Dr. Fischborn and Dr.Waldrop, thank you very much for hosting another fortnight of yeast! This is like Xmas for us.

First I'm sorry to post so "long" and not very distinct questions but if I just made it short there may be misunderstandings, so I try to explain some of the motives and elaborate the questions.

Your and everyone else's comments on these issues are much wanted!

I have some questions regarding yeast that just to set them in proper context are related to my attempt to understand yeast and make a computer simulation of a beer fermentation. I am currently modeling the states, active dormant and dead. And the transitions are considered by transition probabilities (statistically that is), and the transition functions are supposed to be state functions of yeast, wort and fermentor variables. The below question all relate to stress and biomass yield and to an extent the transition probabilities between states. I hope to decompose the stress into some principal stresses, and find how the depress the biomass yield (but also how they cause damage and death, which is another side of it)

Q1 --------------------------------------------------------------

Tracing back to Balling, many formulas in brewing, including alcohol/FG/OG formulas tend to assume a fixed biomass yield of some 5%. As I understand this is an empirically determined value, that I assume is an effective average under "typical conditions". But as far as I understand the biomass should be a dynamic and in an extended dynamic treatment I doesn't seem valid to treat the biomass yield as a constant? Stirplates in starters are but one example.

Note: I am aware that respiration levels does increase the biomass yield too, but that is not what I am after here. I am trying to understand stress depression of the yield. Considering the biomass yield vs. time, during a batch fermentation. Since stresses build up, and especially external sugars drop in the very end I am assuming that the biomass yield must drop during fermentation. For example, as the sugar concentration is low the rate of energy production drops.

Actual Q1)

How low is the cellwise biomass yield at EOF, just before the cells start to tend to go dormant? What do you think about the idea that the biomass yield drops to close to zero? What about correlating the transition from active to dormant with the biomass yield drop? The idea I have is that the biomass yield in turn would depend on the free energy balance. Incomes - expenses. Expenses also including possible stress factors, transport costs etc. If you feel this still is a dim question perhaps you can elaborate about the topics of dynamic biomass yield and biomass - stress correlation?

Q2---------------------------------------------------------------

Actual Q2)

In a normal batch fermentation, how would you rate these different factors that depress the biomass yield? CO2, alcohol, concentration gradients on culture, UFA/sterol drop.

When using a stirplate but *not* aerate, what factor is most important being responsible for the increased biomass yield? i.e. I want to if possible put numbers on how much the CO2 supersaturation depresses the biomass yield. etc.Have these things been quantified, and isolated from other stress factors? What if you stir in an pressure gas chamber of high CO2 pressure, would the benefit from removal of gradient be significant still? Or is it some mechanical excitation of the cells?

Q3 ------------------------------------------------------------

As alcohol tolerance are supposed to relate to pitching rates, sterol levels and also other add-on stress factors, I wonder what the conditions are for the alcohol tolerance numbers that you sometimes find for strain descriptions? It seems clear that there has to be a limit, but it also seems that the limit can be stretched? (i.e. it's not fixed) so the question is thus

Actual Q3)

How does yeast companies typically *define* the alcohol tolerance limit? i.e.. what is the exact experimental setup/conditions and numerical procedure used to arrive at the alcohol tolerance numbers?

-Frederik

RESPONSE:

Fredrik, Thank you for your very interesting questions.

Question 1

First of all we have to admit that we are not qualified to advise you with specifics on the modeling aspect.

You are absolutely right that stresses are going to affect negatively biomass and ethanol production. At the end of fermentation the general yield is very low. But you have never a homogenous cell culture meaning you will find a wide range of yeast generations. Most of the cells at the end of fermentation are still quite healthy but because of the conditions they are in they are not reproducing quickly.

With respect to stress factors it is almost impossible to give specific values. If you just look for one stress under defined conditions (like ethanol concentration) you will get a number. As soon as you add another factor to the ethanol stress like temperature your number will change.

This is unlikely to be a consistent and easy to understand relationship....we are not trying to put you off your project but you can imagine how difficult your task is with all the relevant parameters taken into account.

There was a poster presented at the World Brewing Convention in San Diego this year in which the presenter demonstrated a model for beer fermentation and propagation. But he was focusing on a few key parameters and achieved relatively good correlation with practical fermentation results.

Question 2

It is difficult to rate the different factors because as the environment changes their impact will change. Concentration gradient will probably be most important towards the end of fermentation because the lack of ATP production will make it difficult to transport anything against the concentration gradient.

CO2 and alcohol accumulate together but it is easier to remove the CO2 effect than ethanol so therefore ethanol is probably more important. CO2 concentration is depending on the hydrostatic pressure in the fermenter. The taller the fermenter the more important the CO2 levels become. As for the UFA/Sterol drop, it will happen and the amount of UFA/Sterols at the beginning coupled with the addition of Oxygen at the beginning will determine the impact of the drop.

On a stir plate the removal of CO2 is probably the main improvement to a static fermentation. But improved distribution of the yeast in relation to nutrients and ethanol should be considered as well. As for CO2 supersaturation you could determine the impact on biomass on its own but then again these numbers are probably not relevant under normal brewing conditions.

Question 3

Mainly from experience. We encourage our customers to give us feed back on their experience with our products. For some strains we will do our internal testing under our standard conditions to determine alcohol tolerance. But this is a subjective test. Other producers will have their own tests.

Finally on the growing subject of sugar consumption; Kurt Thorn and Alan Meeker are absolutely correct. To complicate things further the delay in maltose and maltotriose consumption is also, in part, due to induction of genes by maltose and maltotriose presence. Sugars like glucose fructose and sucrose do not require their presence to have their transporters in the membrane, maltose and maltotriose do.

Keep up the good work,
Forbes & Tobias