Oxygenation experiments with hydrogen peroxide

Discussion in 'General Brewing Techniques' started by Adr_0, 19/5/17.

 

  1. Adr_0

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    Posted 26/5/17
    Numbers from yesterday:
    Gravity-25thMay1.png
    Att-25thMay1.png

    #1 is basically 24hrs behind the Control.

    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?
     
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  2. MHB

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    Posted 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
     
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  3. technobabble66

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    Posted 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?
     
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  4. Adr_0

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    Posted 26/5/17
    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?


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

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    Posted 26/5/17
    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
     
  6. tavas

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    Posted 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?
     
  7. peteru

    Here, taste this!

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    Posted 26/5/17
    If it was Goldcross H2O2 then the main stabiliser is phosphoric acid with traces of phenacetin and it is pharmaceutical grade.
     
  8. Jack of all biers

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    Posted 27/5/17
    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.

    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;
    EDIT - Sorry the quote didn't transfer across as a quote, so I have quoted it!
     
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  9. Adr_0

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    Posted 27/5/17
    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.
     
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  10. Jack of all biers

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    Posted 27/5/17
    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).

    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
     
  11. Adr_0

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    Posted 27/5/17
    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...
     
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  12. technobabble66

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    Posted 27/5/17
    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!
     
  13. Adr_0

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    Posted 28/5/17
    Yes, you are probably right.

    Next set of numbers:
    Gravity-28thMay1.png
    Att-28thMay1.png

    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.
     
  14. Adr_0

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    Posted 28/5/17
    Floc seems to be ok - #1 vs Control:
    10 day 1vsC-floc.jpg
     
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  15. Adr_0

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    Posted 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:
    sediment-1-3-2.jpg

    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.
     
  16. peteru

    Here, taste this!

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    Posted 12/8/17
    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.
     

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