# Oxygenation experiments with hydrogen peroxide



## Adr_0 (19/5/17)

Two guys walk into a bar. The first says "I'll have a great big glass of H2O" - he drinks it and walks away. The second guy says "I'll have a great big glass of H2O, too"... but we don't know what happened to him.

Like a few other people, I have been curious to see if H2O2 is a possible alternative to using bottled oxygen or shaking. The cost and simplicity is appealing, but there is possibly a negative impact on yeast health (counter to the point of oxygenation) and flavour/aroma effects. 

There have been some great experiments in the O2 thread, and like these guys I wanted to see for myself rather than endlessly hypothesise. So I started a quick experiment yesterday.

A few points on the beer and common points:
- Wort was base, some German caramels, all Challenger to 1040 and 35-40IBU's. SG was a bit down due to a doughball issue.
- Yeast was frozen 1275, which had been slowly unfrozen and 'revived' with 500mL of weak starter. If ever there was a bunch of cells that needed O2 I would say this would be appropriate.
- The slurry was then added to a 1L starter, mixed for about 15min (by hand) and split in the following manner:
> 500mL onto stirplate into 12L of wort for control batch
> 150mL into 3 x glasses for 3 x tests into 2L test batches
> Yeast in the 3 x test batches was static, expecting oxygenation from H2O2 vs the control which was on a stirplate

I framed the experiment around the following:
- Control, being batch with yeast on stirplate for ~6hrs, hopefully showing oxygenation but not significant growth
- 1, being 150mL yeast starter with 4mL of 1.2% H2O2(12% diluted as 20g made up to 200g with distilled water) straight into the yeast
- 2, being 4mL of 1.2% H2O2 added to the wort, adding 150mL yeast starter 40min later
- 3, being 150mL yeast starter added to wort, 20min later adding 4mL 1.2% H2O2 solution

I used Lyrebird_Cycles' method detailed here

The 4mL of 1.2% was based on approximately 12ppm O2 target in 2L of wort, but also diluted to aid mixing. Test batch 1, given it was added to 150mL of starter, copped 160ppm H2O2 which would either make or break it....

I wanted to evaluate H2O2 impact in four ways:
- Dissolved oxygen measurements
- Visual indicators, largely krausen or flocculation
- Gravity measurements
- Sensory (aroma/flavour) indicators

A 3200ppm methylene blue solution was made up (3.2g diluted up to 1kg with distilled water), which was then diluted down to 1600ppm with 100g made up to 200g, then 160ppm with 20g made up to 200g – which should represent 16ppm O2. This was then serially diluted into 12ppm O2, 8ppm O2, 4, 2, 1ppm O2 standards:




A few introductory pics:
Test containers for each test wort/yeast/H2O2


Test jars for DO samples:


Yeast splits:



Unfortunately my beer was yellowy red (dickhead!) so I should had added some yellow food colouring to make the standards a little more representative. Anyway.
Measurements of the base wort for each one were taken:
- Control was 0-1pppm
- Batches 1, 2, 3 were all 1-2ppm which is likely from transfer to each of the test containers. The below pic shows a sample with no methylene blue indicator (left) vs indicator in, and the 1ppm standard in the background. All three tests were similarly, likely 1-2ppm.



DO measurements were taken every 20min, with events – yeast or H2O2 additions – shown on the graph. In each, a strong methylene blue solution was added – approx. 0.5mL – and lightly swirled, with resulting colour assessed. Colour indication was a little hard, so I also made up a ‘saturated’ sample with 12% H2O2 dumped in, and a ‘reduced’ sample with ascorbic acid dumped in. I also had a ‘no indicator’ vial to illustrate the difference.

Measurements are below:…



A few example pics. Test 1, comparing 20min after pitching H2O2'd yeast starter into the wort vs the reduced sample (left) and the saturated sample (right). I'd say 1ppm or less - basically no blue/green there at all:




Test 2, 40min after adding the H2O2, just before yeast pitch, as compared to the reduced sample (left) and saturated sample (right). I'd say 12-16ppm:



Test 3, 20min after adding H2O2 where the yeast was already in suspension. Left one is 35min after adding H2O2 with yeast, while right is only 20min after. Possibly 4ppm on the right vs <2ppm on the left?




Gravity readings I will update over the coming days, but appear to be similar: essentially all three have gone from 1040 to 1032 in 12hrs, with batch three potentially only at 1033.

Sources of error:
- Sample collection was actually pretty good. My LODO method, post boil, seems to not pick up any O2 and this held on for 60min to not indicate any blue/green tint when compared with the reduced sample:


- Colour indication was a big one. For some stupid reason I decided to make a tasty, malty ale rather than a wit bier. A control for this would have been adding some yellow food colouring to the standards.
- DO measurement would have been very accurate with a DO meter. I could have potentially hired one, but I wasn’t able to arrange one unfortunately.
- Gravity readings are being taken with a refractometer. Light source is consistent (only during the day) but there may be a 0.1% Brix error.
- Mixing in the samples. I did do a decent rock back and forth and try to wait 20min or so for mixing to occur, but it is quite possible that there was not adequate mixing.

Conclusions – so far:
- It seems like adding H2O2 does increase the O2 content. There is an evident spectrum of blue from a completely reduced solution to a saturated solution.
- Yeast does indeed reduce the O2 as every mark past 40min was lower in O2.
- There did not seem to be a visual effect of adding H2O2 directly to the yeast.
- A 160ppm dose to the 150mL starter didn’t seem to kill the yeast – given gravity has dropped in line with the other three batches.
Next points:
- Gravity over the next few days may reveal something
- Sensory tests will be a really big test. Batch 2 had H2O2 added and floating around for 40min before yeast was added and managed to get up to 12-16ppm, so this may taste different to other batches. Inversely batch 1 didn’t really get above 1-2ppm IN THE WORT at any stage, though it is presumed the starter was quite high.


----------



## Lyrebird_Cycles (19/5/17)

I possibly forgot to include this in the original discussion but the way to make the standards independent of wort / beer colour is relatively simple:

Firstly, make up all the standards in distilled water, not wort or beer.

When testing a wort or beer, make up an extra tube of clean wort / beer with no methylene blue addition.

To do the comparison, place this side by side with the sample being tested in front of the light source and place one of the standards behind the no addition tube.

Look through the test sample to the light source. Look through the clean sample plus standard to the light source.

Compare


----------



## Adr_0 (19/5/17)

D'oh! That would have been a good idea. I had varying degrees of break in the samples - primarily the early ones - so there are a few sources of error there.

I'm hoping that the sensory part of the experiment yields some results - hopefully I can get the samples through quickly and into bottles without too many dramas.


----------



## Adr_0 (19/5/17)

So the second round of gravity readings are in:

The control - stirplate with no H2O2 is leading, a few points ahead of the next player. Krausen is starting to form on this one. Krausen is also on #3, where yeast was pitched then H2O2.



1 and 2 - where H2O2 was straight into the starter, or the wort was pre-H2O2'd - don't have krausen yet.

Original gravity 1040
Gravity at +19.5hrs
Control - 1028
1 - 1030
2 - 1031
3 - 1031

Looking forward to the next few days. I've avoided the sensory evaluation so far and will start a few days after terminal gravity.


----------



## Jack of all biers (19/5/17)

Good job Adro. 

I love the fact that the stir plate control is similar (6hrs instead of 4hrs) to the vitality method Mardoo has mentioned in the O2 thread. Agree that the DO measuring instrument will be a better indicator, but you have taken it to the step to prove that in wort conditions O2 is converted and used up by the yeast.

I strongly suspect (not yet fully backed up by my sensory tests) that oxidation with the wort is unavoidable with H2O2, but great experiment. Not wishing to hijack your thread, but I may post my final results here in a few months time, when my oxidation results (for what they're worth) are in.

Cheers for taking it to the level you have.


----------



## Adr_0 (20/5/17)

I'm leaning towards #2 - where H2O2 was added to the wort before the yeast - ending up the worst in sensory indicators. I wonder if #1 will preserve the most malt flavours but have some yeast stress character, and if #3 will be the best balance.

If anything comes up better than the control, I'll be happy.

Next lot of gravities. All have krausen up now, with the Control having dropped a little krausen already. Samples are at 37hrs.
Control - 1.026
#1 - 1.027
#2 - 1.030
#3 - 1.029



Looks like #2 is performing the worst out of everything. I wonder if the H2O2 just oxidised the malt/hops and didn't decompose into O2? This is the only one of the tests where yeast catalase wasn't present to help the decomposition to O2.


----------



## Adr_0 (21/5/17)

Next lot of gravities/attenuation:





So the control is comfortably ahead! Gravity/attenuation isn't everything, but it's still going to take a very clean and flavoursome beer from either 1, 2 or 3 to convince me that these offer a better alternative to the stirplate used in the Control.

EDIT: for those wondering about slightly different shapes, I added another decimal of precision in Beersmith which has shifted a couple up or down a bit.


----------



## Benn (21/5/17)

http://m.ebay.com.au/itm/Oxygenating-Tablets-Provides-Oxygen-in-Aquariums-amp-Fish-Transport-Bags-200g-/191737999412

It's Fish safe...


----------



## MHB (21/5/17)

Pass
I'm not interested in putting Barium Peroxide into my beer.
Mark


----------



## SBOB (21/5/17)

MHB said:


> Pass
> I'm not interested in putting Barium Peroxide into my beer.
> Mark


Come on.. why not?
The following is just an extract from an MSDS



> Extremely hazardous in case of ingestion. Very hazardous in case of skin contact (irritant). Hazardous in case of eye contact (irritant), of inhalation. Slightly hazardous in case of skin contact (corrosive, permeator). Prolonged exposure may result in skin burns and ulcerations. Over-exposure by inhalation may cause respiratory irritation. Severe over-exposure can result in death.


----------



## peteru (21/5/17)

Barium enema and an iodine injection to go with your computed tomography scan?


----------



## Adr_0 (22/5/17)

Well, #2 has died in the ass and #3 isn't looking too much better. Keeping in mind the starting DO of the beer was <2ppm as I had vigorously boiled and then went straight into cubes, then transferred under CO2 - no shaking or pouring from a great height - so there was minimal DO to start with. It does appear pretty evident that throwing H2O2 into a wort environment just throws O-'s around which will oxidise the wort rather than decomposing to O2. Even #3 where there was yeast/catalase available didn't really get enough decomposition into O2 - though there was possibly a bit.

Very interestingly, throwing diluted H2O2 into the starter seemed to be a different story. I'm using a refractometer to measure gravities and you could argue that #1 and the Control are very close - but #1 is still a touch behind. Perhaps multiple trials or multiple parallels may produce different results. But I'm pleased I tried this method, with a much higher proportion of yeast/catalase to wort giving the highest chance of H2O2 decomposing to O2.

I also did a bit of a sensory test with a blocked nose on the Control and #1. I've been fermenting 1275 at 16°C to hopefully promote the lovely cherry and pear, but also potentially dribble a bit of diacetyl in. Cold, I couldn't pick a difference. I had to warm up and shake samples to pick a difference in diacetyl, with #1 possibly showing a touch more diacetyl. It's bloody close though. Hops, sweetness, pear and cherry are basically identical though. I will try again tonight with these two - bigger samples, and see if the wife can pick a difference.

In terms of the other two, I'll wait until bottling to see what they're like. I may just discard #3 but I'm curious to see how oxidised #2 is.

Results below:


----------



## Adr_0 (23/5/17)

Next lot of results. I had bumped up the temperature from 16 to 18°C around 24hrs ago, so there has been a little bit more of a 'kick' across the samples.





Control is now at 1011.2 vs #1 at 1013.6, so a couple of points clear. #2 and #3 have both done their hamstring so are out of this race.

In terms of sensory evaluation, the Control is actually coming across more harshly including a more harsh bitterness, while #1 is quite clean. The expected FG is 1010 though, so with 3-4 points to go #1 will still get drier and bitterness will come forward more. The control could actually be picking up more trub in the sample as the tap is lower than it is in #1, but there are certainly no complaints with the #1 sample which is interesting.


----------



## MHB (23/5/17)

Following this with interest, nice to see someone keeping records and testing an hypothesis.
Mark


----------



## Jack of all biers (23/5/17)

Adr_0 said:


> Control is now at 1011.2 vs #1 at 1013.6, so a couple of points clear. #2 and #3 have both done their hamstring so are out of this race.
> 
> In terms of sensory evaluation, the Control is actually coming across more harshly including a more harsh bitterness, while #1 is quite clean. The expected FG is 1010 though, so with 3-4 points to go #1 will still get drier and bitterness will come forward more. The control could actually be picking up more trub in the sample as the tap is lower than it is in #1, but there are certainly no complaints with the #1 sample which is interesting.


It's interesting that in my test, the 'control' batch (aerated as opposed to your vitality starter) also ended up ahead gravity wise, and didn't taste as good to start off with when compared to the H2O2 treated batch. I found the H2O2 batch had much stronger malt flavours and fruity esters as opposed to the aerated batch. It didn't come through to the packaged product though. 

Other than taste/aroma factors, what about the appearance of your batches. Are some more cloudy than others or any difference at all?


----------



## Adr_0 (24/5/17)

Jack of all biers said:


> It's interesting that in my test, the 'control' batch (aerated as opposed to your vitality starter) also ended up ahead gravity wise, and didn't taste as good to start off with when compared to the H2O2 treated batch. I found the H2O2 batch had much stronger malt flavours and fruity esters as opposed to the aerated batch. It didn't come through to the packaged product though.
> 
> Other than taste/aroma factors, what about the appearance of your batches. Are some more cloudy than others or any difference at all?


Doesn't seem to be much of a difference - except for #2 (H2O2 into wort 40min before yeast) where the yeast has already conked out!
(C 1 2 3)



I might give #2 and #3 a little swirl. This was frozen yeast pitched into wort that evidently contained very little oxygen, so represents a good example of yeast struggling the whole way down. Probably what happens with a lot of kits sprinkling a single pack of yeast on top of the wort.

Flavour wise, the control is actually down to 1007.5 which is a lot lower than I thought it would get. After a quick decant of the trub and hop particles, it tastes lovely, smooth and round so happy with that - 1275 for the win. #1 tastes extremely close, with the difference being more over the difference in gravity rather than a massive change in yeast health (for better or worse).


----------



## MHB (25/5/17)

So if the results mean anything, there would be no good time to add peroxide to a brew.
Hopefully you will get an Oxy kit and repeat the experiment with oxygenated wort, against an unaerated control.

Like I said before, its good to see someone documenting the results of an experiment, and put them up so others can see what happens
Appreciated Mark


----------



## Ducatiboy stu (25/5/17)

I might just keep to a BOSS to oxygenate my wort


----------



## mr_wibble (26/5/17)

MHB said:


> So if the results mean anything, there would be no good time to add peroxide to a brew.
> Hopefully you will get an Oxy kit and repeat the experiment with oxygenated wort, against an unaerated control.
> 
> Like I said before, its good to see someone documenting the results of an experiment, and put them up so others can see what happens
> Appreciated Mark


Here here!

And if you do test pure O2 in this manner, please run a "Waaay too O2" sample.


----------



## MHB (26/5/17)

Good call
Mark


----------



## Adr_0 (26/5/17)

Numbers from yesterday:






#1 is basically 24hrs behind the Control.



MHB said:


> So if the results mean anything, there would be no good time to add peroxide to a brew.
> Hopefully you will get an Oxy kit and repeat the experiment with oxygenated wort, against an unaerated control.
> 
> Like I said before, its good to see someone documenting the results of an experiment, and put them up so others can see what happens
> Appreciated Mark


I think one of the interesting comparisons is #2 vs #1, where #1 shows a distinctly better attenuation profile - why is this? The difference was likely down to the absence or presence of yeast catalase which has been shown to act as a catalyst in the 2H2O2 -> 2H2O + O2 reaction.

The actual dosage into #1 was 320ppm H2O2, or 0.032% in the 150mL starter. I said 160ppm but this was really the maximum potential O2, and it didn't necessarily get there. I gave it 40min in an unmixed situation to break down everything into O2, but a few web references (e.g. this one here, where the lowest was 0.09% or about 3x stronger) point to low concentrations like this taking potentially a number of hours to fully break down. I'm not picking up any yeast health issues at this stage from getting whacked with 320ppm H2O2 either, even though yeast is apparently only equipped to deal with ppb levels....... we will see in future tasting though. I will get a few of the CQ guys to have a taste and will probably throw a few around the Brisbane swap to get some feedback.

I'm leaning towards another experiment comparing stirplate and stirplate + H2O2 in a split lager batch. I would hope that stirring and 4-6hrs would be enough time to decompose the H2O2 to O2, with any excess just leaving as O2 gas. This for me would be the real test as I want something simple to help push lagers along rather than having a wand, stone, O2 regulator and bottle getting used for 5min once a month and being unused in the shed for the rest of the time.

It would be good to see an experiment with gaseous O2 into the wort before pitching to compare wort freshness vs H2O2 on a stirplate, as well as the attenuation comparison. Although H2O2 does have a higher oxidation potential than gaseous O2, I wonder how much wort freshness is being lost with oxygenation prior to pitching?


----------



## MHB (26/5/17)

Try to take this the way its intended, I have said I admire the dedication and work you have done.
Where it breaks down for me is in your interpretation of the results.
When I look at the results as reported my first observation is that in every case where peroxide was added the ferment was slower or even less complete. Clearly #3&4 aren't encouraging, so on the evidence adding H2O2 to the wort isn't a good idea.
Which leaves adding H2O2 to the Starter - comparing control and #1

Looking at the results of C and #1 and your reporting that #1 smells/tastes OK, the attenuation profile looks to me exactly like you would expect to get with a Higher (C) and lower (#1) pitch rate of very similar yeast.
The obvious conclusion being that adding H2O2 to the starter reduced the amount of or vitality of the yeast. Another day or two should tell the story, but I suspect that C and #1 will be fairly similar by the time the smaller yeast population in #1 is finished. Sorry to say this to indicates that there was no benefit from adding H2O2 to the starter - In fact in terms of fermentation processes there is really not much difference between a starter and a ferment - except that the intention in one is to increase the yeast population and vigor, in the other to use the yeast to make alcohol. The metabolic pathways and nutrient demands are the same.

If you want to persist in trying to make Peroxide breakdown into O2 try adding a bit of Silver to the starter (a pinkie ring, ear ring, silver chain, charm... anything that you can pinch from a jewellery box) Silver will break down the peroxide very quickly, silver and its salts are toxic to yeast so I would pull it out before adding the yeast. Just don't add too much peroxide to a closed container with silver, the reaction is very fast and quite exothermic (can get very hot), I was discussing this with a mate (better chemist than me) and we coined the term PeroxieBomb.
I haven gathered up some parts to make a small reactor to convert H2O2 to O2 and deliver it to the wort, its basically an Oxy kit without bottled oxygen, before anyone gets too excited, the upfront cost is less, the cost of peroxide/O2 gas is way higher (TANSTAAFL) but that's life.

When your at the club, ask around I'm sure someone would be willing to lend you an O2 kit, I strongly suspect that there are enough people here interested in the results that people would be more than happy to help. I also think that using O2 will really make a significant difference.

Good luck and I am looking forward to your further results.
Mark


----------



## technobabble66 (26/5/17)

Definitely fantastic to see a well documented comparison. Great job Adr_0
Starting to get interesting with the results!

I'd agree the early numbers seem to indicate the H2O2 idea is not a good one in reality.

However, we need to let it play out to completion and see the *final* results, namely the FG and the sensory perceptions of quality.
And then, ideally, do the same again but with the Control & #1, plus O2 into a starter, and O2 into the FV. It'd complete the full comparison nicely, and confirm the benefits (or lack thereof) of both approaches.

Thanks again to Adr_0 !!


EDIT: as an aside, i'm curious as to why the fermentation curves are all "wobbly" like a flat sine curve, etc? Simply from minor temp fluctuations, perhaps?


----------



## Adr_0 (26/5/17)

MHB said:


> Try to take this the way its intended, I have said I admire the dedication and work you have done.
> Where it breaks down for me is in your interpretation of the results.
> When I look at the results as reported my first observation is that in every case where peroxide was added the ferment was slower or even less complete. Clearly #3&4 aren't encouraging, so on the evidence adding H2O2 to the wort isn't a good idea.
> Which leaves adding H2O2 to the Starter - comparing control and #1
> ...


Looking at the results, yes my method of a quick spin on the stir plate is the best performer. And I agree that the #1 sample had yeast of either less quantity or less vitality.

I guess the million dollar question is:
-was cell count reduced by adding 320ppm worth of H2O2; or
-was not enough O2 produced, meaning the yeast did not have enough vitality?

If the first one is true, perhaps less -
or no - peroxide should have been added. If the second is true, I should have waited longer before pitching. 

I'm not sure that you or I can answer this, and though I've had a quick look through some papers I certainly can't say confidently one way or the other, though I am leaning towards not enough time to produce the O2 - based on how slow O2 production is at low total concentrations. 

What do you you think about #2, where the peroxide was in suspension for 40min before yeast? Do you think the peroxide reduced the yeast count, or was mostly used up oxidising the wort leaving very little to produce O2 once the yeast was added? 




technobabble66 said:


> Definitely fantastic to see a well documented comparison. Great job Adr_0
> Starting to get interesting with the results!
> 
> I'd agree the early numbers seem to indicate the H2O2 idea is not a good one in reality.
> ...


The wobbles would be temperature fluctuations due to the low volumes (2.5L) vs the main 12L batch.


----------



## MHB (26/5/17)

Adr_0 said:


> Snip
> What do you you think about #2, where the peroxide was in suspension for 40min before yeast? Do you think the peroxide reduced the yeast count, or was mostly used up oxidising the wort leaving very little to produce O2 once the yeast was added?


I suspect its moot - either way no gain in fact a loss of ferment vigor, I mean I suspect you could spend a lot of time working out when adding H2O2 does the least harm, but what's the point?
Mark


----------



## tavas (26/5/17)

Hi Adr

Sorry if I missed it, but what grade of hydrogen peroxide did you use? Do you know what stabilising agents were in it?


----------



## peteru (26/5/17)

If it was Goldcross H2O2 then the main stabiliser is phosphoric acid with traces of phenacetin and it is pharmaceutical grade.


----------



## Jack of all biers (27/5/17)

MHB said:


> ...
> When I look at the results as reported my first observation is that in every case where peroxide was added the ferment was slower or even less complete. Clearly #3&4 aren't encouraging, so on the evidence adding H2O2 to the wort isn't a good idea.
> Which leaves adding H2O2 to the Starter - comparing control and #1
> ...
> Sorry to say this to indicates that there was no benefit from adding H2O2 to the starter


This was the conclusion I came to in my comparison brew also (although not as thorough an experiment as Adro's)



Adr_0 said:


> Looking at the results, yes my method of a quick spin on the stir plate is the best performer. And I agree that the #1 sample had yeast of either less quantity or less vitality.
> I guess the million dollar question is:
> -was cell count reduced by adding 320ppm worth of H2O2; or
> -was not enough O2 produced, meaning the yeast did not have enough vitality?
> ...


I would think vitality is the key. I know with my H2O2 experiment there were obvious signs of yeast stress in; greater ester production, less flocculation and a greater amount of time to reach same attenuation. Hence why I came to the conclusion that there was no benefit of H2O2 for the fermentation side of things. My reasoning being that if H2O2 can't beat simple aeration then it is of little use to fermentation full stop. My results of the pure side by side comparison are very similar to your Control V #1. As MHB points out #2 & #3 are clear signs of yeast under strain. Now this maybe due to differing size of your test fermentations or amount of yeast per test, but the consistent sign is that the vitality of the yeast is degraded by the H2O2 addition. Whether the reason is that it is because of H2O2 effecting the yeast or because H2O2 has no effect, but produces too little O2 to improve vitality is mute as MHB points out. The fact that no obvious positive advantage is given is enough indication that H2O2 is of little value in a practical sense.



MHB said:


> I suspect its moot - either way no gain in fact a loss of ferment vigor, I mean I suspect you could spend a lot of time working out when adding H2O2 does the least harm, but what's the point?
> Mark





Adr_0 said:


> I'm leaning towards another experiment comparing stirplate and stirplate + H2O2 in a split lager batch. I would hope that stirring and 4-6hrs would be enough time to decompose the H2O2 to O2, with any excess just leaving as O2 gas. This for me would be the real test as I want something simple to help push lagers along rather than having a wand, stone, O2 regulator and bottle getting used for 5min once a month and being unused in the shed for the rest of the time.
> 
> It would be good to see an experiment with gaseous O2 into the wort before pitching to compare wort freshness vs H2O2 on a stirplate, as well as the attenuation comparison. Although H2O2 does have a higher oxidation potential than gaseous O2, I wonder how much wort freshness is being lost with oxygenation prior to pitching?


I think the stirplate v stirplate + H2O2 experiment is a fairer comparison and I would expect will result in a similar outcome to my aerated v pure H2O2 experiment (but am keenly awaiting you to prove my expectation wrong). Your comment about the wand, stone, O2 bottle etc was exactly why I gave H2O2 a go. I will be borrowing a bottle of O2 and regulator to hook up to my recent purchase of a wand and stone and be giving it a test on my next lager batch to see if there is great improvement (I anticipate due to the experience of others that it will be). I would be very interested in a comparison of the stirplate vitality starter method V Oxygenation, but that will have to wait until I can get my hands on a stirplate.

To look at your question of O2 effect on 'freshness' of the wort - I'll quote someone with experience in the area of the question and whilst I know you know (and most currently participating in this thread) (I did before I began my experiment, which is why a main part of my experiment is the long term sensory testing), but just a reminder of what DrSmurto posted back in Feb on the O2 thread for those looking at this in the future;


> DrSmurto, on 17 Feb 2017 - 11:04 AM, said:
> 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.





> DrSmurto, on 17 Feb 2017 - 1:55 PM, said:
> 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.


EDIT - Sorry the quote didn't transfer across as a quote, so I have quoted it!


----------



## Adr_0 (27/5/17)

Jack of all biers said:


> This was the conclusion I came to in my comparison brew also (although not as thorough an experiment as Adro's)
> 
> I would think vitality is the key. I know with my H2O2 experiment there were obvious signs of yeast stress in; greater ester production, less flocculation and a greater amount of time to reach same attenuation. Hence why I came to the conclusion that there was no benefit of H2O2 for the fermentation side of things. My reasoning being that if H2O2 can't beat simple aeration then it is of little use to fermentation full stop. My results of the pure side by side comparison are very similar to your Control V #1. As MHB points out #2 & #3 are clear signs of yeast under strain. Now this maybe due to differing size of your test fermentations or amount of yeast per test, but the consistent sign is that the vitality of the yeast is degraded by the H2O2 addition. Whether the reason is that it is because of H2O2 effecting the yeast or because H2O2 has no effect, but produces too little O2 to improve vitality is mute as MHB points out. The fact that no obvious positive advantage is given is enough indication that H2O2 is of little value in a practical sense.
> 
> ...


Firstly, I used 12% from Anpros diluted up to 10x weight which should be 1.2%. In 150mL starter this ended up as 320ppm or ~9-10mM. In 2.5L of wort this was 19ppm or 0.5mM.

Based on concentrations I would expect viability of #2 to be significantly higher than #1.

I can understand the cause of the poor performance in #2 being a moot point because it's rooted anyway, but understanding the reason for and how it can be compared to #1 (e.g. comparison of viability/viable cells, cell vitality) is of interest to me. Technically #1 should have copped the biggest hit to viability, given the high concentration - and the hit to viability should have been massive if it were in its exponential growth stage. Conversely #2 should have been insulated to a degree from the H2O2, assuming that it was too busy oxidising wort to challenge the yeast.

Given as well that #1 had no additional oxygenation while the control spent 6hrs on a stirplate, I'm leaning towards O2 having been produced at some point in #1 - otherwise it would have performed a lot worse.

So I'm keen to do a stirplate test, likely with lower concentration and at the end of the growth phase (in the starter). The pre-exposure to oxygen and being in the stationary phase should significantly reduce the lost viability, and the additional time on the stirplate (6hrs vs 40min in my first test) should give it the chance to produce oxygen.


Just a grab from above:
"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."

I'm curious about this, as I thought this was the basis for all oxidation of wort compounds and aeration water treatment. I would expect the reaction rates with gaseous O2 would be slower if the reactions are spin forbidden, but they will still happen. I can appreciate that gaseous O2 is preferred as it is _less_ reactive, but it's still reactive. I guess the phase of adding the O2 - prior to significant growth and fermentation - helps this significantly, but I'm curious about staling before yeast is added. I guess to my mind, yeast should be pitched then the wort oxygenated but I'm not sure that everyone does this.


----------



## Jack of all biers (27/5/17)

Adr_0 said:


> Firstly, I used 12% from Anpros diluted up to 10x weight which should be 1.2%. In 150mL starter this ended up as 320ppm or ~9-10mM. In 2.5L of wort this was 19ppm or 0.5mM.
> 
> Based on concentrations I would expect viability of #2 to be significantly higher than #1.


Please don't take my comments as a criticism. The problem with H2O2 is the randomness in which it will give up the O- and which molecules that O- react with. It could be that randomly the conditions of #2 meant that the O- reacted more with the wort and next time the experiment is repeated with the exact same circumstances instead of reacting with the wort molecules it reacts with other O- and creates O2. I guess this is the point DrSmurto was trying to point out.


Adr_0 said:


> Given as well that #1 had no additional oxygenation while the control spent 6hrs on a stirplate, I'm leaning towards O2 having been produced at some point in #1 - otherwise it would have performed a lot worse.


Absolutely, and was my observations too, but given the stirplate performed better (similar to my experiment only just) then the O2 take up from simple stirplate is better than the best H2O2 sample produces O2 for the yeast. Never said it doesn't work at all, just that it doesn't seem to create better conditions than aeration (given the stirplate is really only continuous aeration).



Adr_0 said:


> Just a grab from above:
> "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."
> 
> I'm curious about this, as I thought this was the basis for all oxidation of wort compounds and aeration water treatment. I would expect the reaction rates with gaseous O2 would be slower if the reactions are spin forbidden, but they will still happen. I can appreciate that gaseous O2 is preferred as it is _less_ reactive, but it's still reactive. I guess the phase of adding the O2 - prior to significant growth and fermentation - helps this significantly, but I'm curious about staling before yeast is added. I guess to my mind, yeast should be pitched then the wort oxygenated but I'm not sure that everyone does this.


My understanding of DrSmurto's quote is that O2 can react with metal ions (iron & copper for example), but won't with the organic compounds in wort. I've emboldened the step above. It is this step that then allows O- to later split off the metal molecules and either form H2O2 which then can react with the organic compounds (wort/beer) or just react with the organic compounds straight up. Thus creating the oxidisation flavours we like to avoid. What he is saying is that with low amounts of these metal ions plus the speed in which yeast consume the more stable O2 makes the likelihood of this random splitting of O2 into a superoxide radical lower and therefore less O- radicals are later produced. This is all very complicated chemistry, which is above my head, so if what I understand from DrSmurto's comments is wrong please feel free to correct me, but I have read similar elsewhere.

EDIT - this article mentions the above O2 to metal oxidation reactions, but only briefly, but it's all I can find that is relevant to brewing.
View attachment 03 - The role of oxygen.pdf


----------



## Adr_0 (27/5/17)

Jack of all biers said:


> Please don't take my comments as a criticism. The problem with H2O2 is the randomness in which it will give up the O- and which molecules that O- react with. It could be that randomly the conditions of #2 meant that the O- reacted more with the wort and next time the experiment is repeated with the exact same circumstances instead of reacting with the wort molecules it reacts with other O- and creates O2. I guess this is the point DrSmurto was trying to point out.
> 
> Absolutely, and was my observations too, but given the stirplate performed better (similar to my experiment only just) then the O2 take up from simple stirplate is better than the best H2O2 sample produces O2 for the yeast. Never said it doesn't work at all, just that it doesn't seem to create better conditions than aeration (given the stirplate is really only continuous aeration).
> 
> ...


Added comments to quote

I'm fine with the high oxidation potential of H2O2 in wort, which is why I'm continuously leaning towards lower concentration and dilution in a yeast-heavy environment rather than into wort. From what I've read as well this should be with the yeast in a stationary state. At low concentrations, with pre-exposure to oxygen and with the yeast in a stationary state the viability is maintained. A yeast-heavy environment such as a starter should be well equipped to handle the challenge of H2O2, and given enough time the yeast should readily take up O2.

We'll see anyway...


----------



## technobabble66 (28/5/17)

I'd agree with your line of reasoning, Adr_0, and would similarly wonder about the possible staling reactions from O2, compared to those of H2O2. However, one thought has occurred to me: the temperature could be a critical factor in the difference. HSA and the reactions involved are (now?) theorized to have a "sweet" spot somewhere ~60-70*C (from memory). Meaning above that O2 doesn't dissolve into the wort much, and below that the reaction rate is too slow. So basically the temp we're talking about when pitching yeast is too low for staling reactions from O2. Whereas H2O2 disassociates fairly well at pitching temps. It would still technically fall under the point you made earlier that O2-derived staling reactions may occur very slowly, but they'd still occur. However, it may help understand why the amount of staling reactions could be so dramatically different. Just a suggestion!
I'll maybe try to get around to going over the details of what DrS & JoaB posted sometime tomorrow. 
I'd definitely be looking at low dilution (ie: weak) additions to a high yeast solution. Also maybe consider multiple additions - eg: instead of 20ml in 1 hit, maybe 5ml first (yeast initiates catalase production) then a few more 5ml additions every 30-60mins thereafter. Just another suggestion! [emoji1]


----------



## Adr_0 (28/5/17)

Yes, you are probably right.

Next set of numbers:





So we've hit terminal gravity, which is nice. I think I'll give it a couple more days at 20°C, but this #2 is being a right royal PITA and starting to hold things up. I think I'll start to gently swish, perhaps daily, to get the gravity down so that I can bottle.


----------



## Adr_0 (28/5/17)

Floc seems to be ok - #1 vs Control:


----------



## Adr_0 (6/6/17)

So, bottling today. Bottling will be done by pre-purging bottles with CO2 and also topping off the headspace with CO2 to minimise the impact of bottling oxidation on the end product.

Firstly, a summary of time vs fG:

Control - 7 days
1......+1day/24hrs
2......+10 days
3......+5 days

I took a shot of the three test batches as well for curiosity. From left to right (1, 3, 2) which I'm guessing suggests decreasing yeast growth - or at least decreasing total yeast cells:



So, in simplistic terms, the resulting samples from this experiment where H2O2 was added did not provide yeast vitality, viability or attenuation benefit vs a control batch aerated for 6hrs on a stirplate. It is unknown whether any benefit could have been provided vs an un-aerated batch.

In terms of dosing, the starter in #1 had around 320ppm/10mM H2O2 once in solution which is considered by most literature to be a fairly detrimental dose, however still significantly out-performed #2 and #3 where H2O2 concentration was closer to 20ppm/0.6mM, suggesting that oxygen was potentially produced in greater volume than in #2 and #3.

The significant difference in attenuation profiles and yeast volume between #2 and #3 suggest that H2O2 is - as per scientific consensus and literature - going to favour wort oxidation reactions rather than decomposition to O2. This is expected to have a doubly negative impact on the beer.

Some tasting in the next 4-6 weeks will hopefully provide further information on the degree of oxidation and yeast health experienced in all the samples.

I think future work will definitely be focussing some attention on the starter - in say a split batch of schwarzbier with both starters on stirplates. We'll then hopefully see if there is a positive or negative impact to an aerated starter... and then after that I promise I'll buy an O2 kit.


----------



## peteru (13/8/17)

Just wondering, did you have a chance to perform any sensory evaluation on the samples yet?

BTW: I think I'm going to pull the trigger on an O2 kit in the next day or two. Got a lager (Cerny Pivo) I want for Christmas, so not about to take too many risks tying up my single bucket fermentation fridge.


----------



## Jack of all biers (12/11/17)

Jack of all biers said:


> I may post my final results here in a few months time, when my oxidation results (for what they're worth) are in..



Forgive my posting this in two threads, but as Adr_0 started this more relevant thread prior to our initial posts in the O2 thread. So because the sensory tests as to whether the beer would last for the long term, being my main focus, I did not complete the H2O2 experiment until a couple of weeks ago when the final two beers were compared. I initially wanted to do a comparison every 2 weeks, but that started to drag out to every month or so (with the remaining beers being consumed in between!

This will be my last post about the H2O2 experiment, as all the bottles are finished. All up, I found that about 6 of the H2O2 bottles showed signs of oxidation, with one friend describing it as tasting like cardboard (I found it tasted like a weird shandy, but not in a nice way). Only one of the aerated bottles (from 25L) showed a similar flavour, which I put down to oxidising from bottling. So 6 from the entire 25L batch 'oxygenated' by H2O2 were less than good. All the others were fairly good and to top things off, after the last few raised the total pf oxidised up to the 6, the very last one was great. So, the older they got the more the oxidation showed (roughly between 3 and 7 months, with most in the 5-7 month period). 

All up the aerated batch tasted better all through, with the H2O2 being more hit and miss. I think this does indicate that, for me at least, the idea was good, but the randomness of the O- factor defecting your beers, is just not worth it.

H2O2 as a ghetto oxygenation method. Nah, you're better off aerating.


----------



## Adr_0 (13/11/17)

peteru said:


> Just wondering, did you have a chance to perform any sensory evaluation on the samples yet?
> 
> BTW: I think I'm going to pull the trigger on an O2 kit in the next day or two. Got a lager (Cerny Pivo) I want for Christmas, so not about to take too many risks tying up my single bucket fermentation fridge.


The control and 1 tasted identical, to be honest both were only 'ok' as they were a bit dry. Number two ended up with an aceto infection, while number 3 was flat (flavour wise) and interestingly had basically no head retention - very much an odd occurrence with my beers.

I still need to do a starter comparison in the near future, but in the meantime my oxygenation method has been vitality starters - working an absolute treat in combination with decent cell count. Who'da thunk it?

PS - I still have not bought an oxygenation kit and at this stage do not intend to.


----------

