yeast, oxygen, growth, and fermentation

[mandoman]

Another 'help me out here' thread.

When yeast are pitched into unfermented wort they begin to bud or reproduce asexually in the presence of oxygen. Once the oxygen is depleted they switch to anaerobic metabolism and create alcohol and co2. So, what I'm thinking is that the 'lag time' prior to what we call fermentation (bubbling, physical activity, etc.) has at least something, if not a lot, to do with the concentration of oxygen at initial pitch. I'm guessing this is why I've read that no lag time is not necessarily a good thing. The questions that come to mind are, 1) is there an ideal 'lag time' or initial o2 concentration for a given pitch rate/yeast health, 2) can this be easily determined given that we often use repitches and don't really know viability, cell counts, etc., 3) if you give a wort 'too much' o2 can this give you too high a pitch rate due to too much reproduction, and if so 4) if you pitch the 'right cell count' do yeast even need the o2 in the first place since they don't really need to reproduce or is this initial reproduction beneficial as the yeast adjust or somehow react to the wort and thus the subsequent reproduction is healthy in that it is specific?

I know, a lot of questions.

 

[Kaiser]

A lot of factors go into the lag time. Reproduction and pitching rate are only two. Others are wort temperature and yeast vitality. When we pitch yeast into aerated wort it will quickly take up the O2 and start building sterols which are an essential building block for their cell walls. It will also start lowering the pH of the beer (wort becomes beer as soon as the yeast is pitched) which will facilitate the uptake of amino acids. And if a high graviy environment is present the yeast will work on increasing the internal sugar concentration in order to keep the osmotic pressure (which tries to suck the water out the the cell) at bay. There will be only little aerobic metabolism because there is an excess of sugars available to the yeast (search for Crabtree effect).

Most of the yeast growth will actually happen once the O2 has been consumed and the yeast will grow as long as it has everying to make a daughter cell: enough amino acids for building proteins, enough sugar for fuel and enough sterols to share for making cell walls. If insufficient O2 was supplied at pitching time the yeast may not have been able to create enough sterols to achieve the desired amount of growth. The fermentation will become sluggish with yeast cells that have thin cell walls because their sterol reserves are stretched.

A O2 level of 8-12 ppm is recommend at pitching time. But unfortunately this is not easily measured b/c DO (dissolved oxygen) meters are rather expensive. The vast majority of home brewers rely on good aeration practices to achieve the necessary level of O2 before pitching the yeast. When wort is saturated with O2 from air it will settle at about 8ppm. Such saturation can be achieved to shaking for a minute or two.

Brewers do want some yeast growth in their fermenters. It is important for the flavor profile of the beer and commercial brewers also rely on the yeast crop for pitching subsequent batches.

Kai

 

[Tonedef131]

Kaiser seems to have addressed all your questions other than number 3. Excess oxygen isn't really a problem on the homebrew scale. In giant commercial conical fermenters they are more careful because with that much weight on the wort it can hold more O2, up to levels that will be toxic to the yeast. But in your carboy that won't be the case, any excess O2 will simply bubble out of the wort. And if you put 10ppm of O2 the yeast don't necessarily need to use it all. They will only reproduce a certain amount before starting to convert sugar into ethanol/CO2, when this happens any extra O2 left over will get pushed out of the wort by the CO2 being released.

So keeping in mind that your "pitch rate" is how much you actually pitch in, not how much yeast is there after reproduction, no the amount of O2 will not effect the pitch rate. Assuming you made healthy wort for them and gave them plenty of O2 they will take care of themselves from there. Reproducing causes many of the flavors that we know as beer...but not starting with enough healthy cells, starving them of oxygen, and getting them beyond their comfortable temp range can cause the off flavors we all try to avoid.

 

[Kaiser]

Yes, I forgot about excess O2. While it not possible if you use atmospheric pressure air as the O2 source it is possible with the pure O2 oxygenation systems that many of us use. But I don’t really know how much is too much.

Kai

 

[mandoman]

very good gentlemen - thank you!

 

[Piotr]

When wort is saturated with O2 from air it will settle at about 8ppm. Such saturation can be achieved to shaking for a minute or two.

Are you sure 2 minutes is enough? Now I'm shaking whole 5 minutes and I will be more than happy to reduce this task to 2 minutes.

EDIT: here:
http://cdn2.libsyn.com/basicbrewing...00&nva=20090513194100&t=0cf70dbd12a53a4a16b77

I have read about those 5 minutes (Figure 1 at the end of the article)

 

[SpanishCastleAle]

Might as well ask it here:
Somehow I got the impression that if possible, it would be beneficial if I were to shake the carboy to aerate the cooled wort, pitch the yeast, shake it again...and then shake it at least once every hour until I see signs of fermentation. So that's what I do (whenever possible...which is 'usually').

Question is: Is it doing anything beneficial? I just figured that during the lag phase I could keep 'adding' O2 until the yeast had their fill and were ready to move on to the next phase.

My lag times are always fairly short but I wouldn't say they're consistent...but then neither is the gravity of my beers (that's all over the place). It might be as short as 2 hours or (rarely) as long as 12 (if I pitch a washed slurry straight from a mason jar from the fridge)...usually between 4-6 hours.

 

[Kaiser]

Are you sure 2 minutes is enough? Now I'm shaking whole 5 minutes and I will be more than happy to reduce this task to 2 minutes.

I have little experience with artration through shaking and was guessing at the time necessary for sufficienct O2 uptake. The paper you mentioned has better data than what I can provide.

Kai

 

[Piotr]

Might as well ask it here:
Somehow I got the impression that if possible, it would be beneficial if I were to shake the carboy to aerate the cooled wort, pitch the yeast, shake it again...and then shake it at least once every hour until I see signs of fermentation. So that's what I do (whenever possible...which is 'usually').

I don't do it primarily becouse I want to be consistent with amount of O2 I put into the wort.

I see anonter drawbacks: if you make too much foam during fermentation, you can have problems with head stability. I've also heard that wort agitation during fermentation encourages ester formation, which may not be desireable, depending on style.

 

[SpanishCastleAle]

Thanks Piotr. I actually thought it was more consistent this way...relative to varying gravity worts that is. I want the higher gravity worts to get more O2. I guess I figured the yeast in lower gravity worts would get what they need and then start fermenting while the yeast in higher gravity worts would keep working aerobically until ready to ferment. But that Crabtree effect throws a big monkey wrench in there.

I've also heard that wort agitation during fermentation encourages ester formation,

I'm not doing it during fermentation...just at the very beginning right after I pitch the yeast. As soon as I see signs of fermentation (CO2 bubbles forming on the surface...not even a layer...just little colonies of bubbles) I'm done. I've read about the head retention issue...it seems the jury is still out on that one.

Now that I'm doing fast ferment tests I will better be able to see what effect on fermentation it has. My first FFT was already a bit of a surprise. Maybe I'll do two FFTs next time and give one the periodic early shake and leave the other one alone.

 

[Piotr]

Now that I'm doing fast ferment tests I will better be able to see what effect on fermentation it has. My first FFT was already a bit of a surprise. Maybe I'll do two FFTs next time and give one the periodic early shake and leave the other one alone.

Interesting, let us know the result of this experiment.
I do FFT since some 10-12 batches, and usualy they reach final gravity.

Do you use airlock? If so, there might not be O2 in the carboy any more, it might be purged with CO2 from fermentation...

 

[SpanishCastleAle]

Interesting, let us know the result of this experiment.
I do FFT since some 10-12 batches, and usualy they reach final gravity.

Do you use airlock? If so, there might not be O2 in the carboy any more, it might be purged with CO2 from fermentation...

Ha, well based on the FG of my first FFT (1.012 from an OG of 1.060) I hope it does reach the limit of attenuation...this is a Dunkleweizen using the Hefe Ale WLP300 yeast (same for FFT) and 1.012 is about where I expected the beer to finish (actually a tiny bit lower). I expected a lower FG for that FFT. Hence the usefulness of the test. Maybe it was the decoction that made it less fermentable than I expected.

I do not use an airlock while I'm shaking the carboy and waiting for signs of fermentation...just a bit of aluminum foil. As soon as I see the bubbles I put the blow-off tube on.

 

[Piotr]

I'm not doing it during fermentation...just at the very beginning right after I pitch the yeast. As soon as I see signs of fermentation (CO2 bubbles forming on the surface...not even a layer...just little colonies of bubbles) I'm done.

Look what an interesting quote I've just found:

"Another method you can use to increase the fruity esters in your homebrew would be to increase the fermentation temperature, which will encourage rapid yeast growth and accomplish the same end result. Of the two methods, raising the fermentation temperature during the lag phase is probably the best choice"
Esters, or Who Put That Banana In My Beer

it was about increasing ester formation via temperature shifts, but I see, that the lag phase is not so neutral and unimportant as we think....

 

[SpanishCastleAle]

Interesting link Piotr. It also says that in addition to yeast strain selection or increased ferm temps:

Another method of increasing ester production in your beer is under oxygenating your wort. Yeast use lots of oxygen building their cell walls (4-14 ppm) and will continue reproducing until all the dissolved oxygen is gone. During reproduction, yeast use oxygen to produce unsaturated fatty acids which also uses up aCoA, a precursor for ester production. Therefore by reducing the amount of oxygen available for the yeast, you will effectively be increasing the available ester precursor acetyl coenzyme A, making it available to produce more of the fruity esters you want.

So it almost seems that if anything, shaking it periodically will reduce esters.

Living in Florida, the first off-flavor/aroma I learned about was banana esters. Most of my earliest beers had it (of course I was also under-pitching at the time too). It seems that as long as I keep it reasonably cool for the first few days (after which it's all but finished) then it doesn't matter much after that. Even as high as 68 F (environment...not contents) doesn't get me much if any banana, then I just it let slowly warm up to room temp and leave it there until it's racked (my Dunkleweizen brewed on Saturday is already @ 73 F).

The Mr. Malty "14 essential questions about yeast starters" (or whatever it's called) really seemed to stress yeast health so I figured periodic shaking contributed to that. Hopefully the upcoming dual FFT will provide info.

 

[Piotr]

Another surprising (for me) finding, about ester production.

In a podcast - Basic Brewing Radio, interview with D.Logsdon, Part 3, about 40 min;
a listener ask what shall he do to maximize ester production.
The answer is: start fermentation cold, and then rise the temp to the warmer end of the fermentation range.

It suprised me, because this is what basically Jamil proposes to reduce ester production; like pitching lagers in 7-8*C, and then rise to 10*C.

link:
http://www.basicbrewing.com/radio/mp3/bbr11-10-05.mp3

 

[menschmaschine]

Another surprising (for me) finding, about ester production.

In a podcast - Basic Brewing Radio, interview with D.Logsdon, Part 3, about 40 min;
a listener ask what shall he do to maximize ester production.
The answer is: start fermentation cold, and then rise the temp to the warmer end of the fermentation range.

It suprised me, because this is what basically Jamil proposes to reduce ester production; like pitching lagers in 7-8*C, and then rise to 10*C.

link:
http://www.basicbrewing.com/radio/mp3/bbr11-10-05.mp3

I think they're both right. Starting a lager colder than the optimum range and letting it warm up to the lower end of the optimal range before fermentation really gets going will keep esters down. Starting an ale in in the low end (or lower than) the optimal range and letting it warm up to the higher end (or higher) of the optimal range after fermentation has taken off will keep fusel alcohol production down while facilitating higher ester production.

 

[SpanishCastleAle]

That's strange because I almost always start my ale fermentations in a water bath with ice to keep cool but I only leave it like that for 2-3 days. Then I just drain the water and let it warm up to room temp (74-ish) for the remainder of the primary. Esters are very low (often too low for style).

Note: when I start the fermentation it's not at or near the low end of the ale range...it's actually still in the high 60s so it's in the middle. So it only increases temp by 7-8 F the whole time.

 

[Piotr]

Starting an ale in in the low end (or lower than) the optimal range and letting it warm up to the higher end (or higher) of the optimal range after fermentation has taken off will keep fusel alcohol production down while facilitating higher ester production.

That's basically what I'm doing with my ales. I don't have problems with fusels, but usualy I don't get enough esters, especially for belgian beers. I think I'll use more agressive warming shemes - so far I elevate the temp 2-5*F, I think can do 10*F or more.

 

[menschmaschine]

There are other things that influence ester production (e.g., pitch rate). For temperature, it is a real balancing act... i.e., getting the timing right to raise the temperature. It may only be a few hours to a day after fermentation has started... I don't really know.

Take Moortgat's Duvel, for example. They pitch low (rate) and start fermentation in the low 60s°F. By the end of fermentation (~5 days), they have the temp in near 80°F and they get wonderful ester production with low fusel alcohol production. The big unknown there is at what point and how quickly they raise fermentation temps.

 

[Kaiser]

Ester production is something that is stronger towards the end of fermentation since the main precursor for esters, the Acyl-coA, is needed for yeast growth. This means as long as the yeast is growing it is consuming the acyl-coA and it will not be available for ester production. Many brewing texts state that techniques aimed at higher yeast growth (e.g. low pitch rates) will limit esters. This is in contrast to what home brewers believe who say that low pitch rates cause more esters.

I believe that it is not as simple and that both might be right but that there are other factors (like O2 levels) that skew the results and create the perception that low pitch rates always yield more esters. And some brewers may also confuse a less clean fermentation resulting from a low pitch rate with increased ester production. I still have to find published data on pitch rate and ester production. I was told that it is not a linear curve but a U shaped one with a minimum and a rise of ester production on either sides (lower and higher pitching rates)

I once did an experiment to test the ester production based on pitching rate and O2 content. While the experiment itself was inconclusive the introduction talks about what I found about ester production in the literature: Experiment Pitching Rate and Oxygenation - German Brewing Techniques

Kai

 

[Piotr]

Ester production is something that is stronger towards the end of fermentation since the main precursor for esters, the Acyl-coA, is needed for yeast growth.

This is great info, that means we have full control on both fusels and esters.

One thing in bothering me: diacetyl rest - we elevate the temperature at the end of fermentetion, does that mean we risk more intense ester production during diacetyl rest?

 

[menschmaschine]

One thing in bothering me: diacetyl rest - we elevate the temperature at the end of fermentetion, does that mean we risk more intense ester production during diacetyl rest?

I agree with you Piotr. Esters being produced more towards the end of fermentation seems to be counterintuitive regarding diacetyl rests. I'll have to check the literature on this. I was under the impression that esters were produced most during about 25-75% of the fermentation time frame... but that's only my inference from what I've read.

 

[SpanishCastleAle]

Is it possible that different worts (or rather 'beers' since we've added yeast) have conditions more/less favorable to hydrolyze esters? I've read that esterification is reversible, esters can be cleaved via hydrolysis and that there is an equilibrium point. But I may be confusing the 'chemical' esterification reaction with the 'biological' esterification reaction. Although intuitively it seems the resultant esters would be hydrolyzed the same, no?

The chemical esterification takes place after the beer is relatively stable but during fermentation things are changing fast (relatively speaking). Are there possible conditions during fermentation where esters are being hydrolyzed as fast as they are being formed (or anywhere close to it)? Could that be a reason why it seems we're all over the map on this one?

 

[Kaiser]

There are two ways of ester production in beer. One is through the yeast the other is simply the reaction of an acid with an alcohol present in the beer. The latter is what happens during aging and brings out the dark fruit notes that we all love in well aged beers. This one takes time. Here we are concerned about the ester production of the yeast. Yeast growth actually stops fairly early in the fermentation. I’m not exactly sure when but I expect it to be near the high Kraeusen stage. After that the yeast enters a more or less stationary phase where they just consume sugars but don’t grow anymore b/c they ran out of building blocks (I assume sterols or amino acids or both are missing). I’m unsure when the Acyl-coA production stops. If it were to continue during this phase we should see much more esters being produced since there is no growth which would consume this Acyl-coA. regardless of temperature.

I think this is why there is not much harm in maturing and finishing lagers at a higher temperature (a.k.a. diacetyl rest).

I do not completely understand the mechanisms of ester production yet. It seems as if there are some pieces of information missing as there are still some contradictions left.

I think the best way to control esters is through the choice of yeast. Once the proper yeast is chosen it should be pitched healthy, in sufficient amounts and at the proper temperature and O2 level. Getting a not-so-estery yeast to produce esters may require stressing it which in turn can produce a lot of other less desirable byproducts like higher alcohols and diacetyl.

Kai

 

[schweaty]

Acetyl CoA is not produced in fermentation but rather aerobic respiration. So once the yeast have switched their metabolic process are esters no longer produced since Acetyl CoA is not either?

Per every molecule of glucose 2 pyruvic acids are produced, then decarboxylized producing 2 CO2 and 2 acetaldehyde, which is then reduced to 2 ethanol. So does the pyruvic acid react with ethanol to produce esters in fermentation?

 

[stoutaholic]

This article from the BJCP's website may answer a lot of these questions:

http://www.bjcp.org/cep/WyeastYeastLife.pdf

It was put together by Greg Doss, who answers your questions when you post them on Wyeast's website. Very intelligent and helpful guy.

Anyway, the experiments conducted in this study look at the relationship between pitching rate and dissolved oxygen on the production of yeast growth byproducts, such as esters.

However, what the experiments DO NOT show is the combined effect of O2 and pitching rate. In other words, holding O2 constant, higher pitching rates naturally produce less yeast growth and therefore fewer yeast growth byproducts. Holding pitching rates constant, higher O2 levels produce MORE yeast growth, and therefore more byproducts.

But what happens when you have a high pitching rate AND high O2 levels? Or high pitching rates of yeast that had already built up sterol reserves in the starter that were pitched into wort with a very low O2 level? My theory was that I would get a very clean fermentation if I highly oxygenated my starter and let the yeast build up sterols, and then pitched into un-oxygenated wort (that is, I did not aerate the wort at all, only the starter). And, as expected, there was practically no lag phase at all (i.e. anerobic growth proceeded almost immediately). However, the resulting beer has a very odd taste to it. I'm not sure if it was due to the fermentation or to something else I did wrong, so I plan to perform the experiment again in the future.