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Old 08-27-2009, 03:56 PM   #21
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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.
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Old 08-27-2009, 04:06 PM   #22
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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.

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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.

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Old 08-27-2009, 04:08 PM   #23
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Quote:
Originally Posted by Kaiser View Post

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
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/J...3_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.
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Old 08-27-2009, 04:55 PM   #24
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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.

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Old 08-27-2009, 05:05 PM   #25
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Quote:
Originally Posted by stoutaholic View Post

----------
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.
Yeah, definitely, I don't have access to those journals where I work unfortunately.
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Old 08-28-2009, 03:24 PM   #26
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Don't know if this is even worth posting but I read the following on the Danstar site in this article:

Quote:
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
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.
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Old 08-28-2009, 04:58 PM   #27
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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.
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Old 08-31-2009, 04:30 AM   #28
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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."

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Old 09-01-2009, 03:11 PM   #29
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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.

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Old 09-01-2009, 04:06 PM   #30
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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.

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