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Gail
11/06/10 07:08 PM  
Can Brett metabolize glucose directly into acetic
I just fumbled about reading scientific abstracts I don't fully understand for about two hours and then the lightbulb went off.

Somebody here will know the practical as well as the scientific scoop:

I added a standard suggested amount of corn sugar to 2 year old plambic that was in the 70F+ temperature range, then bottled (with some unintentional aeration in the racking). (I know it could have been adjusted to be carbed more highly, but I was ok with moderate carbonation in my beer design).

No carbonation! 6 weeks later there is no evidence of CO2 in the bottles. It also tastes more acetic now. Flat and sharply sour. Ouch.

I know the beer was warm and that drives out CO2, and I know the brett is probably pretty stressed. It could be that the brett couldn't make enough to make any pressure, but that seems odd.

Zymurgist question: Is it possible that the brett converted sugars to acids instead of into ethanol and CO2?

Also, would you:

A - Wait a year

B - add a dropperful of more yeast (champagne?) and sugar syrup, re-cap

C - accept this as a sharp flat overly-sour beer

SteveG
11/08/10 11:33 AM  
Re: Can Brett metabolize glucose directly into ace
It may well be relivant to ask ... which Brett(s) were you working with? I can say that I would be amazed if any beer that had sat for two years could carbonate without reintroduction of yeast. If it were me I'd pop the bottles and try again. Personally though I am not all that into still lambics. If you are OK with no bubbles then option C may be for you, though from the way you say it I would gather that option C is not something you find all that appealing. There could also be an option D, if you have the stuff to force carbonate...
dsanborn
11/09/10 10:59 AM  
Re: Can Brett metabolize glucose directly into ace
I would try B (at least with some of the bottles)

Steve - I think you tasted my old lambic during an online tasting/swap several years ago??

Anyway, it was also a 2+yr old and I did pretty much the same thing...it's still flat...it's been 3+yrs...it's still weirdly sour (not really in a good way).

I have poured it with a touch of fruit juice in the glass and that makes it drinkable, and almost enjoyable. But it has no carbonation.

Good luck, scamborn

Gail
11/09/10 11:27 AM  
Re: Can Brett metabolize glucose directly into ace
Yeah, playing with just part of the batch seems appropriate. Thanks!

It was Brett Brux with various dregs. I used the wooden dowel apprach, and opened for tasting a bunch of times, then transfered with an oxygen leak issue, so there was plenty of opportunity to get acedic as well as lactic-sour. I was surprised to see it jump so much after adding the sugar and waiting 6 weeks, however.

I sure wish I had a translator for scientific abstracts on Brett. (I have a feeling that interesting insights could come of all that work, and I'm still struggling with basic terms like "crabtree" and "krebs." I wonder if there's a book I should be reading?)

CASK1
11/10/10 08:31 AM  
Re: Can Brett metabolize glucose directly into ace
Are the Brett abstracts online? Can you provide links? I'd love to take a look...
SteveG
11/10/10 04:57 PM  
Re: Can Brett metabolize glucose directly into ace
Well, of all the bretts to not trust with conditioning carbonation, I think B would be top of the list. Some bretts behave a lot like more conventional yeasts, Brett B is, IMO as far in the other direction as you can go.
Gail
11/10/10 06:45 PM  
Re: Can Brett metabolize glucose directly into ace
Cask1 -- most of the stuff I have wondered about regarding brettanomyces and the other lambic microbiota leads me to results like this:

http://scholar.google.com/scholar?q="brettanomyces"

(I put it in quotes to keep from getting results based on scientists with the first name of Brett -- sometimes google is too smart for its own damned good. Lots of the research is wine-spolilage oriented, but that can shed light on what Brett likes, too)

Here's an example of something I'd love for help interpreting:

http://onlinelibrary.wiley.com/doi/10.1111/j.1745-4530.2005.00393.x/full

" The influence of temperature and agitation on acetic acid production by B. bruxellensis in a glucose medium was investigated at different levels, 26, 30, 34C and 250, 350, 450 rpm, respectively. Temperature and agitation were shown to be decisive factors (P < 0.05) in acetic acid production at 288 Lh−1(0.6 vvm, OTR = 124 mgO2L−1 h). The optimal conditions for a high volumetric productivity were 30C and 250 rpm, respectively. "

So, this may agree with SteveG in that it says Brett B is the big acetic producer. I believe it is saying that Brett B can make acetic acid from glucose. I'm wondering if any CO2 is produced when Brett B takes that pathway instead of making alcohol.

Any biologists in the house?

JWW
11/11/10 09:24 AM  
Re: Can Brett metabolize glucose directly into ace
The key to that excerpt is here "288 Lh−1(0.6 vvm, OTR = 124 mgO2L−1 h)" because that is telling the oxygen sparge rate, which means they are bubbling in O2 into the medium, maintaining an aerobic environment with an oxygen tranfer rate of 124 mg O2 L/h. Anytime you maintain an aerobic condition with Brett you will produce Acetic acid as the main overflow metabolite.
Gail
11/11/10 11:37 AM  
Re: Can Brett metabolize glucose directly into ace
Thank you! So that's 124 mg of O2 per liter of glucose liquid per hour? Quite a lot of O2 and sugar for that Brett to work with.

Obviously that O2 rate is not happening while bottling at home, even at a fairly warm room temperature and some tasty priming solution, but could this happen to homebrewers -- even a little? Does Brett B like to make acetic acid from glucose (instead of alcohol and CO2 may I presume?) whenever it has enough oxygen?

If so, can we take that as Brett functioning counter to the way that Acetobacter works, where the organism converts alcohol to acetic acid? Sounds like Brett makes acetic acid from the sugar directly.

JWW
11/11/10 01:23 PM  
Re: Can Brett metabolize glucose directly into ace
"So that's 124 mg of O2 per liter of glucose liquid per hour?"

Not quite, its not per glucose liquid, its per liquid volume of culture, so its 124 mg of o2 per broth volume in the vessel per hour.

"Does Brett B like to make acetic acid from glucose (instead of alcohol and CO2 may I presume?) whenever it has enough oxygen?"

I am no expert with Brett and there are far more knowledgeable on this forum, but my understanding/experience is if you give brett O2 it will make a mixture of acids and alcohols with acetic being predominate (amounts vary by strain and ferm conditions). So if you just have a bottle open to air vs stir plate, you will have increased levels of acetic acid in the stir plate along with alcohol and CO2.

I am pretty certain metabolites produced by various strains of Brett has been raised on here before, so maybe more detail can be found in the archives.

Gail
11/12/10 11:51 AM  
Re: Can Brett metabolize glucose directly into ace
Thank you. I've had a pretty tough time finding Brett metabolism information that makes sense to me in either research abstracts or forum posts, which is why I decided to wade in and ask about the priming solution to acetic acid possibility. Besides, I'm interested in how the experienced brewers here have experienced specific things, and I really appreciate any insights in a practical context.

(One thing I may do besides continuing to scour forums and abstracts is to try to find a biology text at the level that can help me better understand research papers. Maybe an introductory undergrad text.)

I haven't perceived acetic acid right out of a starter, but I also haven't used a stir plate with Brett. I wonder if older Brett B that has been in an anaerobic, acidic and alcohol-rich environment reacts differently to an increase in O2 and glucose than in a starter, with fewer stressors.

All very mysterious!

JWW
11/12/10 05:03 PM  
Re: Can Brett metabolize glucose directly into ace
I understand completely, I briefly searched the archives to see if anything specific came up and I did find it difficult to locate posts regarding acetic acid specific to Brett brux. But I do seem to remember the topic coming up with technical conversation included.

For carbonation purposes, the little bit of O2 (remember air contains 20% O2) that makes its way in will produce very negligible amounts of acetic acid, probably wouldnt even break the taste threshold.

Don't think of this as a stressor response, its a growth response. If O2 is present with glucose then it will produce acetic acid as a byproduct of growth. If its not present then it wont produce acetic acid, no matter the age. Maybe there is some funky metabolic pathway were an amino acid is present that goes counter to this, but in most cases this holds true. I routinely grow brett on a shaker platform in order to achieve higher cell densities and it always produces acetic acid, this gets decanted and further expanded in a non aerated starter a day before brewing so that the culture is active for pitch. I have also grown starters anaerobically. I havent noticed any differences in quality of beer or growth of Brett between the two. The only difference is that you want to remove the acetic acid produced during cell growth prior to fermenting with it. I just happen to be lucky enough to have a centrifuge to do this and decant off the acetic acid laced broth. My point here is that you shouldn't associate acetic acid and poor health of Brett, you just dont want it in the beer... or maybe you do... Different strokes for different folks.

As for learning the metabolism stuff, check out a basic undergraduate biochemistry book, itll have everything you need. Or you can search online for glycolysis and Krebs or TCA Cycle to start.

hope this helps

CASK1
11/12/10 10:32 PM  
Re: Can Brett metabolize glucose directly into ace
I just found this link through another forum. It's geared towards Saccharomyces, but it still offers some good basic info on acetic acid production by brewing yeast.

http://www.danstaryeast.com/acetate-regulation

ChadY
11/29/10 05:09 PM  
Re: Can Brett metabolize glucose directly into ace
http://www.brettanomycesproject.com/brettanomyces/wp-content/uploads/2010/11/9.pdf

there is a diagram of the dissimilatory pathways during the various phases of growth of B. intermedius during batch culture.

The reason for the lack of carbonation is most likely due to the already low pH of the beer. After a length conversation with Jeff Sparrow he stated the reason a lambic brewer blends 1 year old and 2 year old beer is to temporarily bring the pH up so that the bottle of gueuze will have a high enough pH to allow the yeasts to work and carbonate in the bottle. The pH will then drop back down in the bottle, hence one of the reasons why bottles are stored for 6 months or more before being released.

The amount of oxygen which was taken up during bottling is not enough for the Bretts to produce copious amounts of acetic acid.

Also at what temperature did you store the bottles. Myself and other brewers producing sours (especially if bottle conditioning with Brett) have had good success at 80*F and re-carbonation occurring fully in 2 weeks.

B-Dub
11/29/10 08:13 PM  
Re: Can Brett metabolize glucose directly into ace
Chad Y,

How much effort have you put into keeping the O2 out of the bottle? Purge everything from the barrel to the bottle?

Seems like a good idea to keep the DO to a minimum.

BW

ChadY
11/29/10 08:36 PM  
Re: Can Brett metabolize glucose directly into ace
On a true bottling run absolutely! push beer with CO2, through a line pre-filled with CO2 into the bright beer tank pre-filled with CO2 then CO2 top pressure to push to the bottling line, bottle purging before filling... you name it

But as far as in lab bottling trials where the beer is for observing carb levels for priming, DO is not a consideration. And as such most acetic would be formed at the same time the bottle becomes carbed.. a little after but no noticeable amounts in my opinion (maybe a few mg/l)

A friend at AC Golden has been hand bottling homebrew style no purging of the bottles, he's bottling from a corny, filling the 750 ml bottles with the beer then pipetting in a sugar and yeast mixture... pretty archaic with good results... some over carbed but that was actually with a cider yeast... In the sours it would be impossible to notice a few mg/l increase in acetic acid as its already between 500 and 1000 mg/l, depending... Thats my main reason for thinking that most O2 pick up would be negligible when bottling a sour with bottle conditioning, and shouldn't account for an increase in acetic acid.

For brewers wanting to do bottling trials a blichmen (sp?) bottling gun to purge bottles and then pressure fill from a keg pre-mixed at 50*F with yeast and sugar works well and is negligible O2 pick up... Brett is quick to scavenge any oxygen as it gets dissolved into solution but it would mostly be in the head space and slowly diffuse into the beer.

I can't argue with the dogma that O2 and DO should be kept low!

Gail
11/29/10 10:27 PM  
Re: Can Brett metabolize glucose directly into ace
Thanks for the great links and comments.

I finally got some belated carbonation after over two months. I have learned a lot from this experience.

My batch was very small, a blend from two gallon jugs that were 3/4 full, and which had had wooden dowels in the stoppers for much of two years.

I was bottle-conditioning in room which was averaging about 65 degrees at that time, with some warmer days up into the 80s. (I live in San Francisco, so typically we have no AC and we don't have proper central heating in most of the house, either. Autumn seemed like a good time to blend and bottle!)

Since this batch resulted in only a few bottles, I have been slow to open them. I had opened one (and had one opened by others - how embarrassing!) when I first posted here.

Another month went by, and there is finally slight carbonation - a little opening pfft, but no head on pouring.

So -- looks like NO to the hypothesis of oxygen causing acetic acid. Very interesting comment and link on the acidity situation above! I also found a post by Michael Tonsmeire where he remarked that a sour beer may take more sugar to carbonate because little CO2 is still dissolved in the liquid after so long. The warmest that beer got was probably on bottling day, which was a warm one. It fermented in the basement, which stays cool, but the kitchen got quite warm that day, and I blended and bottled four different small batch sour beers. This was last one, so the gallon containers were warm by then.

I took action, grabbing ten of the twelve remaining bottles and added a new dose of sugar and a wine yeast to each of those. I'm going to give them a couple months before I taste again. I held onto two of the original bottles unopened, as a control.

Lessons learned: Be careful about letting beer warm up before blending. Consider a month a very short time to carb a two year plus lambic with priming sugar. Consider using new brett or acid-tolerant wine yeast. Consider blending with a young beer.

That last item seems appealing, traditional, logical... but daunting. How much young beer - and how do you prevent bottle bombs? How young? What if you used a very young "clean" all brett beer for the blending? Has anybody written practical guidance on the young beer blending approach, and how to calculate the remaining gravity points available for conversion into CO2 ?

 
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