Introduction: Folks, we are fortunate to be joined by Dr. Tobias Fischborn of Lallemand, a colleague of Dr. Cone. Clayton said he may be asking for help from such luminaries in the field as Tobias, as he says "No one can know it all!" (I will resist the urge to phone Dave Burley to tell him this!)
Fredrik, you have taken on a huge task which is beyond what I can help you with in a brief session. The variables are mind boggling in a batch process. The media that the yeast finds itself is dynamic. It is changing from moment to moment: The stress of the osmotic effect is going down as the sugar is converted to alcohol. At the same time the stress of the alcohol is increasing. The number of yeast cells is increasing during the first 1/3 to 1/2 of the fermentation. The individual yeast cells produces 20 - 30 times as much alcohol while it is growing than it does when it reaches the stationary phase. Available nutrients change as the yeast grows. Temperature can change if good controls are not in place. pH and CO2 levels change. In a continuous, steady state process the variables are minimized yet are still many. Each variable effects the rate of fermentation and the resulting CO2 production. Many researchers have worked out good models on separate variables and some variables in combination. I would suggest that you find a good technical library and search through both brewing and wine technical journals.
Since you are interested in transport systems, enzymes and metabolic pathways, I will suggest two excellent text books: "Brewing Yeast Fermentation Performance" 2nd. Edition, edited by Katherine Smart (last name very descriptive) Blackwell Publishing, ISBN 0-632-06498-6. "YEAST Physiology and Biotechnology" by Graeme Walker, Wiley Publisher, ISBN 0-471-96446-8.
Please feel free to ask more questions. I sometimes feel that I am not on the same wavelength as you, so I may not answer the question to your satisfaction. I have asked a colleague to add something to your questions. His comments are under "Tobias." You will find my answers under "Clayton" at each of your questions,
Since some months ago I am attempting something as stupid as a beer fermentation simulation model and I've many questions that I would love to have your and everybody else’s opinion on. I can't choose which are most important questions so I will just list them all. I don't expect to get answers to all of them and I don't even know to what extent all questions are well defined. But in any case all ideas and input on some of them from you would be much appreciated! The background of the question is a quest to find an algorithm that will simulate yeast and the most relevant parts in fermentation, and as accurate as possible predict the CO2 production with time.
Related to yeasts sugar utilization that may be of interest for stuck fermentations, in wort consisting of mono, di and trisaccharides, I've read that the simplest sugars are the first to be transported into the cell and digested and each sugar needs a specific enzyme in order to be broken down to monosaccharides. If the transport proteins are blocked or if for some reason the relevant enzyme to break down the sugar is deactivate the fermentation may get stuck? My questions are,
1) what basic variables could possibly determine the synthesis and activity of these enzymes, as well as the activity of the transport proteins?
Clayton: STRESS. Extended period in cold storage before re-pitching. Inadequate temperature during cold storage. Poor temperature control during fermentation. Nitrogen starvation. Excessive repitching. Vitamin, mineral and oxygen deficiencies. Glucose/maltose out of balance. Glucose represses the maltase activity until the glucose is below about 0.4%. High levels of glucose adjunct can cause a problem special attention is given to the fermentation.
2) What are the possible mechanism for jamming or blocking a transporter or inhibiting an enzyme (like maltotriase) that would in turn cause a stuck fermentation?
Clayton: All of the replies to question 1). If the yeast is stressed for any of the above reasons, often times it can't quite finish the job of attacking the last bit of sugar which happens to be Maltotriose.
3) Also, when does the synthesis of these enzymes occur (ie. maltase, maltotriase, etc)? Does it occur upon request when a new type of sugar is arriving, or is it synthesized during budding or maturation so it only has to activate? Or are they always active?
Clayton: I believe that the enzymes are produced on demand.
4) Also "how sequenced" is uptake of the sugar profile? Is there an overlap in the mono -> di, and di -> tri transitions, or is there possibly a tiny delay or dip in energy production for that transition?
Clayton: There is no delay between the glucose and sucrose uptake. The Maltose fermentation kicks in when there is <0.4% glucose.
Glucose & Fructose is entering the cell by facilitated diffusion (energy-independent). The driving force is the glucose/fructose concentration gradient across the yeast membrane. The translocation is via a permease (carrier protein) and the transport is coupled to the glucose phosphorylation.
Sucrose is transformed by invertase in the cell wall to fructose and glucose and then taken up by the yeast. Maltose is entering the cell by active transport (energy dependent) driven by the proton gradient (H+symport). ATPase expels protons (H+) which then re-enter the cell with maltose. Glucose represses the maltose up-take and its utilization. Glucose concentration higher than 0.4 % w/v impairs maltose uptake by acting both as catabolite repressor and inactivator of maltose permease. The maltose transporter is induced by maltose, repressed by glucose (>0.4 %w/v) and inactivated following growth on glucose or by nitrogen source exhaustion.
Maltotriose is believed to have the same or similar active transport system as Maltose. There is a sequence in uptake since maltose and Maltotriose are inhibited by glucose. The transporters are induced by the sugar.
Also I've got a nutrition uptake related question:
5) I've read that the uptake of nutrition, or at least FAN are restricted to the lag phase that are put into pools used throughout the fermentation. And that nutrients that are added in the middle of fermentation are not utilized. Is this true, and why? Are the transporters blocked or busy? Are there special purpose transporters for amino acid uptake that are never used for sugars?
Clayton: Transports systems for amino acids are entirely different than for sugar uptake and alcohol output. Very little if any FAN is taken up by the yeast during the lag phase. The majority of the FAN is utilized during the growth phase. As the alcohol level builds up towards 5%, the yeast find it more difficult but not impossible to take in nutrients and reproduce. So the growth slows down dramatically and the yeast goes into a stationary phase and require very little fresh nutrients. The best time to add nutrients is during the growth phase. Allow the yeast to consume the nutrients present in the wort then add fresh nutrients during the last half of the growth phase. This is ideal but not practical.
Tobias: The amino uptake mechanism is similar to the maltose uptake (active transport driven by proton gradient) but different transporter are used. The uptake of some amino acids is repressed by ammonium catabolites. The yeast should still be able to use FAN at a later stage of the fermentation.
And a question regarding sterol and oxygen .
6) From books and articles I've read that sterol levels are important for cell permeability. What are the details on this? Do low sterols cause the wall to "leak", or to become impermeable? Or does it simply cause some of the transport mechanism to malfunction in general? What do you think about modeling the efficiency of the transporters as increasing with sterols, so that low sterols would basically be blocked and at high enough sterol levels the permeability would reach the ideal and maximum value?
Clayton: Sterols act as a growth factor and will protect the yeast from alcohol toxicity near the end of the fermentation. Sterols play a role in keeping the cell wall fluid (rather than leathery). During the growth phase, the cell wall must be fluid or elastic so that the bud can form. If the cell wall is not elastic enough, the lack of sterols become a growth limiting factor. Near the end of the fermentation the cell wall will become leathery limiting the transport of sugar into the cell but more important it limits the alcohol transport out of the cell thus the excessive alcohol in the cell becomes toxic and will result in a slow or stuck fermentation.
I do not know how you will be able to control the rate of sterol production or the total amount. Yeast can produce the precursor squalene with no oxygen, then with very little oxygen, 10 - 15 ppm, it can move squalene up to sterol.
And a last but not least, a lag related question:
7) Does the yeast absorb all oxygen and nutrients, or at least until the cellular pools for this are full before starting sugar uptake? Or can the sugar uptake start in parallel even though at slow rate to supply extra energy? Is it possible to set a condition for the start of sugar uptake? Such are "all pools full", or "no nutrients left to absorb"? I have considered the case were the cells are very low on glycogen, and that the energy supplies from glycogen simply are not enough to power synthesis of sterols and other things during the lag phase - in such a case. Without sugar uptake starting in parallel would,
a) yeast would be stuck in the lag phase forever?
b) Would it stop all nutrition uptake and synthesis due to lack of energy, and instead start to uptake sugar - and NOT do any more nutrition uptake?
Clayton: If the pitching yeast is healthy, there is enough of everything that the yeast needs inside the cell to start the fermentation as soon as the lag phase is over. The yeast will do many functions at the same time: metabolize sugar, utilize the nitrogen sources, converting it into cell mass, enzymes, DNA etc. Sugar is a nutrient for the yeast also. During the growth phase it metabolizes the sugar to receive energy and along with minerals, vitamins, nitrogen produces cell mass. The yeast does not have to wait for a period of time and a series of things to happen before it can start to convert sugar to alcohol and CO2.
8) Does yeast synthesize sterols exclusively during the lag phase or can it do so later on if there are enough oxygen and other building blocks in the late part of fermentation?
Clayton: There is very little activity in the lag phase other than acclimating the yeast cell to its new environment. Yeast produces sterols anytime there is oxygen available and there is a need for it.
Tobias: Oxygen uptake is at any time and sterol synthesis as well. That is what we are doing in our propagation.