissolved O2, bacterial contamination, etc...
It’s exactly those factors that affect yeast growth and the final yeast cell count that I’d like to get a handle on. I think one major factor is amount of nutrients available in the wort. But I don’t think that viability plays a large role here. Especially if the amount of growth is significantly more than the initial population. My thinking is that non-viable yeast cells will not consume any of the available nutrients and leave more for the others. That means that while non viable cells may not be able to grow other cells should be able to grow a bit more since they have more nutrients available.

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Old 02-11-2013, 03:35 PM   #55
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Hello Kai!

Let me answer your questions:

Quote:
Let's assume two things:
(1) - 1 gram of extract gows X Billion new cells
(2) - yeast only bud once they consumed enough resources to grow a new cell
The first proposition assumes that 1 g of yeast should have more or less some billions of new cells. However, remember that you will have a mixture of new and old cells compounding this 1 g of yeast biomass. In fact, the proportion of new/old cell depends on the viability of culture and the initial cell concentration inoculated in the wort.

Quote:
This means that if there are more yeast cells per extract than can be grown from that extract not every cell will be able to grow a daughter cell. In an idealized culture (all cells consume nutrients at the same rate) no new cells should be able to grow since none of the cells will be able to consume enough nutrients to grow a daughter cell. But the culture is not ideal which means some cells will be able to consume enough nutrients to grow a daughter cell while others won't. The ones who don't grow buds will consume extract but don't actually contribute to cell growth (though it makes them healthier and better prepared for fermentation). This mechanism also means: the more initial cells are trying to consume the existing extract the fewer will be reach nutrient levels sufficient for budding. That's why I expect cell growth to drop with increased initial cell density.
This is a bit more complicated...in a batch culture, you will have non-reproducing old mother cells (which not bud anymore, but are live and nutrient-consuming cells), budding old mother cells, budding new cells, and naive new cells. All these cells consumes nutrients. Thus, if your initial cell concentration is high, the nutrients of the wort will be fast depleted and, again, the culture will reach the stationary phase of growth. In other words, the number of new cells generated will equalize the number of cells that die by aging or random mutation. After nutrient depletion, all yeast cells enter in quiescence and begins to consume its internal supply of nutrients. The concentration of intracellular nutrients depends on the age of yeast cells (new cells = more intracellular nutrients).

Quote:
What is causing this limit? is it simply the available amount of nutrients or are there other mechanisms at work? I don’t have my data here at work but tonight I want to check the max culture densities that I observed.
Nutrient levels, the age of the yeast cells, genetic mechanisms, among other physiological and environmental factors regulate the upper limit of cell concentration observed in stationary phase. In the lab., we can achieve high cells numbers (>10^9 cells/mL) by using a technique called "fed-batch", which increase amount of culture media is added in a bioreactor, with O2 levels, temperature, and pH being constantly monitored and kept at specific numbers. For homebrewing batch culture, this is more complicated, seems that many environmental points are not finely regulated, influencing the final number of cells in the wort.

Quote:
I’m wondering about that. One limiter might be the amount of alcohol that is produced when the initial sugar concentration is high.
Yes, and ethanol concentration above 15% is toxic for the majority of yeast strains. High sugar concentration leads to elevated ethanol levels in wort, restricting the cell cycle of viable cells

Quote:
It’s exactly those factors that affect yeast growth and the final yeast cell count that I’d like to get a handle on. I think one major factor is amount of nutrients available in the wort. But I don’t think that viability plays a large role here. Especially if the amount of growth is significantly more than the initial population. My thinking is that non-viable yeast cells will not consume any of the available nutrients and leave more for the others. That means that while non viable cells may not be able to grow other cells should be able to grow a bit more since they have more nutrients available.
Viability is an important factor. If you consider that non-reproducing cells are live and consuming nutrients at levels that are more or less similar to reproducing cells, your culture will reach the stationary phase. Only dead cells will not consume nutrients. It is important to note again: the batch culture used by homebrewers will be composed by a heterogeneous population of yeast cells: senescent old cells, reproducing old cells, reproducing new cells, and naive cells. All these cells consume nutrients, depleting the wort fasting if the initial cell number inoculated is high. Is possible to get a culture only composed by new cells in a batch culture? No, because the cells will age with time, originating a heterogeneous population. Is there a method used to obtain only reproducing cells? Yes, but only with special techniques and equipments (chemostats or bioreactors) not easily available for homebrewers.
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Old 02-11-2013, 07:21 PM   #56
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The first proposition assumes that 1 g of yeast should have more or less some billions of new cells. However, remember that you will have a mixture of new and old cells compounding this 1 g of yeast biomass.
I think you misread the statement. I was talking about new growth that one can expect from a gram of extract. It didn’t talk about the amount of cells in a gram of yeast.

Quote:
This is a bit more complicated...in a batch culture, you will have non-reproducing old mother cells (which not bud anymore, but are live and nutrient-consuming cells), budding old mother cells, budding new cells, and naive new cells. All these cells consumes nutrients.
I would expect that the proportion of non budding old mother cells is small compared to the other cells. A given culture should contain about 50% cells with no scars, 25% with one scar, 12.5% with two scars and so forth. There should not be a significant amount of cells with more than 6 scars, for example.
Quote:
In other words, the number of new cells generated will equalize the number of cells that die by aging or random mutation.
So what you are saying is that once the growth rate approaches the death rate the population of healthy cells will not grow. That makes sense and would explain an upper limit for the cell density.

Thanks,
Kai
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Old 02-11-2013, 08:41 PM   #57
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I think you misread the statement. I was talking about new growth that one can expect from a gram of extract. It didn’t talk about the amount of cells in a gram of yeast.
You are completely right! I missed the statement. Thank you for the observation.

Quote:
I would expect that the proportion of non budding old mother cells is small compared to the other cells. A given culture should contain about 50% cells with no scars, 25% with one scar, 12.5% with two scars and so forth. There should not be a significant amount of cells with more than 6 scars, for example.
Depends on the aging of culture. Fresh yeast culture will have less multi-scar cells than old cultures. I like to say that fresh cultures have less senescent (non dividing) cells that old cultures. Its simplify a lot the discussion, because the number of scars is a not a reliable factor when considering the viability of culture (some yeast strains can display a large number of scar before senescence, while others stop dividing early). When and what factors that leads a yeast cell to enter in senescence is a hot topic of research and debate today in the scientific yeast literature (including my research on yeast aging).

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Thanks,
Kai
[/QUOTE]

It was my pleasure!
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Old 02-12-2013, 03:23 PM   #58
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Fresh yeast culture will have less multi-scar cells than old cultures. I like to say that fresh cultures have less senescent (non dividing) cells that old cultures.
I’d like to understand this aspect a bit more. How do the number of scars increase from a fresh to an old culture? If the cells aren’t dividing there should not be an increase of scars and if they are dividing there should be an increase of cells with a low scar count.

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… (including my research on yeast aging)…
That’s what I like about these forums. It gets me in touch with people who may have answers for some of the questions I have.

Kai
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Old 02-12-2013, 08:31 PM   #59
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I’d like to understand this aspect a bit more. How do the number of scars increase from a fresh to an old culture? If the cells aren’t dividing there should not be an increase of scars and if they are dividing there should be an increase of cells with a low scar count.
Remember when I wrote that in stationary phase the number of new cells generated is more or less equal to the number of cells that die? By some reason that is not completely understood, the new cells (without scars) generated enters in quiescence (non-dividing state) faster than old, multi-scared cells during stationary phase. Thus, the fast explosion of new cells observed during exponential and early stationary phase is not observed during the late stationary phase. With time and presence of lower amount of nutrients, the old cells (single-scar to multi-scar cells) divide some times before senescence and death, thus increasing the number of cells with scares. But, again, I do not like to use the scares as an indicative of how old is a culture of yeast cells because the number of scares is very strain-dependent.

In scientific literature regarding yeast aging, there are two major mechanisms that describes how a yeast cell age: (i) the replicative lifespan, which corresponds to the maximum number of daughter cells generated by a mother cell (which can be determined by counting the number of scares) and (ii) chronological lifespan that is associated to the maximum time that a cell can be kept in quiescence mode before senescence and death. For my point of view, I think that chronological lifespan is very important during beer lagering of some styles (lagers). However, replicative lifespan could be important during the active phase of fermentation. Interestingly, there are some hypotheses that discuss that old cells' death release very small amount of nutrients that sustain the quiescent state of new cells until the environment becomes adequate to induces the exponential phase again.

Unfortunately, many of these studies are only performed with lab-conditioned haploid yeast strains in standard culture media. There are not any information regarding the replicative and/or chronological lifespan of brewers strains.

In conclusion, old yeast culture contain more senescent cells, with low viability and vitality, but also contain new cells in quiescent state that can be stimulate to divide when the nutrient levels of wort/beer increase again. In this sense, the starter is fundamental to restore viability and vitality of culture, especially if the original culture is very old.

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That’s what I like about these forums. It gets me in touch with people who may have answers for some of the questions I have.
I have the same opinion! A learned a lot from different people with different backgrounds in this forum.
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Old 02-16-2013, 12:37 PM   #60
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Great thread. I have a question about Woodlands latest blog post. Hope you don't mind me tacking it on here. Woodland discussed an equation showing the number of cells produced as a function of sg and fg. Seeing as most people pull their starters after 24 hours, are we not getting the optimal number of cells out of our starters? I assumed most reproduction occurred during the lag phase, but the numbers seem to indicate that reproduction chugs along until you hit fg.

Also, I've never really measured the sg of my starters. Do most starters hit fg after 24 hours?

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