A theory of fermentation

[jeff.k...@gmail.com]

Hi everyone

I'm piecing together a theory of cider fermentation and welcome comments/critique.

Fundamentally, yeast activity does three things:  It increases the yeast count in the fluid, generates CO2 and alcohol.  

The yeast count is a proxy for the amount of nitrogenous substances that are bound up and no longer available for building further yeast cells.  These substances is can be freed again as the yeast dies and breaks down.  

As for the metabolism of the yeast, how the yeast is distributed in the cider is an important parameter.  Yeast that is suspended and distributed throughout the cider is more active than yeast that is settled on the bottom of the fermentation tank.  So what is keeping the yeast suspended?  I think it must be the turbulence of the CO2 bubbles rising.  So when you have rapid fermentation, the yeast stays suspended, more yeast cells are active and the rate of fermentation increases at the rate of yeast reproduction.  Under ideal conditions, this could be exponential.  When you have slow fermentation, the CO2 production drops off, turbulence decreases and the yeast cells settle to the bottom of the tank and 

So now the question is:  why do the yeast cells at the bottom of the tank behave differently to those that are suspended?  I propose that the density of yeast cells on the bottom of the vessel generates a microcosm of high alcohol and low nutrients at the very bottom of the vessel which inhibits then inhibits yeast.  This effect is probably limited to within the yeast layer itself.  As long as diffusion of alcohol out and nutrients in (including sugars) cannot keep up with the yeast activity, those cells at the bottom will be inhibited.  

This implies that the shape of the fermentation vessel will have a direct impact on fermentation speed.  Tall, narrow vessels should promote fermentation because CO2 bubble will be more concentrated, producing more turbulence.  Shallow, wide vessels should inhibit fermentation by allowing yeast cells to quickly become immobilized by minimizing bubble turbulence and minimizing the distance needed to fall before hitting the bottom.  This  is what the reference from 1935 I posted earlier was pointing out and which Claude confirmed empirically.  This assumes that once yeast have settled to the bottom of the tank they are immobilized and do not get re-suspended.  You can lock in these conditions by racking - which guarantees that the immobilized yeast are permanently removed from the system.   The important point is that in a shallow vessel more yeast will settle out and become immobilized than in a tall vessel.  

This also explains why cold crashing works.

Thoughts?  I think the two biggest questions in this theory are whether CO2 bubble turbulence is responsible for keeping yeast suspended and whether the yeast layer at the bottom inhibits itself by creating a microcosm of high alcohol and low nutrients. 

/Jeff

[Claude Jolicoeur]

Interesting rambling Jeff!

Maybe a few more points to consider:

- Pre-fermentation clarification of the must - I have read that yeast cells will be more in suspension within the fermenting cider when there are also some particles that are in suspension. The yeast cells sort of hook themselves to these particles. Which means if the cider has been clarified prior to the start of fermentation, then there are less particles in suspension, and a greater fraction of the total yeast population will remain on the bottom of the tank.

- I agree that in a higher container, each CO2 bubble will travel longer to reach the surface, hence creating more fluid movement. And this may favorize a higher fraction of the yeast population to be in suspension in the fermenting cider.

- What makes you think that bottom yeast would be inhibited? If it was the case, then the fermentation speed would not decrease after

a racking where the bottom yeast is eliminated and the suspended yeast is kept. My observations do not concur with this. In general, after a racking, the fermentation speed may be cut by a factor of 2 to 3. This means that the yeast in suspension (which is kept in the racking process) is responsible for only half or a third of the fermentation speed, and the yeast residing on the bottom (which is eliminated by the racking) was actually responsible for the remaining of the activity.

[jeff.k...@gmail.com]

My (limited) evidence is from my ice cider fermentations (where the initial SG = 1.160), which I keep below 12 C and which are actively fermenting for 6-10 months.  The speed of fermentation is initially 200-300 FSU (after the yeast is established) and then - with no racking or intervention - diminishes over time while there is still a large excess of sugar.  One of my batches last year stuck at an SG of 1.120 after two months of fermentation.  I had to add 10 ppm Fermaid K to get it to restart.  This year, one of my batches is now down to and FSU of 20 at an SG of 1.10 and is clearing.  

 I have run a test where I take a sample of the slow ice cider and bring it inside to room temperature (20-22 C).  Fermentation picks up immediately and the SG goes down to 1.03 within a few weeks.  I interpret this to mean that a lack of nutrients is not an issue.   

The temperature is inhibiting the yeast, but the temperature is fairly constant.  If the speed of fermentation were a function of temperature alone it ought to be constant as well.

From this (again, admittedly limited) data, the yeast sitting at the bottom of my vessels does not seem to do much.  When I do rack, there is always a couple of cm of yeast sludge at the bottom.

I concede that your observations of fermentation speed dropping by a factor of 2-3 suggests activity in the settled yeast.  But could it also be explained by nutrient recycling?  I don't know how important autolysis is to yeast activity, but if you remove the yeast at the bottom as a nutrient source, could that also explain the reduction in the fermentation speed?

/J  

[jeff.k...@gmail.com]

Rereading my post, I see the apparent contradiction in having to add Fermaid K to restart a stuck fermentation, but having adequate nutrients to maintain fermentation down to 1.03. 

I guess I just have no idea what is going on :-) 

[Claude Jolicoeur]

Le mardi 26 avril 2022 à 07:12:00 UTC-4, jeff.k...@gmail.com a écrit :

I concede that your observations of fermentation speed dropping by a factor of 2-3 suggests activity in the settled yeast.  But could it also be explained by nutrient recycling?  I don't know how important autolysis is to yeast activity, but if you remove the yeast at the bottom as a nutrient source, could that also explain the reduction in the fermentation speed?

My understanding from what I can observe, is that a racking has 2 main effects:

- immediate reduction of fermentation speed, in a ratio equivalent to the reduction of the number of active yeast cells. Hence if that number is reduced by a factor of 2, the speed will also be reduced by a factor of 2 - and this is immediate.

- on a longer term, by elimination of dead/inactive yeast cells and reduction of nutrients, and this will contribute to prevent the active cells from multiplying again - hence this should have as effect a gradual deceleration of the fermentation speed.

[AW]

Are live yeast motile?  As in, does a fermenting yeast preferentially re-suspend itself by precipitating CO2 on its own membrane or by some similar mechanism?  By the same token, do dead or dying yeast tend to settle out preferentially (flocculation)?

It seems from the comments like the lees near the end of fermentation contain most of the yeast biomass but are responsible for <=half the fermentation capacity.  I would consider that rather than the lee environment inhibiting fermentation, that the composition of the lees is biased toward dead and dying cells.  

[jeff.k...@gmail.com]

That is an excellent question.  I have no idea, but assumed they just passively stayed in suspension like colloids, with turbulence helping that process.  

Does anyone know for a fact if yeast are somehow motile?

[jeff.k...@gmail.com]

I wonder if  the nutrients needed for yeast metabolism are a function of temperature.  I don't understand how the fermentation of my unracked ice cider at 10 degrees C can get stuck with a few cm of yeast at the bottom of the barrel - then ferment to near dryness when a small sample (which is in effect racked off) is warmed up to room temp.  

[luis.ga...@gmail.com]

Sorry for this late intervention on the subject.

I make cider since almost 10 years with racking and the intention to maintain a level of residual sugar in the final product. From my observations, I can say that another impact of racking is to actually clarify cider. I have concluded that the effectiveness of fermentation speed reduction after racking is related to the decreased turbidity after racking. Hence, my observations are leaning toward the fact that a significant part of fermentation speed reduction is associated with the reduction of suspended yeast in cider.

I admit I have no idea how a racking can physically or chemically influence the clarification of cider.

Is it a phenomenon that you have also observed Claude?

Louis 

[Love Lindholm]

Claude,

Just a thought regarding the question of whether or not the yeast in the lees at the bottom is inhibited:

Could it be that it is indeed inhibited, albeit not the point where it becomes completely inactive, so that it contributes to the overall fermentation but with a lower sugar consumption per cell than the yeast in suspension? 

Also, how large a fraction of the total yeast count is typically lying at the bottom of the vessel? If, say, 80% of the yeast is lying at the bottom and each of those cells ferment at 25% of the speed of the yeast in suspension, then the overall fermentation speed should be cut in half if the bottom layer is removed and the rest is kept. So the observation that the overall fermentation speed is reduced after a racking, could very well be consistent with different speeds of fermentation in different parts of the vessel.

Best
Love

[Claude Jolicoeur]

Le vendredi 6 mai 2022 à 14:15:23 UTC-4, luis.ga...@gmail.com a écrit :

I admit I have no idea how a racking can physically or chemically influence the clarification of cider.

Is it a phenomenon that you have also observed Claude?

Not really.

What I have observed is that the more clarified the cider is before racking, the more fermentation speed reduction one gets by racking.

And an explanation for this is that a greater fraction of the active yeast cells is eliminated during the racking process, indicationg the yeast residing on the bottom are active.

Now could the bottom-residing yeast be only partly inhibated as Love speculates in the other post? I have no idea really.

Now, one thing is: the dead yeast cells fall to the bottom. So if one counts the total biomass residing on the bottom of the tank, including dead and alive cells, then yes surely this biomass is globally less active than the suspended biomass which is all alive and active.

[Love Lindholm]

Hm. I don't want to push this too far, since I am speculating. But why would dead yeast cells fall to the bottom if alive cells can stay in suspension? If there is a heat convection movement in the tank, plus CO2 bubbles moving upwards, then surely dead yeast could be in suspension as well as alive yeast. Or why would exclusively alive yeast stay in suspension?

The way I was thinking about clarification was that as the fermentation rate approaches zero, the driving forces of motion within the vessel (heat and CO2 from the fermentation) stops, and all solids (dead and alive yeast cells, pectins and whatnot) fall to the bottom. It probably takes a sort of minimum fermentation activity to keep solids in suspension. So if the cider is clear, the total activity is not high enough to keep anything from sinking to the bottom, and any remaining fermentation activity thus goes on at the bottom.

[scott heath]

An active yeast cell will be producing tiny bubbles of gas, which adhere to it for a time and thus carry it upwards in the liquid. Once they stop producing gas, they sink.

[CGJ]

Scott's assessment that tiny bubbles being stuck to the cells is what is
keeping them in suspension is a concept that I've been considering for a
while.

It has been my experience that when I do an early racking, the resulting
cider is as cloudy as before racking, but experiences a quick additional
significant yeast precipitation (in less than a day). I have attributed
to this in mind to mechanically knocking loose the adhered bubbles,
making the yeasts suddenly less buoyant. This seems particularly
noticeable with my go-to commercial yeast (Wyeast 4766).

I have wondered if I could combine this with a chilling (to cause the
yeast to slow down) AND a brief stirring (to pre-unstick the bubbles)
and then rest period (to allow settling) prior to the early racking to
maximize the racking effect... something that I may try some cold
morning next season.

Carl
West Barnstable
Massachusetts

[Love Lindholm]

It could definitely be that each yeast cell gets buoyancy from its own CO2 bubbles and nothing else. I am not entirely convinced, though:

1. If you open a vessel with a very active fermentation, it can look almost like a jacuzzi. The motion in the liquid is definitely strong enough to stir up particles the size of yeast cells, dead or alive. I would assume this happens to some extent also when the fermentation is less strong.

2. If yeast cells surf their own bubbles, it seems likely they would gather at the surface. I myself, at least, have not seen this happening.

3. If active yeast cells adhere to their bubbles and float, why is there active yeast at the bottom of the vessel?

[AW]

"Current paradigms assume that gas bubbles cannot be formed within yeasts although these workhorses of the baking and brewing industries vigorously produce and release CO2 gas. We show that yeasts produce gas bubbles that fill a significant part of the cell. The missing link between intracellular CO2 production by glycolysis and eventual CO2 release from cells has therefore been resolved. 

"We suggest that intracellular CO2 may eventually be secreted by pressure through the yeast cell wall to affect pressure homeostasis. This in turn should result in vigorous bubble release under diminished pressure (decompression) of the external environment, resulting in their coalescence and enlargement to visible bubbles of millimeter and centimeter size as is generally experienced in products of fermentation such as leavened bread, traditional beer, and champagne. Internal cell pressure is probably needed in these yeasts to keep bubble size at a minimum to decrease any adverse effects on cell function. Alternatively, these bubbles may be in nonpressurized transit as they are shipped out of the cell upon production."

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3503256/

"Two major kinds of fermentation viz. top (ale) and bottom (lager) fermentation, display a difference in their yeast distributions inside a sugar broth. The reason for this difference is believed to be yeast–bubble adhesion arising due to surface hydrophobicity of the yeast cell wall; however, the physical mechanism is still largely a mystery. In this report, through in vivo experiments, we develop a novel theoretical model for yeast distribution based on the general conservation law. This work clarifies that bubble-induced diffusion is the dominant transport mechanism in bottom-fermentation by lagers whereas, yeast–bubble adhesion plays a leading role in transporting ales in top-fermentation, thereby corroborating the centuries-old belief regarding distribution difference in yeast population in two kinds of fermentation."

https://royalsocietypublishing.org/doi/10.1098/rsif.2020.0735

[Love Lindholm]

Cool!

First study:
It seems like yeasts are a little bit like fish, then: they have a swim bladder. They do not discuss, though, if CO2 in the cytoplasm makes the cell float in the medium.

Second study:
So both adhesion to bubbles and convection induced by bubbles is involved in moving yeast around in the tank. At least for S. Cerevisiae. And there is yeast at the surface. Cool!

I am a bit confused as to the type of yeast they use, though. They say they compare ale yeast and lager yeast. However, they only mention S. Cerevisiae, but lager yeast is S. Pastorianus, right? They say the lager yeast they use is called Fermentis, SAFLAGER S-24, but Fermentis does not seem to have yeast with that name; they have one called SAFLAGER S-23, which is a Pastorianus. So I think there is typo in the name and that they somehow overlook that the lager yeast is a different species.

[Miguel Pereda]

My observations in a 200 L, 80 cm high stainless steel cask tell me the following:
Cider with finished fermentation, no gas release and virtually no dissolved gas. It had lost all the gas.
1/ It was still quite cloudy. It clarifies worse in steel than in wood.
2/ The yeast in suspension at the top third was around 2,106 CFU/mL.
That is to say that there are still yeasts in suspension but they were practically all dead.
3/ Huge amount of yeasts in the bottom lees. Most of them were also dead.
These observations are what they are and I cannot extrapolate them to tanks of, for example, 25000 L.
Miguel A. Pereda