A question about the role of aeration.

[bernardsmith]

Not a chemist and don't pretend to be but I have a question about aeration. The cell process known as ATP suggests that while yeast are in the presence of O2 they will not in fact produce ethanol (alcohol) but they need to be in anaerobic conditions to produce alcohol much like our muscles produce lactic acid when we starve them of oxygen by working the muscle "too hard" in relation to our ability to provide that muscle with oxygen. Is this the case?

If this is so (and I may be completely off base here) then what is the value of frequent aeration of the must during active fermentation?. Is this only to ensure that the yeast cells remain healthy and are able to reproduce (but when budding will they be fermenting?) . When brewers create a starter they aerate constantly (using magnetic stirrers , for example) but they are not producing alcohol. They are ensuring a healthy population of cells...(though many mistakenly believe that they are increasing the number of yeast cells) What is it that I am missing? What is the precise function of daily aeration during active fermentation?

 

[Squatchy]

If you buy Chris WHites book it has tons of info on aeration. I'm not sure what you said is true about an anaerobic state being the only time yeast make ethanol. Yes can respirate with or without oxygen. They start making ethanol as soon as they get out of lag phase and they are in the presence of plenty of O2. The book claims, and supports with references that O2 helps them make better cell walls, makes them less prone to make fussels, finish faster and reproduce better when pure O2 is added.

 

[HeidrunsGift]

My take on it:

-Yeast certainly prefer respiration (necessarily aerobic) over fermentation (can be aerobic OR anaerobic). Together, these are the three forms of catabolism (breaking down molecules for energy) a yeast can choose to use during glycolysis (catabolism of a glucose molecule) From what I remember, this is because 28 molecules of adenisine triphosphate (ATP-- molecules that have potential energy stored in their bonds that when broken apart can be used by the yeast sell for anabolism, or biosynthesis/growth [cataobolism and anabolism are the two major parts to metabolism]) are created during respiration as opposed to 2 ATP molecules during fermentation. So why does yeast do choose fermentation? It is true that oxygen is required for respiration, but due to the Crabtree effect yeast will shut down almost all respiration in presence of simple sugars. In other words, you can aerate the hell out of must/wort, but since it has a high concentration of fermentable sugars, it represses the respiratory pathway and will choose fermentation (either aerobic in the presence of O2 or anaerobic when there is no O2). As a side note, apparently a negligible amount of respiration will still occur during fermentation so the yeast can release carbon atoms to be used for biosynthesis.

-As you stated, respiration produces primary H2O + CO2 (no ethanol). Fermentation produces CO2 + Ethanol (yay!) So we want our drops in the hydrometer to be from fermentation, not respiration.

-It is important to note that oxygen is NOT NEEDED for fermentation. In the presence of oxygen, yeast can perform aerobic fermentation (or respiration if there is a low enough concentration of simple sugars). In the presence of ZERO oxygen, yeast can perform anaerobic fermentation. The glucose molecule is still oxidized (presumably inside the mitochondria) and this sounds contradictory since there is no oxygen molecule, but from what I understand it just means that the stripped electron is going from the glucose molecule and being accepted by a molecule other than oxygen (as opposed to aerobic fermentation when the electron is accepted by an O2 molecule).

-So why is O2 so important? While its not important for fermentation, it is required for lipid synthesis. During aerobic fermentation, yeast biosynthesize (not sure if thats a word of not) unsaturated fatty acids and sterols into lipids *in the presence of O2*, and it are these lipids that are responsible for the rigidity and fluidity of the cell membrane. In wort, lipids can be found in wort, but it is better for the yeast to create their own. And more is better, which means it is difficult to over aerate the must. As alcohol levels increase, it becomes difficult for cells to safely take in nutrients/vitamins in the wine/mead without also taking in hydrogen ions and alcohol molecules (which can stall the fermentation/kill the yeast cells). The lipids help regulate what goes in and out of the cell, and therefore are crucial to fermentation as ABV increases. This is why (I suspect) that mead (typically 14-18% ABV) is aerated more than wine (12-14% ABV), which is aerated more than beer (typically 4-7% ABV). Lower ABV beverages simply don't have the toxic environments from alcohol that our typical meads do.

-So when might respiration happen? We know that the fermentable sugar concentration has to be low (I'm not sure exactly the number in terms of SG/Brix, though I'd be curious to know that) in order for the Crabtree effect to not take place. Respiration also requires O2, so I *think* it would only happen if you have a mead nearing end of fermentation -- getting near dry levels -- and perhaps you degas it too vigorously/splash it, introducing enough O2 with a low enough SG to induce the respiratory pathway over fermentation pathways.

This is one of the best articles I have read on the subject: https://www.morebeer.com/articles/how_yeast_use_oxygen

Which was posted by none other than the Sasquatchy on this thread: http://www.gotmead.com/forum/showthread.php/26358-Current-Thoughts-on-Degassing-and-Aeration/page2

Many thanks to Ryan for finding/posting that article! Also, full disclaimer: I'm not a chemist or biologist, so if anyone out there is and notices some errors, please chime in. What I posted above is a summary of what I have read on the subject over the past couple years.

Skal!

Edit: I'm not sure that I would say *frequent* aeration is needed, if by that you mean continuous. Zainasheff's book recommends waiting for the second dose for at least 12 hours, because this is how long it takes them to do a cell division (page 84). Once that division has happened and the cells are able to synthesize more lipids, then the colony can benefit from another aeration and in that presence of O2 create more lipids. Presumably it would continue to benefit in multiples of 12 hours until harmful oxidation can occur....which I'm not sure exactly when that happens (probably past the 1/3 break) but I suppose thats a topic for another thread.

 

[Squatchy]

Shit Charlie. Good job. One would think your a microbiologist for sure. Good job!

 

[Dadux]

Well gonna chime in since this is in my area of expertise. Basically what was said is mostly right. Yeast can do one of two proceses. Celullar respiration requires O2 and provides a greater amount of ATP molecules (energy) per glucose molecule, using the oxigen in a series of reduction reactions. The result is 6CO2 and 6H2O. in the fermentation process glucose is broken onto 2 ethanol molecules and 2 CO2, and requires nothing that is not inside the cell. This process is faster and genereates much less energy but the yeast choose to do this while they can in a high sugar solution.
The O2 introduced by aereation is quickly absorbed by the yeast and used in a number of different processes, one of which is indeed lipid synthesis. Certain lipids will need O2 to be made (but not all, dont get confused. Still for a cell to be healthy they need different lipids and in different ammounts).
So they dont need to be in anaerobic conditions to do fermentation. The ammount of oxigen you add by aereating goes to other processes rather than glucolysis (and not only lipid formation).
That said the conversion rate is not 100%. that means not 100% of your glucose molecules will be turned into alcohol, for a number of reasons.
So based on this
1- The value of aereation is providing the yeast with a molecule they have a hard time obtaining otherwise, thus making it easier on them to live (and ferment!)
2- Yeast budding is a proccess that requires a lot of energy so yes, when they reproduce they ferment, and actually a LOT more than in the stationary phase
3- When you do a starter the yeast are actually fermenting too. They will also be increasing the number of cells and making them healthy.
4- Aereation is not done daily (usually not), once the stationary phase starts it is not needed (or maybe just not much)
5- Fermentation will happen during ALL phases (lag, growth and stationary) of yeast life unless they are inactive/dead. This is because they always need energy.

Also a couple of things not related to fermentation (in case anyone is interested): ATP is a molecule produced by a number of processes (not a process). that molecule is the "energy coin" of the cell. It is broken to generate energy. And the same way that fermentation, you dont need to be in anaerobic conditions for your muscles to do anaerobic respiration. Some of your muscle cells do that regularly, even when they have oxygen.

Hope this helped

 

[bernardsmith]

Thanks to everyone who responded but I guess I am still a little confused. My basic assumption is that with yeast
1. Survival of cells is ONLY through ATP
2. ATP can occur in both aerobic and anaerobic conditions.
3. ATP is far more efficient (allows for more energy with less effort) under aerobic conditions
4. With yeast , when ATP occurs anaerobically alcohol is a byproduct.
5. In the presence of O2 virtually no alcohol is produced.
If 1 -5 is true, then I ask what in fact is the value of introducing air (or O2) into the must once the lag stage has ended and yeast has begun to ferment IF the length of the active fermentation is about 7-10 days... (If you invoke yeast health then note that brewers avoid aeration for good reasons while fermentation of the wort is ongoing and they don't seem to have any problem with yeast health although admittedly , typically, their SG is lower and their pH is higher).

Thanks

 

[Dadux]

Welp seems like i did a crap job explaining

Fermentation is just a way of obtaining energy. In cells (yeast cells in this case) energy is stored in molecules called ATP. For easy references ATP = energy. ATP is NOT a process, but a way of having a reservoir of energy that can be used anytime for anything.

Energy is obtained by breaking glucose (sugars). Respiration breaks glucose into smaller pieces in a SLOW proccess that gives MORE energy, and it NEEDS O2. Fermentation is a FASTER process that gives LESS energy, and leaves alcohol, and it does NOT NEED O2. Both processes give energy to the yeast (and that energy is converted into ATP) but since in your must there are a lot of sugars, they will chose the faster process (fermentation) as long as they can, even in presence of O2.

O2 has other roles in the cell, and the yeast will use it for that. Thus, aereating gives O2 to the yeast and they use it for other processes not linked with respiration. Those processes are mostly related to reproduction, and so you want to aereate during lag phase and growth phase. Once the stationary phase starts, aereation is not necessary.

I can explain it further but dont want to complicate this too much, at least until you undertand the basics.

 

[Dadux]

And yeast health IS a thing. Just so happens that most beverages are made with things that get spoiled. O2 makes things get rotten. Honey does not rot nor do spices and most herbs (at least not so easily). but fruit rots easily and thats why you dont aereate in wine. In beer with an ABV so low you dont need to aereate either, but many times starters are used for the purpose of making the yeast grow healthy. In mead, the benefits usually outweights the problems of aereating (there are some exceptions such as melomels/pyments) so that is why its recomended

 

[HeidrunsGift]

Hi Dadux and thanks for the responses! Those are very helpful and they did bring up some additional questions:

The O2 introduced by aereation is quickly absorbed by the yeast and used in a number of different processes, one of which is indeed lipid synthesis. Certain lipids will need O2 to be made (but not all, dont get confused. Still for a cell to be healthy they need different lipids and in different ammounts).

Can you list the uses for some of these other lipids that are produced (that presumably are not used to maintain cell membrane's rigidity/fluidity)?

So they dont need to be in anaerobic conditions to do fermentation. The ammount of oxigen you add by aereating goes to other processes rather than glucolysis (and not only lipid formation).
That said the conversion rate is not 100%. that means not 100% of your glucose molecules will be turned into alcohol, for a number of reasons. [\QUOTE]

-Would you please list out some of the other processes that O2 aids in (if they are ones that we would be concernced in)?
-As the glucose molecules are catabolized, what else are they turned into and in what amount? What I'm getting at is, are they in significant enough quantities that our hydrometer readings are not accurate indications of ABV? In the link I posted below, the article states that some respiration does takes place even when the Crabtree effect is induced, but it is so neglibile that we don't need to worry about that affecting our hydrometer readings.

1- The value of aereation is providing the yeast with a molecule they have a hard time obtaining otherwise, thus making it easier on them to live (and ferment!) [\QUOTE]

-I agree, but would emphasize that the PRIMARY importance of O2 is for anabolism (biosynthesis of lipids), and not catabolism (fermentation).
-This does lead to a question: after the respiratory pathway is repressed, do yeast prefer aerobic or anaerobic fermentation? In a different answer you stated that O2 is not needed during stationary phase, but, if they prefer aerobic fermentation, then it would make sense to make it easier on them and continue to aerate until negative affects of oxidation can take place. As I mentioned below, I don't know at what point that occurs. Do you know when that starts to happen? Anecdotally, it is past the 1/3 sugar break.

I can explain it further but dont want to complicate this too much, at least until you undertand the basics.

So I understand this might not be a good thread for the Newbee section. I for one would like to continue to delve into the science of it though, but if others do not maybe we can start a separate thread or continue on a PM? Thanks again!

Skal!

 

[HeidrunsGift]

In the presence of O2 virtually no alcohol is produced.

I think Dadux hit these all, but I'd re-emphasize that what you are missing is the Crabtree effect: in the presence of enough simple sugars (ie, dissolved honey) the respiratory pathway is repressed *regardless of how much O2 is present.*

In the presence of enough O2 AND a LOW ENOUGH simple/fermentable sugar concentration (again, not sure exactly how low, but I suspect nearing dry levels) THEN respiration will take place over fermentation. At that point, CO2 and H2O are primarly produced rather than primarily CO2 and ethanol

 

[Dadux]

Well imma try to answer those no problem. Altough this is gonna stray a bit from the fermentation topic...Dont know if all of it its gonna interest you, but you asked for it! ;D
STOP READING HERE IF YOU ARE NOT INTERESTED IN BORING BIOLOGY STUFF

1-Lipids have numerous activities within the cell. It is a HUGE family of molecules. Their praimary function is forming the lipid bilayer that is present in all celular membranes. In there they provide structural support as well as mainiting a fluidity and forming specialized regions. Those are the more important funciotins for yeast health. Some lipids are actually also involved in celular signaling and signal transduction. That is, they bind to other molecules and that helps the cell (not necesarily a yeast cell, but those too) comunicate to other cells and integrate outside information. Some of them could be sphingolipids for example (but that is just one of many lipid families). Keep in mind, most lipids are in the membranes (not only the plasmatic one, also organelles) , and some have one function some have others. All of them are important for fluidity and rigidity as you say but that does not mean they dont have other funcitions. Its also worth mentioning here that the outer layer of lipids of the plasmatic mebrane does not have the same composition as the inner layer, and the different lipids can have different functions. All types are needed for a healthy membrane

2- Well im guessing giving O2 to the cell helps forming any molecule that has oxygen in them. Not only lipids have oxigen atoms. I was just trying to explain not everything is about lipids and sugars.

3- Some sugars are used to form glucolipids and glucoproteins. Probably not a significative ammount. winemaking has been going on for thousands of years so the conversions are more or less ok. glucose is broken into pyruvate that can be used to make other molecules, such as aminoacids, or it can be transformed into ethanol. diferent monosacharides (glucose, fructose) also can be bound together to make oligosacharides and polisacharides, or modified to make other monosacharides. All of those have cell functions.
About the ABV i have some doubts myself. Some of the ethanol can evaporate even at room temps and specially if you ferment in open buckets or something like that. That could be a significant altough small amount (plus or less 1% ABV, depends on temps, and how long you are fermenting in open conditions). I have also read that different yeast strains give a different ammount of alcohol (usually the ABV is between 0.58-0.63 x Brix degrees. More info about that here http://www.gotmead.com/forum/showthread.php/26441-SG-versus-BRIX) And also it has been said that the SG-ABV relation is not linear, but i dont know how much of this is true or proven or significative (i mean that if you have an SG of 1.120 the alcohol produced from 1.120 to 1.110 is not the same than from 1.010 to 1.000)

4- Yes you are right, its more important in anabolism (as long as we are talking about yeast cells and anabolism vs fermentation)

5- MMMM as far as i know fermentation is the same. Some organisms just dont tolerate O2. That means they are only capable of anaerobic fermentation (that is, they ferment and dont tolerate O2). Aerobic fermentation is just that, fermentation is pressence of O2. Yeast does tolerate O2. As far as i know, there is no difference in the fermentation process, but if you add O2, some respiration can ocurr (and used for other processes if neccesary, as already mentioned).
The point when yeast stop the growth phase and enter stationary phase is dependant on the sugar in solution and the population of cells, if im not wrong. Thus, the moment when aereation is not needed anymore is different in every must (I admit i might be wrong on this one, not 100% sure). When yeast no longer absorbs the O2 it remains in the must and ends up binding to things there

Hope this clears some of your doubts and does not leave you bored as hell. If you want more specific info ask, or PM me, no problem. Also sorry for any typo or error, English is not my native language

Edit: also, just for kicks I'll let you know that my end of degree project was about how some molecules alter lipid metabolism. And guess what were some of those molecules? Yep, tannins.

 

[Dadux]

I think Dadux hit these all, but I'd re-emphasize that what you are missing is the Crabtree effect: in the presence of enough simple sugars (ie, dissolved honey) the respiratory pathway is repressed *regardless of how much O2 is present.*

In the presence of enough O2 AND a LOW ENOUGH simple/fermentable sugar concentration (again, not sure exactly how low, but I suspect nearing dry levels) THEN respiration will take place over fermentation. At that point, CO2 and H2O are primarly produced rather than primarily CO2 and ethanol

Yes. As said, when they have spare food they go for the fast method, that is, fermentation. Respiration is much more efficient but needs O2 and takes longer and requires enzymes that are costly too. Why waste time and energy when you can just go the easy way? For yeast, fermenting is a much more competitive process, as long as they can afford it.

 

[HeidrunsGift]

Thanks for the replies, that's exactly what I'm looking for I think there is a lot more science WRT O2 related to the fermentation then we realize, or perhaps are taking advantage of. Here are a couple things I was thinking of when I read through your reply.

Some lipids are actually also involved in celular signaling and signal transduction. That is, they bind to other molecules and that helps the cell (not necesarily a yeast cell, but those too) comunicate to other cells and integrate outside information.

I wonder if this is how the cells "sense" that it is time to transition from lag to growth phase? I have also heard that when the biomass concentration is great enough, they "tell" each other to start exponentially growing (believe the professor at UC Davis told that to Squatchy when he went to the mead seminar there). Also, could be how they sense a high sugar concentration and signal to each other to induce the Crabtree effect?

And also it has been said that the SG-ABV relation is not linear, but i dont know how much of this is true or proven or significative (i mean that if you have an SG of 1.120 the alcohol produced from 1.120 to 1.110 is not the same than from 1.010 to 1.000)

Very fascinating, one of the first times I looked at a hydrometer scale chart I recognized that it was not linear and wondered why it was not. Intuitively it should be, but I suppose yeast dont always act in an intuitive manner.

MMMM as far as i know fermentation is the same

That agrees with the protocol of only aerating at pitch and then around 24 hours later. Just wanted to make sure that we weren't missing out on anything by not continuing to aerate past that point, sounds like we are not.

Hope this clears some of your doubts and does not leave you bored as hell. If you want more specific info ask, or PM me, no problem. Also sorry for any typo or error, English is not my native language

Edit: also, just for kicks I'll let you know that my end of degree project was about how some molecules alter lipid metabolism. And guess what were some of those molecules? Yep, tannins.

That does help, thanks! I find it very interesting, and your English is very good! How did the tannins alter lipid metabolism? In a good way? Bad way?

 

[Dadux]

No problem. most people are not interested in the deep science of it and i get, it its complicated.

All cells, including yeast have a lot of molecules (called receptors, mostly proteins i must add) that give them information about the outer world. But sure. lipids are involved too.
And yes, they also secrete certain molecules. Those bind to receptors. When the ammount of binded molecules reaches a certain point it generates a series of changes in other molecules inside of the cell, that change the genetic expression of it. To put a simple example, yeast usually metabolize glucose. When the levels of glucose go down in a must, more and more fructose is internalized. That is a signal the cell uses to create enzimes that are used to convert fructose to glucose, and other proteins related to fructose metabolism. In the process you mention, when the cell senses a lot of certain molecules that means too many yeast cells, and so it will inhibit reproduction. When there are not many, probably reproduction is instead induced.
Or maybe if a cell finds the enviroment good enough to divide, it secretes certain compounds that signal other cells that is ok to do so too

About the crabtree effect, i think its more an independant process. They sense the high ammount of sugars, but dont signal that to each other (however I am not sure, but it would not make much sense since all can "realize" the ammount of sugars in the must)

About the non linear proggresion, i guess its because during growth more sugars are used to different processes, apart from fermentation and generating energy. But there may be other factors involved

Well I did the research on rat cells, but yeah, they did alter the metabolism. Certain lipids were favoured over others. However tannins probably affect yeast metabolism too!

 

[bernardsmith]

Not at all boring.. and very clear.