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Magpie
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[*] posted on 23-4-2013 at 10:47
isoamyl alcohol


I have read for years that the fusel oil produced during fermentation to produce drinking alcohols (brandy, whiskey, etc) is mostly isoamyl alcohol. A portion of the fusel oil remains with the product and is in fact what determines its flavor.

I obtained a liter of fractionation still bottoms from a local distillery, ie, the liquid left in the pot after distilling a batch of corn whiskey mash. Assuming that the fusel oils might be floating on the top I had asked the owner to draw a sample off the top, which he did. He drew off a 50-gallon drum full. I pulled my 1-liter sample off the top of the drum using a turkey baster. I was surprised how pleasant smelling this residue was.

To a 17mL aliquot I added NaCl to saturation (~4.5g). Then I extracted the decantate with 5mL of dichloromethane. This extractate was then placed in a sunlit window sill in a beaker to evaporate off the DCM. To my surprise, what remained were small 2mm square translucent plates that were soluble in water.

So, I was not successful in isolating anything that looked like isoamyl alcohol (bp = 132°C).

Comments?




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[*] posted on 23-4-2013 at 10:58


Quote: Originally posted by Magpie  
To my surprise, what remained were small 2mm square translucent plates that were soluble in water.

Are you sure you didn't accidentally get some of the salt in with the organic phase?




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[*] posted on 23-4-2013 at 11:53


If it was pleasant smelling it is not fusel oil.....

Fusel oil is usually distilled off at the very very end, when the measured ethanol content is about 10% and lower. A cloudy liquid is obtained which never settles. At distilleries I guess they stop way before the percentage drops that low, due to the savings in heating as there is no point to distill the last drops of ethanol out.

I'd say you got some salt in your organic phase, though it may be some very big aldehydes or ketones, or condensation products of said stuff in your beaker.

IIRC high pH fermentation gives a lot of fusel oil.




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[*] posted on 23-4-2013 at 12:17


Quote: Originally posted by DraconicAcid  
Quote: Originally posted by Magpie  
To my surprise, what remained were small 2mm square translucent plates that were soluble in water.

Are you sure you didn't accidentally get some of the salt in with the organic phase?


That was my first thought also. That's probably what it was. The phase separation was plagued with a lot of emulsifiers and foam and not at all clean.

===============================

The pH is about 5. It is a cloudy liquid, a sort of brown color with a very thin white film floating on top.

[Edited on 23-4-2013 by Magpie]




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[*] posted on 23-4-2013 at 19:20


@Magpie

Fusel oil I obtained from a distillary is a dark foul smelling liquor. It is a cocktail of all lower alcohols (except methanol). It also contains generous amount of water.

The approx. Composition is as under:
Water: 15 %
Ethanol : 25%
Propanols:15%
Butanols:5%
Pentanols:30%
Residue:10% Butanols are predominently n-butanol & Pentanols are about 25% iso & 5% n.
It is the residue which gives foul smell.

Only practical way of separating IsoAA is by salting out the water and then fractionation using an efficient packed column.
1st take out the fraction upto 120 DegC and then take out IsoAA as top fraction @132 DegC. I have managed purity of 98+ (by GC)

Let me know if you need any more inputs.

Gsd
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[*] posted on 23-4-2013 at 22:03


Very nice work gsd! And thanks for the detailed information on how to isolate the isoamyl alcohol from the fusel oil.

I'm presuming that you received your fusel oil as such from a large distillery. My problem is that my local distillery is tiny and does not recover the fusel oil as such. He just sewers his bottoms from each batch. IIRC he said the bottoms amount to about 130 gallons/batch.

So, if I am going to be able to extract any isoamyl alcohol I'm going to first have to learn how to get a sufficient quantity of fusel oil, say at least 100mL, I imagine. Do you have any suggestions on how I might do this? The distillery owner is interested in learning how to do this also. So I feel he will work with me. Being a very small distillery (he only has a 2-man crew and one still) he is also very flexible in what he can do and has plenty of time to do it.




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[*] posted on 23-4-2013 at 23:00


Fusel oil is a very tiny fraction of a regular alcohol distillary. I don't have my data & literature handy at the moment, but IIRC 1000 lit of distilled alcohol leaves about 10 lit of fusel residue. So it constutes about 1%.
The lower alcohols have notorously close BPs and Relative Volatilities so separating them individually is impractical on smaller scale. However you can make a good use of that bottom fraction by converting it into mixed esters of Acetic acid. It should find a good market as a cheap paint thinner.

BTW 130 gallons per batch is a large quantity. At the very least ask the distillary owner to use it as a boiler fuel.
It may result in substantial savings.

Gsd
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[*] posted on 24-4-2013 at 07:57


Just get a 55 gal drum of it, and then quickly distill batches for low, med and high BP stuff. Once you have done that a few times, you can combine the lows, meds and highs, and start fractionally distilling those into components. You will need to go very slowly, with a vac jacketed vigreux column. But you could probably separate out at least some of the alcohols to a reasonable purity. And if you can redistill the ethanol fraction to some purity, it might be worth the trouble for that alone. It is not worth THEIR time, as they are doing it for profit and can make new ethanol via yeast cheaper than recovering the last bits, but for you, it might be worth it. I would love to set up a still for that, but there are not many distilleries that I know of around here. If anyone has one in NC, let me know. I happen to know where I can get some nice fractionating columns...
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[*] posted on 24-4-2013 at 08:46


I don't think I can ask the distillery owner to do any redistillations to recover fusel oil from the bottoms. And I don't want it bad enough to process it through my 24/40 - 1liter pot equipment, even if I had a 24/40 sized fractionating column. Right now I only have a 19/22 sized column.

What I was hoping for is an easy way to decante off the fusel oil from the bottoms of his batch of corn whiskey. This fall he will likely be making some brandy from pinot gris but I assume that similar technical hurdles would exist there too.

I know that industry deals in fusel oil as an item of commerce in lots of 1000 liters or more. So large distilleries must have the economical recovery of fusel oil built into their processing. What I'm really trying to find out is how this might be done on a much smaller scale with more of a manual labor input from me.

================================

Here is a good paper on the subject which answers most of my questions:

http://www.chemicalprocessing.com/assets/Uploads/FUSEL-OILpa...

[Edited on 25-4-2013 by Magpie]




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[*] posted on 22-11-2014 at 11:54


Based on a US Patent by Felix Ehrlich I tried to make some isoamyl alcohol by fermentation. Processing the wash was very tedious and I ended up not finding any alcohol. This was over a year ago. I'm posting this result and the procedure used because of interest by a forum member. I will be glad to answer any questions and welcome your critique and recommendations:


Attachment: Fermentation of Molasses for Fusel Oil.doc (23kB)
This file has been downloaded 408 times




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[*] posted on 22-11-2014 at 13:47


Hi
As discussed in u2u I will post in the reference the papers that might be of interest to you. I do ask others not to post them openly as I have no idea which can be publicaly shown.




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[*] posted on 22-11-2014 at 14:03


I obtained some isoamyl alcohol from alkaline de-esterification of the ethyl ester, aka 'banana oil'. Boy, that stuff STINKS [the ester]. But the alcohol is pleasant.



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[*] posted on 22-11-2014 at 14:10


Ok have posted a couple of them in references, They took ages to upload, so will try and get some more up with them later.
Most except the Ehrlich pathway as the excepted route for this in yeast, but the Dickingson paper cast a little doubt that this is the sole pathway,the Hartle paper (posting later) Proves there at least 2 pathways for this in yeast. The starting sugar is critical for a good yield, Glucose is by far the best. Those that use sucrose and others often have other nutrients and a mash in them, this can alter the sucrose and side reactions etc.
This is why Breweries can have a problem, but if you seek it then Glucose is the cleanest way




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[*] posted on 22-11-2014 at 14:18


For those that cant see it then this screen shot of part the results is the bit most relevant, when you read the paper and match the genotype with highest level you realize why sucrose and other carbon sources dont work very well without other factors at play.



results.png - 70kB




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[*] posted on 22-11-2014 at 14:24


I used sucrose (molasses) as that is what Ehrlich specified in his patent. I can certainly try again with glucose. I still have the Lillemand yeast in the refrigerator, guarding it from the throwaway wishes of Mrs Magpie. The leucine, which I buy locally from a bodybuilding store, is expensive so I can't tolerate too many failures.

[Edited on 22-11-2014 by Magpie]




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[*] posted on 22-11-2014 at 14:32


If you're willing to order online, I just noticed that this site has leucine for 50% off. http://purebulk.com/l-leucine-powder/ I haven't ordered from them, but I have heard others have been pleased.
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[*] posted on 22-11-2014 at 14:50


Thanks - I may do that if the shipping is reasonable.

The worst part is separating the alcohol from the wash, or fermentation product. I used a lot of diatomaceous earth and numerous tedious filtrations.

Little Ghost is going to have guarantee me a reasonable amount of isoamyl alcohol, say 5mL/gallon of wash, before I'll want to try this again.




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[*] posted on 22-11-2014 at 14:59


Quote: Originally posted by Magpie  
Thanks - I may do that if the shipping is reasonable.

The worst part is separating the alcohol from the wash, or fermentation product. I used a lot of diatomaceous earth and numerous tedious filtrations.

Little Ghost is going to have guarantee me a reasonable amount of isoamyl alcohol, say 5mL/gallon of wash, before I'll want to try this again.


LOL as its part of something else I am doing, as agreed before in return for help from you I will try out the other methods and guarantee you much higher than 5ml a gallon ;).
I need to re read all his notes etc and go back through the papers. Most try and avoid isoamyl alcohol in the wash, but when you get it right (or I guess to most people wrong) The yield is very high.
I will be in touch Via email etc with full details and some the papers I cant post online.




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[*] posted on 22-11-2014 at 15:12


Quote: Originally posted by Magpie  
Thanks - I may do that if the shipping is reasonable.

The worst part is separating the alcohol from the wash, or fermentation product. I used a lot of diatomaceous earth and numerous tedious filtrations.

Little Ghost is going to have guarantee me a reasonable amount of isoamyl alcohol, say 5mL/gallon of wash, before I'll want to try this again.


Ask Aga to see his centrifuge, it should work well for yeast and save alot of filtering. To be honest its pretty clever the way he does it, cheap and easy!

Please dont buy anything yet!! you dont need pure Isoleucine to do this, there is a cheap supplement you can get with enough of it in, most of everything else in the mix wont hurt at all, its what they used at the uni in the end.
I need to ask my Dad what it was called etc, but from memory it was £6 for 500mg. I cant remember how much Isoleucine was in it, but there was more than enough.
Look at this paper as well
http://www.ncbi.nlm.nih.gov/pubmed/9341119/
Sorry my connection timed out before I could get the full file, I have the paper in paper form not electronic.
The long and short of it is, it mentions the other possible pathways, 2 have now been proved incorrect, but the point is the excepted
Ehrlich pathway isnt the main or only pathway to this.
I have some the immobilized yeast out the fridge and getting it ready to culture up. I should be able to inoculate a broth by Tuesday (Latest). A quick look under the scope shows it viable (methyl blue).

[Edited on 22-11-2014 by Little_Ghost_again]




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[*] posted on 22-11-2014 at 17:02


Slightly off topic, I have a'Dutch planted' tropical aquarium, I have had this for years and feed the plants supplemental CO2, This is now done by a sophisticated JBL Micro controller system and CO2 bottle. When I first did it I used yeast and plain sugar to create CO2 and bubble it into the water at a slow rate to feed the plants more carbon.
Eventually what you get at the end of the pipe in the aquarium is a white filamentous like material attached to the tube. This was often described on tropical fish forums as a 'Bacterial growth'. However it eventually transpired it was more likely to be yeast in nutrient poor conditions with little carbon source (the plants were competing), one of the main components found for feeding the yeast in the aquarium was Isoamyl Alcohol that had been ejected through the tube (probably as a vapor).
I found a paper two years ago that mentions yeast being able to grow into filamentous forms, it was on reading that paper that I set up a old system to look at the organism, it did indeed turn out to be yeast at the end of the tube. The paper is
PMCID: PMC14767 Characterization of Alcohol-induced Filamentous Growth in Saccharomyces cerevisiae.
It mentions the complete process and Isoamyl Alcohol as one of the carbon sources that can cause this. Yeast is a fascinating organism that is still to a greater extent not fully understood.
Sorry if its slightly off topic but I cant remember all the paper details now and no longer have a copy, I will try and dig it up and post in research, from a vague memory it contains some useful information that might help with getting the substrate right.

EDIT
I found it on the old pc! so have hopefully attached it here, if your into yeast its a pretty interesting paper, it might also help anyone looking into Isoamyl Alcohol and yeast



[Edited on 23-11-2014 by Little_Ghost_again]

Attachment: Characterization of Alcohol-induced Filamentous Growth in Saccharomyces cerevisiae.pdf (534kB)
This file has been downloaded 307 times





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[*] posted on 22-11-2014 at 17:40


I have also found another paper I havnt seen before, although using a different yeast strain, it indicates semi aerobic or aerobic conditions favored Isoamyl production!!! I will try and upload with some papers in the morning.
The paper is
Effects of Fermentation Temperature and Aeration on Production of Natural Isoamyl Acetate by
Williopsis saturnusvar.saturnus

Hindawi Publishing Corporation
BioMed Research International
Volume 2013, Article ID 870802,

I havnt read it fully yet myself




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[*] posted on 23-11-2014 at 05:47


I cant open DOC formatsat the moment Magpie, do you have it as a PDF?



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[*] posted on 23-11-2014 at 06:53


for Little Ghost:

Fermentation of Molasses for Fusel Oil
5/7/13


The purpose of this procedure is to produce by fermentation a wash high in fusel oil. Specifically desired is isoamyl alcohol, which is the major component of fusel oil.

This procedure is based on the 1908 patent (US 885,591) of Felix Ehrlich, a German chemist. Molasses is the substrate, with leucine added to promote the production of isoamyl alcohol. The scale will be 1:1000 based on the proportions given in the patent.

Chemicals
1. 1 kg of molasses (80° Brix, ~50% sugar).
2. 80g leucine
3. 2.5 liters of spring water
4. dry distiller’s yeast
5. 6N sulfuric acid

Equipment
1. 2 gallon ss pot
2. 1000mL beaker
3. large plastic mixing spoon
4. plastic 1 gallon fermenter
5. fermenter lid equipped with rubber hose for bubbler
6. 400 mL beaker water seal/bubbler
7. thermometer
8. pH paper
9. constant temperature bath with PID control
10. large plastic funnel

Procedure
1. Place 2.5 liters of spring water in the 2 gallon ss pot.
2. Add 1 kg of molasses (Grandmas’s “Original” molasses) and mix well.
3. Add enough 6N H2SO4 to slightly acidify (pH 3.5-6.0). Shoot for pH = 5.0.
4. Heat for awhile to drive off the volatile fatty acids (formic and acetic).
5. Allow to cool to fermenting temperature, ie, 25-30°C.
6. Poor off 1/3rd (~850mL) of the mash into the 1000 mL beaker and allow to cool to 20-23°C.
7. Mix 80 g of leucine (GNC) into the large portion of the mash.
7. To the smaller portion of the mash mix in the yeast (0.9g of Lallemand Super Start dry distiller’s yeast) and let it set for 3-4 hours at 20-23°C.
8. To the large pot add the leavened smaller portion of the mash and mix.
9. Using the funnel place the mash in the fermentor.
10. Keep the fermentor at 25-30°C until fermentation is complete as evidenced by the ceasation of CO2 evolution as indicated by the bubbler.




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[*] posted on 23-11-2014 at 07:33


Hi
Thanks magpie, I am still having trouble getting some the papers posted, can you u2u me your email? It might be easier that way. one thing that really struck me was 80g leucine, from what I have read that seems extremely high.
I am going to run a series of experiments on this and also follow your procedure to compare.
From memory (shaky) I have a feeling that leucine content per Kg was something in the mg per KG. While the guy that you took your procedure from is widely credited with this pathway (it has his name after-all), most up to date research and the last paper I posted in research, shows that his pathway is far less important than first thought.
The paper with the genetic info in it shows that yeast that have that pathway disabled still produce the alcohol and in good quantity.
Whats really interesting is there is still much unknown about the exact respiration pathways with yeast. take something away from them and they respire with a different pathway. Even looking at the ATP and ADTP pathways is more complex than expected. While much of it is still the basic Krebs cycle and electron transfer chain, yeast have a knack of using some pathways that are still not fully understood.

Also I note the pH and comment to shoot for 5
I would have said aim for 6.5 and keep above 5.7, I certainly wouldnt drop to 3, the other thing thats often overlooked is water hardness, did you measure the GH? Spring water can be really soft (low GH) therefore the pH bounces all over the place. I need to dig out the papers with the water parameters. I will post them here when I have.
LG

P.S for the GH etc I normally use my aquarium test kit, but I have some MSDS of the kits somewhere, so will post the reagents used in the test kits. most are really simple but as I have aquariums I am lazy and use those

[Edited on 23-11-2014 by Little_Ghost_again]




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[*] posted on 23-11-2014 at 11:39


added paper to research section, this one to do with valine added to brewers wort. Like most papers they are looking to get rid of what your trying to get, so its worth while keeping that in mind. I will add more as I can get them uploaded.

The yeast I took out the freezer has thawed out ok so now is plated up and should be ready for inoculation into broth next day or so.




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