Crabtree Effect and Over-aeration

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stoutaholic

stoutaholic

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I keep running across what appear to be conflicting statements about yeast behavior in an aerobic environment. One statement is that in a solution of at least 0.4% glucose, which wort always has at the time of pitching, yeast metabolism is fermentative rather than oxidative (Brewing, Hornsey, 1999, p. 117-118). That is, it utilizes the EMP pathway and does not engage in respiration, even if there is abundant oxygen in the solution. This is the well known Crabtree Effect.

However, in the same texts that mention the Crabtree Effect, I also find statements that say that over-aeration can lead yeast to respire instead of ferment, and thereby utilize the valuable sugars in the wort for the production of CO2 and water instead of ethanol. For instance, in the same book referenced above:

"Yeast strains have widely differing oxygen requirements and it is important that there is sufficient oxygen for rapid initial cell growth (especially in terms of membrane constituent synthesis) but not enough for the yeast to undergo aerobic respiration via the oxidation and decarboxylation of pyruvate and ultimately the Krebs’ cycle. (Brewing, Hornsey, 1999, p. 117)"​

Can anyone help explain this discrepancy? If yeast utilize all the oxygen they need in order to synthesize sterols and there is still oxygen left over, will they engage in respiration, or will they simply begin fermentation? If they begin fermentation, what happens to this excess oxygen? Does it just stay in solution, contributing to staling reactions?

The only explanation I can think of is that the yeast initially are fermentative, obeying the Crabtree Effect, but this initial fermentation, which consists of little yeast growth and primarily involves the synthesis of sterols, uses up enough GLUCOSE (since glucose is metabolized before more complex sugars) to reduce the glucose % below 0.4, and at that point there is no longer any catabolic repression for the utilization of oxygen, and the yeast begin respiration if oxygen is still available.
 
stoutaholic said:
The only explanation I can think of is that the yeast initially are fermentative, obeying the Crabtree Effect, but this initial fermentation, which consists of little yeast growth and primarily involves the synthesis of sterols, uses up enough GLUCOSE (since glucose is metabolized before more complex sugars) to reduce the glucose % below 0.4, and at that point there is no longer any catabolic repression for the utilization of oxygen, and the yeast begin respiration if oxygen is still available.
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I like that explanation, although I've never researched this with yeast. I also think that none of this is black and white. Even if yeast don't ferment glucose before respiration, there is surely a certain percentage of yeast cells that begin some fermentation, particularly of simple sugars (besides maltose) virtually immediately upon pitching... just not enough to be noticeable.
 
Apparently this is a topic about which there is a fair degree of confusion. I just read a book called "Froth! The Science of Beer" by Mark Denny that was published this year (May, 2009). It appears that just about every assertion he makes in the chapter on "Yeast Population Dynamics" is wrong. He seems to think that the primary purpose of aerating wort is to allow the yeast to grow via aerobic respiration. He realizes that they need to use oxygen to synthesize sterols, but he actually thinks that most yeast growth in a fermentation occurs while there is still oxygen in solution, and that once the yeast transition to anerobic metabolism, most of the sugars in the beer have already been used up. How does this kind of mis-information get published by a reputable university press?

Anyway, can anyone confirm that yeast do transition to respiration if there is excess oxygen in solution after most of the glucose in solution has been consumed? I've searched through quite a few books and haven't found any source that directly addresses this question.
 
Gah! I was chatting with a phD specialising in fungi microbiology and a 'master brewer' from the IBD for about 2 hours, and we STILL hadn't come to a conclusion at the end of it. The question we were debating was; does yeast actually respirate, or simply perform a metabolic function that happens to utilise oxygen? And what impact does gravity have one these actions?

I've been taught that there is a lag phase at the beginning of fermentation when the yeast is mostly just reproducing - this is propagation, and when most of the O2 is being used for sterol production. After that, the real party gets started with the anaerobic process of fermentation, which will take care of that pesky sugar and turn it into sugar. Now, there is still fermentation going on during the lag phase, and still reproduction going on during the fermentation. It's not like the yeast goes 'ok, there's enough of us now - let's make beer!'. The way I understand it, MOST of the yeast will be creating sterols and budding at first, and MOST of the yeast will be fermenting after that, but both process will continue throughout.

What I can say is that the way I understand it, the crabtree effect (Crabtree effect - Wikipedia, the free encyclopedia) has pretty much nothing to do with our yeasty buddies in practical brewing. What the CT effect applies to is when there's enough sugar that yeast doesn't need oxygen in order to 'feed' itself, 'cause there's enough sustenance in the sugar without using O2 to convert things into available foodstuff. This, consequently, means that yeast will use less O2 in high-glucose environments because it's EASIER to just use the sugars instead. Note the word 'less' - yeast will still be using oxygen, but for different metabolic pathways than they would with less glucose around.

In relation to your specific questions, stoutaholic;
If yeast utilize all the oxygen they need in order to synthesize sterols and there is still oxygen left over, will they engage in respiration, or will they simply begin fermentation?
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See above - the process are simultaneous, but follow general 'phases' where more yeast will do one or the other. 'Respiration' (ie propagation/reproduction) first, fermentation once they're comfy.

If they begin fermentation, what happens to this excess oxygen? Does it just stay in solution, contributing to staling reactions?
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Again, the O2 is still being used, just less of it. The beauty of live beer instead of filtered or pasteurised stuff is that the yeast will continue to 'mop up' small amounts of oxygen introduced to the beer due to it's normal metabolic functions.

I hope that this helps, and I'll try to find the official sources if you'd like. If anyone has different knowledge though, I'd love to hear about it! This question still makes my head ache. In the end, I decided that yeast does what it needs to do and we provide it with the things that it needs, and that's enough for me - the specific process and names and effects are just more than my brain can handle.
 
I have basically resolved these questions. Hornsey's quote above is misleading, or just plain wrong, depending on how you view it. Respiration does NOT occur in a brewery fermentation unless oxygen is added during the period after the vast majority of sugars have been consumed (i.e. at the end of fermentation). Here are two, of many, quotes that confirm this:

Brewing yeasts do not develop respiratory competence under the conditions encountered in fermentation. Thus, in the aerobic phase of fermentation, respiratory pathways are repressed because of the presence of sugars. In late fermentation when the sugars have disappeared and their repressing effects are relieved, anaerobiosis prevents the induction of the respiratory enzymes.
(Brewing: Science and Practice, section 12.6)
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All brewing yeast strains have limited respiratory capacity and are subject to carbon catabolite repression. In a brewery fermentation, irrespective of the presence of oxygen, metabolism is always fermentative and derepressed physiology never develops (see Section 4.3.1 for further discussion). Thus, the major products of sugar catabolism are inevitably ethanol and carbon dioxide. Respiration, in the true sense of complete oxidation of sugars to carbon dioxide and water, coupled to ATP generation via oxidative phosphorylation does not occur.
(Brewing Yeast and Fermentation, p. 70)
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As to what happens to excess oxygen, that is a question that is still unresolved. Sources differ on exactly how long it takes for the oxygen to disappear from solution. Obviously this will differ, depending about the amount of oxygenation. Some sources say "within 24 hours," other say "within 30 minutes". And they are also uncertain as to what actually HAPPENS to the oxygen:

The fate of most of the oxygen utilized during the aerobic phase of fermentation is unknown. Theoretically 10% is utilized for sterol formation and 15% for the biosynthesis of unsaturated fatty acids (Kirsop, 1982). More than 50% is unaccounted for.
(Brewing: Science and Practice, section 12.6)
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This is one of many reasons why I believe it is preferable to pre-oxygenate your yeast, rather than oxygenating your entire batch of wort.
 
Looks like I missed this one the first time around ;)

My understanding is that one should only add enough oxygen as is necessary to sustain the yeast growth and health needed to ferment the batch of beer. Any additional oxygen may lead to excessive yeast growth which "wastes" carbohydrates that could have otherwise converted to alcohol. With oxygenation of the wort there is also concern that you can create oxidation products (just like HSA but slower since the temperature is lower). It is my understanding that brewers try to avoid the latter by supplying sufficienct O2 to the yeast before the yeast is pichtched. That way the yeast already has all the sterol reserves it will need to grow during fermentation.

This being said, it is diffcult for a home brewer to determine the exact O2 requirements of the various yeast strains that are used. And it is also difficult to control the O2 level w/o a DO meter. That's why for us it might be best to err on the side of caution and live with the potential "waste" of sugar due to unnecessary yeast growth.

Brewing Yeast and Fermentation is a nice book and I have learned quite a bit from it. But it is a very dry read.

Kai
 
Kaiser, hey, thanks for replying, I've been wanting to pick your brain on this topic, as you appear to be one of the few people on this forum who really understands this stuff.

Yeah, I've also read about how over-oxygenation leads to over production of biomass at the expense of ethanol. However, I am still trying to pin down exactly WHY this occurs. Boulton and Quain did not clarify this for me, and the best answer I have found so far is from "Essays in Brewing Science":

The primary flavor impact of yeast lies in the formation of a broad spectrum of flavor compounds present in small amounts that arise as products from metabolic pathways that lead to yeast growth (anabolism). Many of these materials are literally the end-products of metabolism that are of no further use to yeast; some simply leak from the metabolic pathway because they occur in excess, and some are products of metabolism that are degraded to make them less toxic to the cell and/or more easily excreted. In general, therefore, there is a positive correlation between yeast growth (requiring more metabolism) and formation of end-metabolites (flavor compounds). However, the correlation that is more to the point is between production of flavor compounds and metabolic flux (similar to, but not the same as, yeast growth). Thus, in a brewery fermentation, the rate of fermentation can be hugely affected by temperature, pitching rate and wort-dissolved oxygen without exactly parallel changes in yeast growth (rate or amount); there is therefore a disconnect between metabolic flux (catabolism, leading to flavor compounds) and synthesis of cell mass (anabolism). (Lewis, Essays in Brewing Science, p. 118)​

I interpret this to mean that the excess yeast growth (at the expense of ethanol production) is due to the fact that excess oxygenation increases the RATE of anabolism with respect to catabolism. In other words, suppose that you have two fermentations with exactly the same pitch rate, gravity, and temperature. You then oxygenate one batch to the precise extent necessary for yeast to fulfill their sterol reserves. You oxygenate the other batch beyond the extent necessary to fulfill this requirement. My interpretation of the research is that the excess oxygen will cause an inefficient fermenation because this excess oxygen somehow changes the rate of anabolism, perhaps because the yeast sense that they will be able to replenish their sterols after several cycles of budding. So, just as temperature increases the proportion of metabolic by-products per yeast cycle, excess oxygen increase the proportion of anabolic growth per yeast cycle. Is that your understanding of the phenomenon, or is this completely wrong?
 
stoutaholic said:
My understanding of this question is that when fermentation begins and even before when CO2 is produced, that some O2 will be driven out of the fermenter by the rising action of the CO2, O2 being lighter than CO2. Perhaps all that is in excess of what the wort will use.

The crabtree effect to my knowledge has to do with less O2 than desired in the wort where the yeast stops respiration and starts fermentation prematurely either by not pitching enough yeast for the gravity of the wort, or not enough O2 to satisify the yeasts needs.
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stoutaholic said:
Is that your understanding of the phenomenon, or is this completely wrong?
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I haven't done much work with respect of controlling yeast and most of what I know is based on the literature. What I get is that yeast's primary desire is to multiply. That's why they ferment in the first place and if there is enough O2 to make more yeast they'll do that even if it is on the expense of the amount of alcohol produced. And even if that increased growth means more byproducts. Jamil for example mentioned increased higher alcohols as the result of too much oxygen.

I know that unless I own a DO meter, I won't be able to accurately control the O2 content of the wort and right now I just do what has worked for me before. But once I have that DO meter I'll definitely will look into the affects of O2 content on fermentation performance and flavor profile.

Kai
 
Kaiser you comments helps clarify the question, which I didn't state completely. Up to a point, the more oxygen we provide to yeast, the more growth. But as you said, the meaning of life for yeast is not to produce ethanol, but to divide and conquer ... i.e. to grow and bud. But until recently I did not believe that cell growth could be separated from the by-products of that growth. In other words, I had always read that more growth meant more by-products. In other words, every time a yeast cell progresses through the cell cycle, it engages in both anabolic and catabolic metabolism. So it uses wort sugars to produce cell mass, daughter cells, and metabolic by-products, including ethanol. So my assumption was that the more growth you have, the more ethanol would be produced. This assumption only holds, however, if the rate of ethanol production increases or decreases in concert with the rate of biomass production. If both rates are in sync, then the more growth, the more ethanol, and you don't get an inefficient fermentation by creating factors that enhance yeast growth -- on the contrary, you get a more efficient fermentation, because the yeast are able to more fully ferment the wort.

So, what is confusing here is why "excess" oxygen, which I take to mean any oxygen beyond what the yeast require to fulfill their initial sterol and unsaturated fatty acid reserves, would change the balance of yeast metabolism between anabolic and catabolic processes; why it would make the yeast utilize each sugar in such a way as to produce more growth and fewer by-products?

I'm planning to do some experiments regarding oxygenation with my DO meter in the near future, so I'll post the results once I have them. "Brewing Yeast and Fermentation" describes a method to determine when the yeast have maximized sterols and minimized glycogen -- the point at which their oxygen uptake rate drops below its maximum. So I plan to measure the rate of oxygen uptake and then test the resulting pre-oxygenated yeast against non-oxygenated yeast pitched into worts oxygenated in the normal fashion to varying degrees of ppm.
 
Nice topic, I read about this in "Principles of Brewing Science" (George Fix, PhD) the other day. Referring back to that passage and a quick trip to Wikipedia, and this is how I understand it:

Yeast are facultative anaerobes, meaning they can produce energy using two different metabolic pathways, respiration (via the Krebs cycle - O2 + glucose -> CO2 + ATP) or fermentation (glucose -> CO2 + ethanol + ATP). The pyruvate formed by glycolysis is the common input into each of these pathways.

In a well-aerated wort, yeast will tend to metabolize glucose more slowly b/c of the higher energy efficiency of the Krebs cycle (more ATP produced than fermentation: 2 moles ATP per glucose for fermentation versus 38 moles ATP per glucose for respiration). Essentially, the yeasties don't have to gobble up the glucose like its going out of style, because they're getting a big net energy gain for relatively small intake of glucose. Also, the ATP produced by respiration acts as an allosteric inhibitor to the glycolysis mechanism.

The uptake of dissolved oxygen occurs very rapidly after pitching, usually within a few hours (according to Fix), at which point the less efficient fermentation pathway takes over. In this anaerobic environment, the yeast start to gobble up the glucose a lot quicker b/c of the lower energy efficiency of the fermentation pathway. Also, yeast growth and multiplication subsides b/c the Krebs cycle pathway is no longer favorable. Thus, the shift from biomass production from the efficient Krebs cycle pathway to ethanol production from the less efficient fermentation pathway.

Then, however, if oxygen is introduced during fermentation, yeast cells will tend to revert to respiration, a process called the Pasteur effect (the reverse of the Crabtree effect). Again, I think this is because of the higher energy production efficiency for the Krebs cycle pathway (respiratory) versus the fermentation pathway.

Anywho, that's how I understand it. Does that explanation make sense?
 
With regards to the passages from Brewing; Science and Practice that suggest brewers' yeast do not undergo respiration, I've never heard that. I've always learned that brewers' and bread yeast undergo respiration first to build up energy reserves
and synthesize the building blocks necessary for reproduction and fermentation. This is why under-aeration is such a problem and can lead to poor or stuck fermentations. Yes, the sterol production is important, because it gives the cell wall the proper permeability, but I've always thought the yeast are undergoing respiration in this initial stage as well. Maybe I'm wrong, but there certainly seem to be some contradictory statements out there.

With regards to over-aeration of wort, the majority of negative effects that I've heard about tend to discuss oxidative stress of the yeast because of free radical formation that damages cell walls. And this is usually suggested to only really be a problem in propogation systems where oxygen is continually delivered into the propogation tanks. Not where wort is initially aerated and then left alone, as is typical in a standard fermentation.
 
Hi moti mo, thanks for the well-researched response. I haven't acquired "Principles of Brewing Science" yet (it's on my list), so it is good to know what Fix has written on the topic. I think he may be one of a number of authors (Papazian, Noonan, Janson, Hornsey, to name a few) who didn't quite understand the process correctly, however.

Yeast are facultative anaerobes, meaning they can produce energy using two different metabolic pathways, respiration (via the Krebs cycle - O2 + glucose -> CO2 + ATP) or fermentation (glucose -> CO2 + ethanol + ATP). The pyruvate formed by glycolysis is the common input into each of these pathways.
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Yep, that's correct.

In a well-aerated wort, yeast will tend to metabolize glucose more slowly b/c of the higher energy efficiency of the Krebs cycle (more ATP produced than fermentation: 2 moles ATP per glucose for fermentation versus 38 moles ATP per glucose for respiration).
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This would be true if the wort did not possess sufficient glucose levels. But at the beginning of fermentation, glucose levels would be sufficient to prevent yeast from engaging in respiration. The events relating to the Krebs’ cycle take place within the mitochondria (Brewing, Hornsey, p. 117), and in a solution with glucose concentrations above 0.2 - 0.4%, mitochondrial development is arrested (Brewing: Science and Practice, section 12.5.5). This is the "carbon catabolite repression" I mentioned previously. So no respiration occurs, even when there are profuse amounts of molecular oxygen available, as long as the solution contains sufficient glucose levels.

The uptake of dissolved oxygen occurs very rapidly after pitching, usually within a few hours (according to Fix), at which point the less efficient fermentation pathway takes over.
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It is correct that dissolved oxygen is taken up very rapidly after pitching, but this not due to utilization of oxygen via the Krebs cycle, but for sterol synthesis and other unknown processes (about 50% of the oxygen usage is unknown and unaccounted for (Brewing: Science and Practice, section 12.6)).

Then, however, if oxygen is introduced during fermentation, yeast cells will tend to revert to respiration, a process called the Pasteur effect (the reverse of the Crabtree effect). Again, I think this is because of the higher energy production efficiency for the Krebs cycle pathway (respiratory) versus the fermentation pathway.
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Yep, if oxygen is re-introduced late enough in the fermentation, when repressing sugars are no longer available, metabolism shifts and mitochondrial development becomes "derepressed", allowing the yeast to respire (Brewing: Science and Practice, section 12.5.5). However, the Pasteur effect, which is a reduction in the rate of glycolysis under aerobic conditions, cannot be demonstrated in S. cerevisiae, though it does occur very dramatically for other yeast strains such as C. tropicalis (Brewing: Science and Practice, section 12.5.7 and Brewing Yeast and Fermentation, p. 84).

One might point out, however, that if all the authors that I mentioned appear to believe that respiration does occur in a fermentation, then why should we believe the particular sources that I have cited? There seems to be a divide between scientific professionals, writing for the professional brewing and scientific community, and brewers/hobbyists writing for the homebrewing community. The former tend to reference studies published in peer-reviewed journals, while the former either lack references or predominantly refer to previously written brewing texts. And because the misunderstanding about respiration in a brewery fermentation is so widespread, it happens that many authors keep repeating this misunderstanding. For instance, books by Michael Lewis and Charles Bamforth (professors at UC - Davis) are likely to agree with published research, while authors such as Papazian and Noonan are less reliable. The best and most reliable source on brewing yeast is "Brewing Yeast and Fermentation" by Boulton and Quain. Practically ever statement in the book is backed published research or the authors' own unpublished experimental data.
 
moti_mo said:
Maybe I'm wrong, but there certainly seem to be some contradictory statements out there.
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Yes, there are contradictory statements out there. But from what I have come across, the more scientific literature agrees that brewers yeast doesn’t respiate even in the presence of O2. The abundance of sugar is the reason. But they do utilize the O2 that is available to them. It is used for making sterols and other compounds that require molecular oxygen.

As you dig deeper into yeast and other brewing topics you’ll come across more contradictions to common home brewing knowledge. In the end knowing one or the other explanation doesn’t change much with respect to your brewing practices. Both theories will explain why you should add O2 to your wort. They only differ in how well they stand up to explaining other observations which are not necessarily seen in home brewing.

Kai

 
If there's a misunderstanding within my response, I would chalk it up to me and not Fix. I would put him in the camp of authors where the majority of their statements are backed up by published research.

Fix mentions that the Crabtree Effect, the inhibition of respiration in favor of fermentation, although it occurs to some degree in normal beer worth, is exacerbated in high dextrose wort.

Also, the Pasteur effect has been seen in S. cerevisiae, but the mechanisms are complicated. When growing with an excess of sugar and nitrogen source, it does not show a noticeable Pasteur effect. However, nitrogen depletion allows the mechanism to be quite efficient - resting S. cerevisisae with depleted nitrogen reserves were shown to respire as much as 25% to 100% of the catabolized sugar. (J. Bacteriol. 1982 October; 152(1): 19-25. So

Anyway, I'm enjoying the research and learning more about this process.
 
Both theories will explain why you should add O2 to your wort. They only differ in how well they stand up to explaining other observations which are not necessarily seen in home brewing.
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Yeah, unless you have the ability to precisely measure DO, which typically is not something homebrewers are capable of, most of this research is not relevant to you, because your best bet is to always aerate to 8ppm. This is a known constant that you can achieve, so then pitching rate becomes the only variable that you have to worry about controlling.

If you have the ability to measure DO, then the research becomes significant, because you then have the ability to precisely control two variables: pitching rate and oxygenation. In my particular case, I am trying to limit the wort's exposure to any oxygen other than what is required to oxygenate the yeast. Also, I don't want my pitching rates to be based upon estimates from a pitching rate calculator that assume certain amounts of growth in a starter. I want the number of yeast in my Activator pack to correspond to the number of yeast pitched, and I want to be assured that they have a consistent physiology for each batch. So my goal is get back to the original goal of varying only pitching rate, but to be assured that the yeast which are pitched will perform as expected. If respiration occurs, as many believe, then my method of achieving this goal would change, because I would have to account for yeast growth in my starter (which is used not for propogation, but for pre-oxygenation). Also, I am trying to understand the concept of "over-oxygenation" so that I can avoid the purported "inefficient" fermentations. Unfortunately, exactly what "over-oxygenation" means and how it produces an "inefficient fermentation" is still a mystery to me.
 
OK, back to respiration in a high glucose environment. I've been stuck on this, and looked up a couple more references. (Yes, I'm procrastinating heavily today, but I'm a bit burnt out at work right now).

This is from Advances In Microbial Physiology, Volume 28, by David Tempest. In the chapter "Regulation of Carbon Metabolism in Saccharomyces cerevisiae and Related Yeasts". On pages 188 - 192, he discusses the originally proposed Crabtree mechanism and new insights that have added to the interpretation of this mechanism:

"Although Holzer (1967) pointed out that the glucose effect may consist of a variety of different repression effects, repression of respiratory enzymes by glucose has come to characterize the Crabtree effect in yeasts....

B. New Aspects of Regulation Gained by Means of Improved Cultivation Techniques

If the regulation mechanism outlined above (Crabtree mechanism) is correct, it should be comprehensive and explain all types of metabolism observable when yeast cells are cultivated with glucose as the carbon source. The first metabolic state contradictory to this claim was observed at low dilution rates in a continuous culture. At low substrate-feed rates, glucose is degraded oxidatively... It follows that the presence of glucose per se is not the primary cause of aerobic ethanol formation and enzyme repression supposed to be the underlying regulatory mechanism. Beck and von Meyenburg concluded that not the presence of glucose but the rate of glucose consumption has a regulatory effect on enzyme [i.e. respiratory enzymes] activities.

Furthermore, a detailed quantitative analysis of the overall metabolism of growing yeast cultures indicated that respiration is not repressed completely by glucose. A significant oxygen uptake by cells is measured both in the first phase of batch culture as well as in the corresponding metabolic state at high dilution rates in continuous culture.

It follows that there is a branched glucose breakdown when cells exhibit aerobic ethanol formation. Depending on the terminal electron acceptors, part of the glucose is catabolized respiratively (electron acceptor oxygen) and part fermentatively (electron acceptor acetaldehyde). Consequently, the type of metabolism corresponding to aerobic ethanol formation is respiro-fermentative. "

This would seem to imply that both mechanisms occur in S. Cerevisiae in aerobic conditions, even in presence of high concentrations of glucose, even though the respiratory pathway is surpressed. Anyway, more data...
 
Thanks guys for the research on this process. Since my lastname is "Crabtree", I've always been interested, but haven't expended the effort to understand it to the level of detail expounded upon here.

Cheers!

Patrick
 
OK, so one more long post. I hope I haven't hijacked this thread, but I get really intrigued by contradictions in the literature, in whatever field I'm researching. Procrastinating more, and doing a search in Google on "Crabtree effect in breweries", I came across a synopsis by Dr. George Fix himself on the division between aerobic respiration and anaerobic fermentation.

Beer and Loafing in Las Vegas - From the HBD Archive

This seems to be a response to a debate (similar to the one we're having now) on a Home Brew Digest forum from 1992:

"From: [email protected] (George J Fix)
Subject: Yeast Cycles (George Fix)
Date: 1992-10-09 20:45:43 GMT
There has been a lot of interesting discussion concerning yeast
cycles on HBD, and I can not resist inserting my two cents worth.

I have really enjoyed the insights Pierre Jelenc has shared with
us. He is absolutely correct in asserting that the classic division
between aerobic respiration and anaerobic fermentation greatly
oversimplifies what is actually going on in individual yeast cells.
Nevertheless, the aggregate behavior, where the total collection of
cells is viewed as an enzyme system, does display regular behavior.
In fact, the differential equations of enzyme kinetics are derived
from such models much in the same way thermodynamics is derived from
particle models via ensemble averaging. It is important to stress
that the equations of enzyme kinetics are valid only as a description
of the system as a whole. They do not predict the behavior of individual
yeast cells, since the eccentricies of the latter have been averaged
out.

The kinetic models give the following picture of the fermentation. In
the early stages there is a net consumption of dissolved O2 as well as
a reduction of wort lipids such as oleic and linoleic acids. There is a
net increase of metabolic energy, which can be characterized as an
equivalent amount of ATP (adenosine triphosphate). This is accompanied
by a net increase in the cell density N(t) with time t. The biochemistry
is involved, but the associated mathematical description is simple, since
the kinetic equations are linear in this regime. Solution of these equations
shows an exponential growth in N(t) with time t, which is usually written
in terms of logarithms as follows:

log(N(t)) = C*t,

where C is the growth constant. I feel it is valid to call this regime
the aerobic respiratory growth phase, even through individual cells may deviate
from the aggregate behavior.

As the cell density increases and the dissolved O2 level decreases, the
nonlinear regime is approached. Things get really interesting here from a
mathematical point of view. Ironically, the biochemistry is straightforward.
What happens in the aggregate is carbon splitting of elementary sugars such
as glucose (G) and fructose (F) followed by formation of pyruvic acid and
then acetaldehyde. The final step is the reduction of acetaldehyde to
ethanol by yeast enzymes. Since O2 is not involved and since alcohol is formed,
I feel it is valid to call this phase anaerobic fermentation. It is important
to note that there is a net expendure of metabolic energy during this phase.
This is why the ATP buildup in the aerobic phase is so crucial to obtaining
a complete fermentation. Also the curve for N(t) vs. time flattens out.

There are a number of mechanisms that have been identified for inducing the
transition to the nonlinear regime (i.e., from respiration to fermentation).
One of the most important as far as practical brewing is concerned is the
Crabtree effect. It has been shown that a sufficiently high cell concentration
of G and F sugars will strongly induce anaerobic fermentation. Brewing yeast
take G's and F's directly into the cell. Sucrose (G-F) is broken up outside the
cell, and then the G and F fractions are then transported inside. In contrast,
maltose (G-G) and maltotriose (G-G-G) are taken intact into the cell, only
later to be broken down into G units. In an all grain wort, maltose is the
major fermentable sugar followed by maltotriose, the others being under 10% of
the total. This has important practical implications for respiration, for
the maltose concentrations will not induce the Crabtree effect (at least in
the levels that exit in normal beer wort) until the maltose is broken into G
units. At this point a proper respiratory cycle will have occured, assuming
of course that a sufficient amount of O2 is dissolved at the start of the
fermentation.

Because of this, I am in complete agreement with the general principles put
forward in Micah Millspaw's post on yeast propagation. I am less inspired by
the use of dextrose (which is the same as glucose) and sucrose as a substrate
for propagating yeast. The reason centers on the Crabtree effect. I am not
suggesting his methods will not work. Micah has a wall full of ribbons to prove
the contrary. I am suggesting, however, there may be a better ways to go.

Over 85% of commercial propagation and those done in research labs involved
with brewing strains use dilute wort (SG ~ 1.020). Paul Farnsworth has an
excellent discription of this procedure in his article that appeared in the
yeast issue of Zymurgy. I belong to the minority that propagates with full
strength hopped wort. The reasons for this and a description of the procedure
can be found in my article that appeared in Vol. 6 of BREWERY OPERATIONS
published by Brewers Publications. I also use an O2 feed during propagation
to induce the Pasteur effect, which is the exact opposite of the Crabtree
effect. Here fermentation is repressed in favor of respiratory cell growth.

I was working only with half a voice during the AHA conference in June, and
likely many points I was trying to make did not get across. I hope this is not
the case with the point about the practical value of testing yeast that have
been aerobically propagated. Minor technical errors can lead to major problems,
not only with aerobic bacteria but with mutation as well. Both should be
checked. Interestingly, the Wyeast strain 1056, which is the same as Siebel's
BRY-96, does particularly well with aerobic propagation. In fact, I have found
the much discussed tendency of this strain to mutate (something that has
happened with samples from both Wyeast and Siebel) is closely related to the
lack of a proper respiratory cycle. Thus, brewing procedure is the culprit,
not screw ups in Chicago or Portland.

George Fix

George Fix"

So he seems to make several points that directly contradict the sources that claim S. Cer. do not respire in normal brewery fermentations, and instead suggests that:

1) yeast do respire in the initial stages
2) this early stage respiration is not repressed by a Crabtree effect b/c maltose is the primary sugar being consumed and not glucose
3) once the Crabtree effect does become relevant, a significant phase of respiration has occurred
4) the Pasteur effect can be utilized for S. Cer. to inhibit fermentation in favor of respiration (i.e. the Crabtree effect is effectively microscopically reversible)

So, again, I can't say with 100% certainty that Fix is right about everything within this description, but it seems like a sound argument to me. I haven't read any of the sources that claim S. Cer. do not undergo respiration (or can't be subjected to the Pasteur effect) in typical beer worts, so I can't speak about them, but...if Fix is right, then those sources are way off base, and if those sources are right, then Fix is certainly way off base (and maybe he is, he's a Ph.D in math, not biology - but you would think someone would have directly called him on it by now).

Hmmmm....
 
moti_mo said:
1) yeast do respire in the initial stages
2) this early stage respiration is not repressed by a Crabtree effect b/c maltose is the primary sugar being consumed and not glucose
3) once the Crabtree effect does become relevant, a significant phase of respiration has occurred
4) the Pasteur effect can be utilized for S. Cer. to inhibit fermentation in favor of respiration (i.e. the Crabtree effect is effectively microscopically reversible)
Click to expand...


This is how I see it:

1) I would have to read the whole thread, but the consumption of O2 is not necessarily an indication of respiration. The absence of ethanol production while O2 is consumed and energy is created is.
2) glucose and fructose are consumed first. Then maltose. His point was that by the time glucose is so low that it doesen’t cause the crabtree effect anymore the O2 is gone and the yeast doesn’t have much choice between respriration and fermentation anyway
3) see 2). The Crabtree effect is caused by an abundance of glucose and fructose

Kai
 
Thanks for all the research. This good information. Could you post a link to the "Regulation of Carbon Metabolism in Saccharomyces cerevisiae and Related Yeasts" article? I found a related article by the same researchers, and they seem to specialize in this topic. I think we need to be careful about interpreting precisely what they are saying, and so I am still working through the meaning of their statement that "the yeast Saccharomyces cerevisiae switches to a mixed respiro-fermentative metabolism, resulting in ethanol production, as soon as the external glucose concentration exceeds 0.8 mM."

As Kai points out, Fix does seem to be saying that the Crabtree Effect occurs. And I think the question of whether maltose does contribute to carbon catabolite repression is unclear, as the sources I have read are inconsistent in this regard. Since maltose, unlike sucrose, can be broken down into gluclose intra-cellularly, I could see how it could maintain a state of repression.

Anyway, I think it is very unclear what Fix is actually trying to say about "aerobic respiration" here. We really need to decompress his post if we want to try to draw any conclusions from it. For instance, he writes:

"There are a number of mechanisms that have been identified for inducing the transition to the nonlinear regime (i.e., from respiration to fermentation). One of the most important as far as practical brewing is concerned is the Crabtree effect. It has been shown that a sufficiently high cell concentration of G and F sugars will strongly induce anaerobic fermentation."​

He then goes on to say that he doesn't believe that maltose sugars, specifically, will induce the Crabtree effect.

So we know that by the "non-linear regime" he is talking about fermentation, and he also seems to be saying that there is a "transition" from "respiration" to "fermentation". And he credits the Crabtree Effects with causing the transition to fermentation.

So this leads to the question, if Fix believes that the Crabtree Effect applies, and that it causes a transition from respiration to fermentation, then when and how does respiration occur?

Well, he says previously "In the early stages there is a net consumption of dissolved O2 as well as a reduction of wort lipids such as oleic and linoleic acids."

This is the lag phase, and this statement is consistent with the idea that yeast utilize oxygen for the synthesis of sterols, etc.

He then states:

"There is a net increase of metabolic energy, which can be characterized as an equivalent amount of ATP (adenosine triphosphate). This is accompanied by a net increase in the cell density N(t) with time t."​

Now, if he is saying that the lag phase involves an increase in cell density, then this is simply not correct. Many studies demonstrate that there is practically no change in cell count or ethanol levels during the lag phase, and that the overall cell mass declines, due to the breakdown of glycogen reserves to fuel cell membrane synthesis. Similarly, it is unclear what he means by a "net increase of metabolic energy," unless he is referring to the expediture of that energy via glycogen.

He continues:

"The biochemistry is involved, but the associated mathematical description is simple, since the kinetic equations are linear in this regime. Solution of these equations shows an exponential growth in N(t) with time t, which is usually written in terms of logarithms as follows:

log(N(t)) = C*t,

where C is the growth constant. I feel it is valid to call this regime
the aerobic respiratory growth phase, even through individual cells may deviate from the aggregate behavior. As the cell density increases and the dissolved O2 level decreases, the nonlinear regime is approached."​

Now, this is a very ambiguous statement, from my perspective. He calls "this regime" "linear", but he gives us a logarithmic growth equation. Logarithmic growth is the opposite of linear -- it is exponential. The exponential growth phase is not the lag phase, it is the "log" phase. But he seems to be saying that the "linear" regime involves logarithmic growth, and that this growth declines as O2 levels decline. And as O2 levels decline, the yeast begin to transition to the "non-linear" growth phase, which he previously referred to as "fermentation".

In any case, whatever Fix is trying to explain, there are many studies that show that by the time the yeast reach the logarithmic (exponential) growth phase, that there is little if any O2 left in solution, and that growth is anaerobic. Also, there is substantial research to show that cell counts and ethanol levels change very little during the lag phase of fermentation. There is also research that shows that ethanol production occurs at the fastest rate during the logarithmic phase. So all these different observations point to the idea that the logarithmic growth phase is anerobic and is the phase during which ethanol is produced, not a period of aerobic growth.

But, as I said, Fix's language is hardly crystal clear, and so it is hard to tell if he is really saying that the "aerobic respiratory" phase is truly the logarithmic growth phase. If he is saying that, then there is substantial research to contradict that idea.

However, based upon the article you cited before, it sounds like there may be some degree of aerobic metabolism that occurs during fermentation. If I can find additional articles by these researchers, we may be able to clarify that question.
 
Guys, I found the link to the artice that moti mo found. Here it is:

Regulation of carbon metabolism in Saccharomyces cerevisiae and related yeasts

I'l post once I've "metabolised" it.

----------
EDIT: Actually, this article at this link is incomplete. Because it is a Google preview, several pages of the article are redacted. So, again, a link to this article would be useful, as it seems to be the basis of the claim that, in a brewery fermentation, metabolism can be both respirative and fermentative.
 
Kaiser said:
This is how I see it:

1) I would have to read the whole thread, but the consumption of O2 is not necessarily an indication of respiration. The absence of ethanol production while O2 is consumed and energy is created is.
2) glucose and fructose are consumed first. Then maltose. His point was that by the time glucose is so low that it doesen’t cause the crabtree effect anymore the O2 is gone and the yeast doesn’t have much choice between respriration and fermentation anyway
3) see 2). The Crabtree effect is caused by an abundance of glucose and fructose

Kai
Click to expand...

OK, this is how I'm beginning to see it:

1. ethanol production in the presence of oxygen does not indicate the absence of respiration, it simply indicates that fermentation is happening. From the Tempest reference I quoted, even in high glucose media, detailed analysis indicates that S. Cer. is both fermenting and respiring (not just utilizing O2 for sterol production, but actually respiring), therefore they use the term respiro-fermentative to emphasize that during aerobic ethanol formation, S. Cer. undergoes BRANCHED glucose breakdown (both respiration and fermentation)

2. maltose consumption is not completely repressed by glucose consumption, especially for brewers' yeasts. Growth and propogation of yeast in high concentrations of maltose (as brewers' yeasts are grown) cause adaptations that increase the yeasts' ability to utilize maltose, and may also cause the repression of glucose uptake in the early stages of fermentation. http://www.scientificsocieties.org/JIB/papers/1993/1993_99_1_067.pdf

3. again, from the Tempest reference emphasizes that the PRESENCE of glucose is not the primary reason for inhibition of respiratory enzymes, but the RATE of glucose consumption is more important. Thus, if a strain of brewers yeast has any adaption for increased utilization of maltose (at the expense of glucose utilization), then the extent of respiration repression should be lower.

I don't think that ONLY respiration is occurring in the early stages, but I also don't think that ONLY fermentation is occurring. I lean towards the supposition that both are occurring, even if fermentation is the primary mechanism.
 
Thanks Stoutaholic. I agree that Fix's language is fairly ambiguous, and ironically he is trying to clear up "over-simplification" with this monologue. I based my current (could change upon reading more) thinking in that last post on the articles by Tempest and the one I found by the research group out of Labatt. I'm also trying to metabolize it all, its a fun topic.
 
stoutaholic said:
----------
EDIT: Actually, this article at this link is incomplete. Because it is a Google preview, several pages of the article are redacted. So, again, a link to this article would be useful, as it seems to be the basis of the claim that, in a brewery fermentation, metabolism can be both respirative and fermentative.
Click to expand...

Yeah, definitely, I don't have access to those journals where I work unfortunately.
 
Don't know if this is even worth posting but I read the following on the Danstar site in this article:
Yeast need a trace amount of oxygen in an anaerobic fermentation such as brewing to produce lipids in the cell wall. With out O2 the cell cannot metabolize the squalene to the next step which is a lipid. The lipids make the cell wall elastic and fluid. This allows the mother cell to produce babies, buds, in the early part of the fermentation and keeps the cell wall fluid as the alcohol level increases. With out lipids the cell wall becomes leathery and prevents bud from being formed at the beginning of the fermentation and slows down the sugar from transporting into the cell and prevents the alcohol from transporting out of the cell near the end of the fermentation. The alcohol level builds up inside the cell and becomes toxic then deadly.

Lallemand packs the maximum amount of lipids into the cell wall that is possible during the aerobic production of the yeast at the factory. When you inoculate this yeast into a starter or into the mash, the yeast can double about three time before it runs out of lipids and the growth will stop. There is about 5% lipids in the dry yeast.

In a very general view:

At each doubling it will split the lipids with out making more lipids (no O2). The first split leaves 2.5% for each daughter cell. The second split leaves 1.25% for each daughter cell. The next split leaves 0.63%. This is the low level that stops yeast multiplication. Unless you add O2 the reproduction will stop.

When you produce 3-5% alcohol beer this is no problem. It is when you produce higher alcohol beer or inoculate at a lower rate, that you need to add O2 to produce more yeast and for alcohol tolerance near the end of fermentation. You definitely need added O2 when you reuse the yeast for the next inoculum.

If you prepare a starter culture you will need added O2 in the starter and perhaps in the main mash as a precaution. You will need to follow the precautions as mentioned above. If the mash is designed to produce 3-5% alcohol you may not need added O2. Brewing above that needs added O2.

Regarding your comment about growing your own yeast that will not need added O2 in the fermenter; The Lallemand yeast factory grows yeast under a different metabolic pathway than you will have in your starter culture. We feed the media to the aerobic fermentation at a rate that will keep the sugar levels below 0.2% at all times to maintain the Pasteur Effect. This builds cell mass with minimum to no alcohol production. As the sugar level rises above 0.2% the Crabtree Effect begins and no matter how much air you feed the fermentation, alcohol + CO2 are the main by-products. Your starter culture will have a much higher level of sugar. You will produce some cell mass but mostly alcohol and CO2 no matter how much air you add by stirrer or bubbles.

Dr. Clayton Cone
Click to expand...
Maybe there's something in there even though it's in layman's terms. If nothing else it's yet another source...and evidence that Dr. Clayton Cone might answer a question.;)
 
I have been researching this some more and I believe that I can clear up the inconsistencies in the research that we are finding.

First, as moti mo surmised, it IS possible for yeast to engage in respiro-fermentive growth. In other words, they can respire and produce ethanol at the same time. The amount of glucose channeled to each metabolic path is dependent upon glucose levels and oxygen levels.

When oxygen is not available, metabolism is completely fermentative. When both oxygen and glucose, fructose, or maltose are available, then many factors come into play in order to determine whether metabolism is purely respiratory, respiro-fermentative or purely fermentative.

If glucose levels are very low, below dillution rates of about 0.38 h-1 (this is strain dependent), then metabolism is purely respiratory. Glucose levels above this rate cause the yeast to channel sugars progressively more towards the fermentative route. That is, as glucose levels increase above this point, metabolism becomes increasingly fermentative.

The following articles describe this phenomenon:

Characterization of the metabolic shift between oxidative and fermentative growth in Saccharomyces cerevisiae by comparative
13C flux analysis

Enzymic Analysis of the Crabtree Effect in Glucose-Limited
Chemostat Cultures of Saccharomyces cerevisiae

However, this effect can only be demonstrated for yeast grown in an aerobic non-fermentable (or very low glucose) medium. Under these circumstances, the yeast develop functional mitochondria and are thus able to subsequently respire.

For yeast grown on high levels of glucose, or yeast in their stationary phase that are exposed to high levels of glucose (as happens at the start of a fermentation), carbon catabolite repression, or even inactivation, occurs and mitochondrial development is supressed. So the ability to respire never develops. As long as these yeast are supplied with sufficient hexose sugars, respiratory competence will remain repressed. Only when the supply of sugars is removed and the yeast are exposed to oxygen, will repiratory capability become de-repressed.
(Signal Transduction in Yeast and Malting and Brewing Science, Volume 2, p. 594)

Therefore, this explain's Boulton and Quain's statement in "Brewing Yeast and Fermentation" that:

All brewing yeast strains have limited respiratory capacity and are subject to
carbon catabolite repression. In a brewery fermentation, irrespective of the presence of oxygen, metabolism is always fermentative and derepressed physiology never develops (see Section 4.3.1 for further discussion). Thus, the major products of sugar catabolism are inevitably ethanol and carbon dioxide. Respiration, in the true sense of complete oxidation of sugars to carbon dioxide and water, coupled to ATP generation via oxidative phosphorylation does not occur. (p. 70)​

So in summary, whether yeast are able to engage in respiro-fermentative metabolism depends not only on the environment that that they are currently in, but on the environment that they have adapted to. Yeast that do not have a glucose-repressed physiology can respire and ferment, provided that both glucose and oxygen are available, but yeast that have previously undergone glucose repression are not able to respire if glucose is still available, even in the presence of large amounts of oxygen.

In a brewery fermentation, oxygen is only transiently available, and the cells being pitched are in their stationary phase. Therefore, these cells never develop respiratory competence, because by the time the glucose is consumed, the environment is anaerobic and they have no choice but to ferment.

This still does not explain, though, how a brewery fermentation can become inefficient by the provision of too much oxygen. I am still researching that.
 
Cool, I can dig that in a realistic brewery fermentation, the conditions are such that the majority of metabolism occurs by fermentation and respiration is minimal, or more likely never occurs at all. Glad we found lots of good data to clear up some of the murky waters (in my own understanding, if not others)

With regards to your question about over-aeration and inefficient fermentations, I go back to one of my former posts regarding oxidative stress: "With regards to over-aeration of wort, the majority of negative effects that I've heard about tend to discuss oxidative stress of the yeast because of free radical formation that damages cell walls."

I've looked into it a bit more and found the following article which seems tailored to your question:

Yeast Genome-Wide Expression Analysis Identifies a Strong Ergosterol and Oxidative Stress Response during the Initial Stages of an Industrial Lager Fermentation -- Higgins et al. 69 (8): 4777 -- Applied and Environmental Microbiology

Applied and Environmental Microbiology, August 2003, p. 4777-4787, Vol. 69, No. 8

There is some good stuff in this article, especially since it is a study done under the conditions of an industrial lager fermentation. Some of the salient points which speak to your question are:

"Ergosterol is an essential lipid component of yeast membranes, and its biosynthesis involves over 20 reactions (16).

Ergosterol has been shown to have vital functions in Saccharomyces cerevisiae cells affecting membrane fluidity and permeability and providing the "sparking function" that is thought to be involved in the progression into the G1 phase of the cell cycle (5, 15, 33).

Altered sterol production renders yeast cells hypersensitive to oxidative stress.
The contribution of ergosterol to protecting cells from oxidative stress was determined by measuring the sensitivity of ergosterol biosynthesis mutants (erg3, erg4, erg5, and erg6) to constant exposure to oxidative stress.

The up-regulation of genes in the first hour of fermentation involved in the thioredoxin and GSH cell functions was a strong indication that the cells were experiencing an oxidative stress response. The similarity in kinetics of induction of the ergosterol and oxidative stress response genes pointed to a possible interaction between these two cell functions (Fig. 4). This interaction was confirmed with results showing that yeast mutants unable to produce ergosterol were hypersensitive to oxidative stress (Fig. 5). This is consistent with observations by Bammert and Fostel (4) that perturbation of ergosterol biosynthesis heightened an oxidative stress response in S. cerevisiae. Additionally Schmidt et al. (51) suggested that proper ergosterol biosynthesis may be involved in cellular protection against oxidative stress, since erg3 mutants are sensitive to paraquat and H2O2.

The presence of oxygen can also cause the production of reactive oxygen species (ROS) within yeast, causing damage to cellular components (56, 66). These results emphasize the importance for tight control of aeration in industrial fermentations. High levels of oxygen caused by overaeration can decrease the expression of ERG11 (ergosterol biosynthesis) and OLE1 (unsaturated fatty acid synthesis) to very low levels (68), further escalating the effects of oxidative stress. Using electron spin resonance methods, Uchido and Ono (60) found that the oxidative capacity of the final beer product was highest following fermentation regimes using lower oxygen levels during the initial stages of the fermentation. Apart from oxygenation regulation, high-gravity brewing techniques have the additional problem of decreased ergosterol biosynthesis due to high osmolarity. Perhaps this problem could be reduced by a modification of the procedure described by Devuyst et al. (18). Preincubation of cropped yeast in low-oxygenated wort with a low concentration of sugar before pitching would provide a yeast higher in sterol and unsaturated fatty acid levels and subsequently better able to endure the rigors of high-gravity wort brewing."

The last paragraph sums up quite nicely why over-aeration can be a problem. Not only do you have the expected effects of increased levels of reactive oxygen species, which are the main culprits of cell damage and aging in any cellular environment (yeast, red blood cells, etc.), but in yeast the excess oxygen also decreases the expression of genes critical for sterol and fatty acid production. Since sterol plays a critical role in protecting the cell against oxidative stress, this further exacerbates the cells' susceptibility to oxidative stress - a double whammy.

I'm sure some of the articles cited in this article can provide even more information. I think the last sentence of that excerpt from above is a really good one for all of us homebrewers:

"Preincubation of cropped yeast in low-oxygenated wort with a low concentration of sugar before pitching would provide a yeast higher in sterol and unsaturated fatty acid levels and subsequently better able to endure the rigors of high-gravity wort brewing."
 
Of course the main question after knowing WHY over-aeration can cause inefficient fermentations, is quantitatively, what levels of oxygenation correspond to this regime of over-aeration. And do your techniques of oxygenation ever actually come close to putting you in this regime.
 
What is confusing about over-oxygenation, though, is that it is supposed to lead to a disproportionate amount of cell mass. The fermentation is "inefficient" not becuase yeast growth in impeded (by over-exposure to oxygen), but because excess biomass is produced at the expense of ethanol. In other words, the wort sugars that could have been metabolized to by-products (ethanol) are instead used for catabolism (cell mass). Nothing I have read would explain why this is the case.

Boulton and Quain actually developed a patent for a pre-oxygenator in order to avoid the problem of over-oxygenation. They suggested that if yeast are exposed to oxygen accidentally, during storage prior to pitching, they could synthesize sterol reserves during this time, and so when pitched into a batch oxygenated to normal levels, they would be "over-oxygenated" because they would not require the same levels of oxygen they normally would (i.e. had they not be unintentionally exposed during storage).

There is a limit to the amount of sterols that yeast can synthesize, and you can determine this limit by monitoring the rate of oxygen uptake of yeast. Once the rate of uptake starts decreasing, sterol levels are maximized. This is essentially what Boulton and Quain's pre-oxygenation technique accomplishes. So this raises the question -- if you pre-oxygenate yeast and then pitch them into oxygenated wort, why would this "extra" oxygen produce excessive growth? Once sterol levels have reached their maximum, oxygen should not facilitate growth past that point, and therefore should not lead to excess cell mass.

There seem to be two possible explanations. Either the excess oxygen in solution somehow effects cell metabolism so that the balance between cell growth and ethanol generation is affected (which is my theory, based upon the quote from "Essay in Brewing Science" that I posted), or that as the cells divide, they replenish their sterol reserves from the excess oxygen in solution (thereby allowing more cycles of cell budding). But this second explanation wouldn't completely clarify the situation, because more growth would just lead to a more COMPLETE fermentation (i.e. a higher proportion of sugars fermented), and therefore it would not be an inefficient fermentation. Somehow cell growth has to be advantaged at the expense of ethanol production.
 
Synthesis of some membrane components requires oxygen. Desaturases - the enzymes responsible for introducing double bonds in unsaturated fatty acids - use a sort of electron transport chain with oxygen as the terminal electron acceptor.
 

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