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Old 08-26-2009, 05:55 AM   #11
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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?

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Old 08-26-2009, 04:11 PM   #12
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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.

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Old 08-26-2009, 04:30 PM   #13
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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.

Quote:
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.
Yep, that's correct.

Quote:
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).
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.

Quote:
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.
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)).

Quote:
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.
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.
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Old 08-26-2009, 04:37 PM   #14
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Quote:
Originally Posted by moti_mo View Post
Maybe I'm wrong, but there certainly seem to be some contradictory statements out there.


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.

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Old 08-26-2009, 04:58 PM   #15
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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.

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Old 08-26-2009, 06:02 PM   #16
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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.
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.
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Old 08-26-2009, 08:15 PM   #17
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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...

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Old 08-27-2009, 12:36 AM   #18
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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

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Old 08-27-2009, 05:46 AM   #19
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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: gjfix@utamat.uta.edu (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....

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Old 08-27-2009, 03:13 PM   #20
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Quote:
Originally Posted by moti_mo View Post
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)


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
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