Get into O2 guys, if you're serious about nicer beer

[Moad]

I was thinking today the thread title needed to be changed, thanks to whoever fixed it up!

I will volunteer to run side by side but won't be able to do it for a month or so

 

[Jack of all biers]

We have one volunteer. Everyone loves a volunteer. Good on you Moad. It's all good re a month or so. We're not getting paid for this and we all have commitments. My results may not be fully known for some months (re any oxidation) unless of course it happens quickly.

 

[Jack of all biers]

Correct. I'd be more tempted to add the H2O2 more like 10mins after adding the yeast, so it has a chance to wake up a little.

Will do.

Now in terms of your out-lined experiment, a few points spring to mind.
1) Good choice of recipe. (Unlike Brulosophy,) it's seems smart to use a simple recipe that isn't hopped to the hilt, to allow any flaws to be easily apparent.

My thoughts also. Keep it simple and less to influences on the end result.

2) Use a liquid yeast or yeast cake. I'd agree with Galbrew's comment above, it's suggested/recommended (by the manufacturers) that dry yeast is prepared in a way that it doesn't need oxygenation, and in fact oxygenation is likely to impede the yeast.

Yep. Already posted I'd use 2 liquid yeast packs.

3) i'd be tempted to use a lager yeast, as a simple lager is likely to show flaws more readily. However, it also means more other stuff could go wrong, so maybe the first comparison should be kept simple as an ale. Either would be fine, really.

Yeah, I really wanted to keep it as simple as possible with less variables. I see doing a lager comparison maybe more appropriate with an O2 comparison. That way it is comparing apples with apples, as it can't be said that the comparative sample didn't have enough dissolved oxygen from shaking.

4) Why limit the estimated addition of O2 to 10 or 11ppm? I believe (from memory) the saturation limit is 12ppm, so why not take it to that? (so basically i'd take it to 11-12mL of your 6% solution.

Purely because a 100ml bottle will make 10 brews. Given it's an Ale, maybe I should go for less to make it a fair comparison to the purely aerated batch, that might if I'm lucky get to 6ppm. But then if I push the H2O2 higher, there is more chance of things going wrong with the yeast or oxidation of wort. So with the 11ppm O2 conversion level, I will more likely detect any faults easier. So my logic is that if I can get away with a value higher than the ideal 8ppm for Ale, and don't taste any faults, then we will know we can scale it back for the different needs of top or bottom fermenting yeasts. Make sense?

Everything else seemed fine, i think.

Not sure what the best choice of yeast would be. I'm thinking the options would seem to be either a simple neutral finish, like WLP-001 or 1056; or go for one that has more character (to show the yeast performance more?) and possibly is a little harder to get to attenuation, such as the English yeasts like 1469/1187/1028/1275/wlp023/wlp005. One other that springs to mind that might suit the recipe well is a kolsch like WLP-029. Sorry, my breadth & depth of knowledge on yeasts is limited

After a bit of thought, I'm thinking Wyeast 1469-West Yorkshire. Not used it, but purely because of the description of creating malty balanced beers and is highly flocculent. I may also go with Maris Otter as I can get it cheaper than the Vienna.

 

[peteru]

The last couple of trials I have done were all with reasonably simple SMaSH beers. Gladfield American Ale + US Centennial and Simpsons Maris Otter + US Columbus.

The one thing that I have been keeping constant is the yeast. It's always US-05 that has been harvested from a yeast cake back in June 2016. I have two 1L bottles of rinsed yeast. Each time I do a batch I pick one of the bottles (alternating between them), pour off the clear(ish) liquid from the top then add the white healthy yeast layer to some starter wort (saved from whatever I'm about to ferment) on the stirplate. I then add water to the remainder of the yeast and trub in the storage bottle, agitate well and sit it in the fridge for about an hour or two. This lets the trub settle and I pour the remaining yeast in suspension into the stirplate vessel. I then top up with more wort. I usually run the stirplate for around 10-20 hours, then turn it off and let it settle until I can see a distinct layer of trub vs suspended yeast (sometimes I also get a clear layer on top). If it looks like I have a healthy dense starter, I'll pitch most of the yeast, but save some in the bottom of the starter flask to step up and store in the fridge for future use. If the starter looks a bit thin, I'll fridge it so that I can separate the "beer", trub and yeast layers and then step up the yeast layer with another stirplate session. I no-chill/cube all my wort (because I hate using and cleaning the Grainfather counterflow chiller), which gives me plenty of flexibility as to when to pitch the yeast.

Totally unscientific, but it actually works fairly well in practice. One of these days I'll have a hop and yeast freezer for long term storage of yeast that has gone through fewer generations, but so far what I have has exhibited no flavour degradation or drift. I guess that as a result of this yeast pitching practice, I probably overpitch, or at least start with a fairly good and active yeast population. I fully expect that the addition of H2O2 could cause some potential yeast cell death, but I also suspect that the catalase reaction will work in favour of the yeast and wort, causing the H2O2 to split into O2 + H2O fairly rapidly, thus increasing the DO and effectively getting me to the same point that adding O2 directly to the wort would get me.

I find it strange that people are questioning the oxidative reactions in wort when using H2O2, but not O2. Surely if O2 in the wort is going to cause issues, it doesn't really matter if it got there by bubbles from an airstone or hitched the ride on a molecule of water. Sure, the chemical reactions can be complex (the chemistry force was never strong with this one, and biochemistry is just black magic), but the proof is in the beverage served at the end of the process. And from that point of view, adding 10-12mL of 6% hydrogen peroxide to a 22-25L batch of beer has caused no harm. Now I just need to convince myself that it provides a benefit. It could just be that I'm doing everything else right and the hydrogen peroxide is a placebo! :unsure:

Or it could be that I've actually done the ground work on an alternative oxygenation method for home brewing purposes, in which case I call the naming rights.

P.S. - Although I am a scientist (I do have a B.Sc.), I am also a pragmatic. My approach is that rather than dismiss an idea because there is no literature to confirm that it should work, I'd rather figure out why something appears to work. Sadly, my scientific background is in neither, chemistry, biochemistry nor biology. But hey, if my tinkering can narrow the search space so that the experts can look deeper at the relevant factors, then I'm happy to be a part of the discovery process.

And the beer is good. :chug:

 

[manticle]

We have one volunteer. Everyone loves a volunteer. Good on you Moad. It's all good re a month or so. We're not getting paid for this and we all have commitments. My results may not be fully known for some months (re any oxidation) unless of course it happens quickly.

I'd be willing but don't yet have an oxygenation setup and I really feel measuring DO levels of the same worts using nothing, shaking, h202 and 02 injection is required to get a good indication.

Then blind triangle tasting.

Not sure how seriously expensive DO meters are but maybe if smurto were encouraged to be involved somehow we could get some quantitative analysis.

 

[technobabble66]

...I find it strange that people are questioning the oxidative reactions in wort when using H2O2, but not O2. Surely if O2 in the wort is going to cause issues, it doesn't really matter if it got there by bubbles from an airstone or hitched the ride on a molecule of water....

OK, i can chime in here for the nay-sayers. There is a difference in the reactivity between O2 gas and H2O2 delivered oxygen.
The issue is not the O2 itself so much as the reaction of H2O2 --> O• + H2O.
That single oxygen atom is more highly reactive than O2 and cause oxidative reactions quite easily, until in binds to another O• to form O2 gas. This should happen very rapidly, but the tiny fraction of time in between involves a more oxidative state. O2 gas is also highly reactive, but not quite to the same extent as the single oxygen radical.
THIS is why i'd argue that the yeast needs to be added to the wort before the H2O2, because i'd be fairly confident in assuming catalase works by decomposing 2 of the the H2O2's together to allow the smooth transition to 2x H2O molecules and an O2 molecule.
The reason i'd assume this is that if it didn't, there'd be little point in having the catalase in the first place, in fact it'd be quite dangerous for the microorganism as it's suddenly going to be creating a truckload of O• radicals around itself if the catalase simply decomposes a single peroxide by itself (does that make sense?). Furthermore, the creation of O•'s near the catalase enzyme would likely cause the rapid destruction of the catalase. Again, a fairly pointless strategy having an enzyme that destroys itself (in a random manner - given the unpredictability of what the radical would attack). This is obviously an assumption currently on my part as i haven't found a paper explaining the exact reaction kinetics of catalase yet. However, i'm fairly happy with the reasoning behind it: evolution generally doesn't tolerate stupid :lol:

I'm also happy to acknowledge this downside of the peroxide method - that the O• radical could damage both the wort and the yeast (and agree with peteru's comment about it above). However, i'm also happy to think there's a very good chance the damage is negligible if the catalase is allowed to quickly decompose the H2O2 and there is only a tiny amount of H2O2 being used in the first place. Both of which are the case here.

Again, i'm happy with the sciencey stuff, it now really needs to have empirical evidence to indicate the veracity of the process. Namely, the production of clean and fully fermented/attenuated beer, preferably with yeast that can hold true in multiple generations without mutations.
-----------------------------

On a slightly separate note, peteru, that process of your's above sounds totally fine to me. Generally, I think if you're getting successful, clean fermentations from many generations of a yeast that holds true to characteristics of the strain, then you're doing it right.
-----------------------------

FWIW due to the ease of it, another bonus of the peroxide method is i generally try to do a 2nd addition of H2O2 ~8-12hrs after pitching (~1/2 to 2/3 the initial volume). Takes ~60secs, including time to go to the fridge to get the bottle: Whip off the glad wrap, quick squirt, 5 second stir, back on with the glad wrap. Job done.

 

[Killer Brew]

Absolutely. Any volunteers? Gold cross H2O2 only costs about $4. Or alternatively, anyone know someone willing to lend an O2 setup for experiments?

I'm in adelaide and have an O2 set up. Happy to get involved.

 

[GalBrew]

On the topic of catalase, there is no need to guess at the stoichiometry of its reactions, it's pretty well documented. Just google free radical detoxification pathways and it will take the guesswork out. I'm still not convinced by any measure that yeast based catalase which is located intracellularly will get much of chance to do much before oxygen radical react with the wort and or the cell membrane of the yeast themselves.

 

[technobabble66]

The papers I've read indicate the catalase is released extracellularly by the yeast to neutralize it's surroundings.

 

[GalBrew]

The papers I've read indicate the catalase is released extracellularly by the yeast to neutralize it's surroundings.

But will it neutralise oxygen radicals before it oxidises wort components?

 

[technobabble66]

From the internets:
"Catalase breaks down and destroys hydrogen peroxide in two steps. The first step involves the catalase removing and binding one oxygen atom and releasing the rest of the hydrogen peroxide molecule as water. The second step is the catalase breaking down another hydrogen peroxide molecule by releasing oxygen gas and water."
So yes, it breaks down the H2O2 in a way that prevents the O• radical from interacting with other components of the wort or yeast.

The million dollar question actually is whether the H2O2 reacts *significantly* with components of the wort to oxidise them *while waiting* to be decomposed by the catalase.
The couple of studies i've seen indicate most H2O2 should be mainly neutralised within 20-30mins, and completed within 1 hr. So it's a small window for anything detrimental to occur.
However, the window is definitely there and it seems unknown as to whether anything significant would occur between the 0.0005L of H2O2 and the ~20L of wort. Given the time frame and the tiny quantity, i'm inclined to assume it'll be minimal. Real testing and comparisons are needed to go from here.

 

[Jack of all biers]

The million dollar question actually is whether the H2O2 reacts *significantly* with components of the wort to oxidise them *while waiting* to be decomposed by the catalase.
The couple of studies i've seen indicate most H2O2 should be mainly neutralised within 20-30mins, and completed within 1 hr. So it's a small window for anything detrimental to occur.
However, the window is definitely there and it seems unknown as to whether anything significant would occur between the 0.0005L of H2O2 and the ~20L of wort. Given the time frame and the tiny quantity, i'm inclined to assume it'll be minimal. Real testing and comparisons are needed to go from here.

And it is for this reason I have included the painful process (for me) of only testing a bottle of each every two weeks. Imagine if the H2O2 beer oxidises after 2 months! That's going to hurt when the majority of 25L tastes like cardboard. But in the name of science, I'm willing to take that risk :drinks:

 

[Jack of all biers]

I'm in adelaide and have an O2 set up. Happy to get involved.

Hi Killer Brew. You able to do a comparison yourself between O2 and H2O2 or only willing to assist with supplying an O2 squirt to someone else's brew (ie me)?

 

[MHB]

Been doing a bit of reading up on peroxide and yeast - I wont be putting any into beer any time soon!

The enzyme Catalase is a part of the yeasts defence against naturally occurring peroxides that can harm the yeast, like any enzyme it has an optimum temperature (~37oC) and an optimum pH 6.8-7.5. Both of these conditions are well outside the brewing conditions where anyone using peroxide would be working, like any other enzyme it wont work very well outside its optimum range.
Some of the enzyme might be excreted into the environment (the wort) but most is found in either the cell membrane or in the cell mitochondria where it can defend vital cell functions.

As someone mentioned above, natural selection is a very important and harsh master, yeast (and most other cells) have evolved Catalase to defend themselves from harm done by naturally occurring peroxides. The amount of peroxide that yeast could be expected to encounter is in the range of 1-0.1ppb, call it 0.5ppb give or take. we are talking about adding 10ppm (10,000ppb) or so. there is no conceivable way yeast will be equipped to handle 20,000 times as much as it evolved to cope with.

There is a wealth of difference between peroxide and oxygen.
Similarities are always suspect, but lets see if this puts it in perspective; we want some Chloride in our wort lets say 50ppm is a good thing, if we added 50ppm of Chlorine in the form of bleach we wouldn't expect the same result, in fact we can be fairly sure the yeast would die.
We also know peroxide is a steriliser, it reacts with most any organic chemical like a free radical ion (think O-), the breakdown product of the reaction is water and Oxygen. The result of adding bleach to chalk will leave chloride ions in solution, like the reaction with peroxide it isn't the end product that scares me it the process that takes us there that does all the harm.

There at threads on avoiding HSA, reducing O2 in packaging... All of them have in common the benefits of reducing Oxidative harm to the beer, after a lot of study I think adding peroxide is the exact opposite and will have all the negative effects associated with Oxygen harm.

I notice some commentary on the response on other fora, where the "Experts" jumped in and rained on the parade - from all I can gather very sensibly.

Mark

 

[Dave70]

Sooo..
Did we settle on a 5 or 2 micron diffuser?

 

[MHB]

Nah you need a 3.5um stone - someone should start making them.
M

 

[technobabble66]

I believe 2 was meant to be fine. I assume the 5 you mention should've been 0.5 micron?

 

[drsmurto]

I've only very briefly skimmed the discussion on H2O2.

A few points from a scientist whose research area involves H2O2 and O2 in juice/wine; similar situation holds with wort/beer.

Firstly, the reason why O2 is used and not H2O2 is that O2 can't react with organic compounds. This is what is called 'spin forbidden' and is a very fundamental principle of chemistry relating to the configuration of the electrons in the atom. It first reacts with transition metal ions to produce the superoxide radical which can then react with organic compounds. Yeast can mop up the oxygen very rapidly and in a medium that contains only trace amounts of reactive transition metals in the required oxidation state, it will do so before much, if any damage is caused by oxygen via the superoxide radical. We have measured the speed in which yeast uses up all the oxygen in juice and it is very fast, in the order of minutes. We can also measure oxidation markers to see what chemical effects are being caused by O2 introduction and the result is negligible. Our oxygen sensors measure down to ppb levels of oxygen.

This is not the case with hydrogen peroxide, it is very capable of reacting with organic compounds or just about anything it comes into contact with. This is not a selective process, you can't expect yeast to protect the wort from H2O2 as it cannot prevent every single molecule of H2O2 from colliding with other molecules in the wort. Adding an incredibly powerful oxidiser to your wort is not a particularly well thought out method of adding O2.

In regards to double dropping as a method of introducing oxygen into wort after fermentation has commenced, this is not something I have seen data on. I have, however, seen published data in a winemaking setting during what is referred to as a pumpover. This is done when a red wine is actively fermenting and is done to wet the cap (floating grape skins/seeds) to increase extraction of colour/tannin etc. To get any decent amount of oxygen in, a range of in-line devices were required to effectively draw oxygen into the liquid as it was passing through the pump, think spargers/venturis or even just 'cracking the fitting'. The physical act of splashing wine back into the ferment is very ineffective as the liquid isn't picking up any oxygen and the splashing impact is mitigated by the layer of CO2 protecting the wine.

 

[technobabble66]

Thank you for your explanation, DrS. Namely in the way O2 gas is cleaved and then processed/mopped up by the yeast
I'd have to concede that O2 gas seems a (much) superior way to add oxygenation.

... We have measured the speed in which yeast uses up all the oxygen in juice and it is very fast, in the order of minutes. We can also measure oxidation markers to see what chemical effects are being caused by O2 introduction and the result is negligible. Our oxygen sensors measure down to ppb levels of oxygen.
...

Just out of interest, have you done these tests for H2O2 additions?
I'm simply curious as to how significant that oxidation of wort by the peroxide is, considering the small amount used (~0.5mL in ~21L) and the variety of other elements of potential oxidation in our homebrewing process.
Call me stubborn or persistent, but I'm merely wondering whether there could still be use of the peroxide as an option secondary to something better like O2 gas, given its simplicity of use & setup.
Also whether the oxidation consequences would be enough to be detectable.
Clearly the O2 gas is best/better practice, but I'd be keen to more conclusively know that the peroxide is not a viable ghetto option that'll still provide some benefits for minimal costs and flaws.

Edit: posted before adding half the text [emoji52]

Edit 2: if the only concern is oxidation of wort, could peroxide still be an easy option for oxygenation of starters, where the supernatant/liquid is decanted off and discarded?

 

[rude]

Might change my sanitiser as I use Hydrogen Peroxide

Usually hit the fermenter with it just before I pitch

 

[drsmurto]

Sulfite/sulfur dioxide is added to wine for 2 reasons. To protect the wine from microbial spoilage, which in combination with the low pH of wine provides protection (sulfite exists in 3 forms depending on the pH - sulfur dioxide, bisulfite, sulfite). The other reason is it is an anti-oxidant. It is there to protect the wine from oxygen. The vast majority of wine is bottled with no yeast present at all so a defence against O2 is needed. As I mentioned previously, O2 get's converted to the superoxide radical via transition metals. This is converted to hydrogen peroxide. At this point, sulfite reacts with it to remove it from the wine. If there is no sulfite present, the superoxide is further reduced by another transition metal to an even more reactive molecule, the hydroxide radical. This, for example, reacts with ethanol to produce acetaldehyde. The last step in this reaction is known as the 'Fenton Reaction'.

So if you are adding hydrogen peroxide to wort you have skipped one reduction step, also known as the rate limiting step, and provided the system with a highly reactive molecule that otherwise would not exist in any substantial quantity if you had added oxygen. We don't measure H2O2 directly, we use the reduction in sulfite to measure the amount of hydrogen peroxide produced and the kinetics of the reaction. There is plenty of research in this area and it is ongoing.

I can't stress this enough. H2O2 is a very powerful oxidant, it will react with any molecule it comes in to contact with. I use it to 'digest' wine (it breaks apart the complex molecules and within hours, red wine looks like the palest white wine), clean glassware. In combination with concentrated sulfuric acid, it's used to etch glass. It is used to power rockets. It's one of those chemicals you treat with the utmost respect, particularly if you are using 30% or greater concentrations.

Thank you for your explanation, DrS. Namely in the way O2 gas is cleaved and then processed/mopped up by the yeast
I'd have to concede that O2 gas seems a (much) superior way to add oxygenation.

Just out of interest, have you done these tests for H2O2 additions?
I'm simply curious as to how significant that oxidation of wort by the peroxide is, considering the small amount used (~0.5mL in ~21L) and the variety of other elements of potential oxidation in our homebrewing process.
Call me stubborn or persistent, but I'm merely wondering whether there could still be use of the peroxide as an option secondary to something better like O2 gas, given its simplicity of use & setup.
Also whether the oxidation consequences would be enough to be detectable.
Clearly the O2 gas is best/better practice, but I'd be keen to more conclusively know that the peroxide is not a viable ghetto option that'll still provide some benefits for minimal costs and flaws.

Edit: posted before adding half the text [emoji52]

Edit 2: if the only concern is oxidation of wort, could peroxide still be an easy option for oxygenation of starters, where the supernatant/liquid is decanted off and discarded?

 

[nosco]

I'll get to the point of my extended question that may not have an answer. Does yeast use o2 more slowly at colder temps?

So if I have slowly chilled a beer to lager temps, say 4c, and then want re-use the yeast slurry for another ferment after racking off the beer. So as not to shock the yeast I keep it at 4c and re pitch it into some oxygenated 4c wort (is that necessary to pitch @4c?) and let it free rise to ferment temp. Will the o2 come out of solution before the yeast has woken up enough to use the o2? o2 later at a higher temp? Also considering that the o2 will dissolve (?) more easily in colder wort?

Edit: If its used in minutes as you say Dr S is it still the case at lager temps. Free rising to ferment temp could take hours (in this heat at least).

 

[MHB]

nosco - there are a couple of interesting points in there.
Basically as a rule in chemistry and biology everything happen slower colder and faster warmer, with the solubility of the O2 being the obvious exception.

I would be very tempted to add nutrients, including Oxygen, at or close to the temperature you are planning on brewing at. For lager this is generally going to be in the 8-12oC range. yeast hates sudden changes in temperature and everything we can do to keep yeast healthy and happy is a good option.
I would only oxygenate a starter in the presence of other nutrients (wort) and when the yeast is at or near pitching temperatures, same if pitching the yeast slurry directly into the wort.

You would be way better off lagering and storing yeast at 0oC (give or take one).

 

[nosco]

Thanks MHB. Good to know Im thinking along the right lines. Hopefully my brewing will follow suit :unsure: . My current practice is to pitch and then oxygenate. It makes sense to me but I have improved my o2 setup so I have a few options now.

Ill go to 4c to get the yeast and then 0c for the beer. I dont want to risk freezing the yeast.

 

[nosco]

So to recap on that question. Subtle changes (up or down) in temp are good for yeast but there is no real reason to pitch at 4c. Its maybe better to let the decanted yeast free rise to about 7c, pitch into 7c wort and then oxygenate. That's what Im going with.

Edit: Hopefully my fridge, equipment, kids, wife and lack of sleep will let me achieve that

 

[Killer Brew]

Hi Killer Brew. You able to do a comparison yourself between O2 and H2O2 or only willing to assist with supplying an O2 squirt to someone else's brew (ie me)?

Happy to help out with the O2. Might leave the experimentation to those who have paved the way.

 

[Jack of all biers]

Happy to help out with the O2. Might leave the experimentation to those who have paved the way.

Thanks. I will crack on with the experiment with the Ale as per my earlier post and see if the oxidation hits taste thresholds by the time it's all finished. If that experiment shows no discernible oxidation flavours, then and only then would I be keen to conduct an O2 H2O2 comparison. If that happens, I'll PM you to see if you are still keen to help out (maybe months from now).

DrSmurto clarified exactly what I suspected would be likely with the breakdown of H2O2 to H2O and O. Whilst, I know some oxidation would be likely, I am still keen to do this comparison to see just how much oxidation occurs and how long it takes to show in the beer. Call it curiosity of a potential ghetto method or what ever you will, but it's going to cost me a few bucks and time doing something I enjoy.

EDIT - If the H2O2 beer gets oxidised quick then I can always use it as slug/snail bait in a bowl or two in the garden. They love it. Too DEATH. Haaaaa, haa, haaaa, haa.

2nd EDIT - I don't like slugs okay. Drowning them in beer seems like the most humane way to me. :drinks:

 

[Stouter]

Some great discussion going on here, good too see, sensational effort.
When just asked, I explained to the wife about how there are blokes on his forum that are scientists, leaders of various industries, and philosophers discussing the merits of putting air in beer. Makes little old, average joe me look pretty special oxygenating my wort hey!

 

[Lionman]

Just ordered a reg and stone off eBay. Will oxygenate my next batch with Tradeflame O2 from Bunnings.

 

[sp0rk]

Finally biting the bullet and enquiring about a Coregas O2 cylinder, if my local supplier doesn't sting me too much I'll try and pick the gear up this week

*edit*
Nope, they don't do Coregas swap and go like Bunnings does and I'm not paying rental
Looks like I'll get a Brewman setup