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Old 07-15-2009, 05:40 PM   #1
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Default Crabtree Effect and Over-aeration

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.


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Old 07-15-2009, 07:23 PM   #2
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Quote:
Originally Posted by stoutaholic View Post
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.
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.


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Old 07-17-2009, 08:48 PM   #3
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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.

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Old 08-22-2009, 08:56 AM   #4
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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;

Quote:
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?
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.

Quote:
If they begin fermentation, what happens to this excess oxygen? Does it just stay in solution, contributing to staling reactions?
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.
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Old 08-24-2009, 08:28 PM   #5
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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:

Quote:
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)
Quote:
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)
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:

Quote:
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)
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.
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Old 08-24-2009, 08:40 PM   #6
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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.

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Old 08-24-2009, 09:34 PM   #7
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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?
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Old 08-24-2009, 10:14 PM   #8
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[quote=stoutaholic;

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? /quote]

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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Old 08-25-2009, 01:59 AM   #9
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Quote:
Originally Posted by stoutaholic View Post
Is that your understanding of the phenomenon, or is this completely wrong?
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.

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Old 08-25-2009, 02:19 AM   #10
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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.



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