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Little_Ghost_again
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[*] posted on 22-11-2014 at 08:26
Clean Ethanol from OTC Yeasts and chemicals


Hi
This thread is where I am going to post a series of experiments, these hopefully will lead to a procedure to obtain the cleanest Ethanol from OTC products.
When finished I intend to use this thread as the basis of an article for prepublication. Feel free to add any comments on experimental procedures etc or ways to improve the experiments. The overall experiment will be broken down into much smaller steps, the best results from each step will go on to become the basis of the next step and series of experiments.
My overall objective is more involved than trying to get the cleanest highest volume Ethanol from OTC products, but that is the part I wish to present later in prepublication.

According to the rules I need to provide references, so this first part will describe the initial experimental set up and procedure for the first experiments.
To begin with I am looking for the best way to test yeast cell viability via Microscopy staining. The first first stain Will be methylene blue [Quantification of Saccharomyces cerevisiae viability using Bac Light,
Tong Zhang etal, Biotechnology Letters 26:989–992, 2004]

I shall list the other stains in the next post after I have sorted out the citations properly,I made an error in the citations so have left the other stains out until I have sorted that out.

I will be using custom made reaction vessels for most of the experiments, I am able to run 9 vessels at a time using a Jenway 9 station stir plate, initial temperature will be the same for all vessels therefore I will place the stir plate in a custom built incubator with temp control to two 12 bit resolution and an accuracy of 0.3%C.
More details to follow in next post

If anyone has a particular yeast type that they want included, then please state now, if its not UK available and your willing to send it,I shall include it.
LG

[Edited on 22-11-2014 by Little_Ghost_again]




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[*] posted on 22-11-2014 at 09:58


Most brewing yeasts give the best temperature and
The alcohol content they are viable to on the package.
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[*] posted on 22-11-2014 at 13:40


Quote: Originally posted by macckone  
Most brewing yeasts give the best temperature and
The alcohol content they are viable to on the package.


And all give other products other than ethanol, most are just different bred strains of bakers yeast, My intention is to find how to get the cleanest ethanol not the most or the quickest.
21% ABV is no good if 15% isnt pure Ethanol. My next post should be much much clearer, I will also explain why I have chosen each strain and what genes they have or lack that are of interest to me.
Any 3rd Gen viable yeast cells I isolate and look promising I will immobilize and store separately at low temp, some might be useful to cross breed with different cultures.
I favor tube and agar immobilization rather than broth. I just find for me it takes less messing with
LG




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[*] posted on 22-11-2014 at 14:33


Good brewers yeast produces mainly ethanol.
High amounts of methanol or fusel oil are frowned upon.
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[*] posted on 22-11-2014 at 21:50


What about salting the alcohol out before further processing? Granted, some ethanol will be lost, even in the saturated salt solution, but I suggest that this would be more efficient than having to distill the entire volume of solution.

I might test...to what extent (in terms of alcohol:water, v:v) salting out works. Basically, a percent yield could be calculated for salting out for solutions of ethanol/water 10:90, 20:80, etc.




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[*] posted on 22-11-2014 at 21:59


Salting out does not work with ethanol and commonly
available salts. Industry is very efficient at separation
processes and distillation is the method of choice.

As a side note distillation of ethanol is heavily regulated
and generally illegal without the correct permits in most
countries. I believe NZ is an exception and there are
others. The US is not one of them.
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[*] posted on 22-11-2014 at 22:06


I've formed two layers in a similar case, one being mostly ethanol with some dissolved organics, and the other being sodium carbonate in water. IIRC, it's happened on several occasions. I will test my memory tomorrow, after I get some sleep.

As macckone said, distillation is generally illegal, and I do not particularly condone breaking the law. Take from that what you will, your judgement is better than mine.




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[*] posted on 23-11-2014 at 03:25


1
If distillation is not permitted, just buy ethyl acetate and add sodium hydroxide, heat it up and the end products are:
- sodium acetate (I believe it's a waxy solid)
- pure ethanol


2
With yeast it simply don't work without breaking the law in your case: you have to add sugar (sucrose) to Turbo yeast, fractional distillation (twice).
The yield after Turbo yeast is only between 15-20% EtOH.




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[*] posted on 23-11-2014 at 06:10


I am not breaking the law! I am conducting research under a company umbrella. These experiments dont require distillation at this point anyway.
Part of the point of these experiments is to put to bed the myths surrounding turbo yeasts etc, The vast majority of turbo yeast is plain yeast in higher amounts. Most tests people do for alcohol show total content not what is actually Ethanol. The testing methods I now have access to should finally put a things to bed.
Some of the reputable turbo yeast people even tell you that normal sugar wont work, these experiments are a kind of spin off for something that dads company is doing for a distillery.
Most breweries and distilleries dont want pure ethanol, they dont use just sugar and water and yeast, they have recipes so the mash ends with a distinctive flavor etc. What I want to do is find a way to make Ethanol that is fermented as clean as possible, I cant find the paper and therefore the reference to it, so I wont say how many alcohols are normally contained in a water sugar yeast mash.
The main problem people have is they make 'Ethanol' via fermentation and then use tests that dont differentiate the different alcohols, so yes you might get lucky and get 15% Alc but its likely only 9-10% of that is Ethanol.
But rather than get into that now, I will conduct the experiments and post the results.
II will also do the normal tests and titrations etc, But the GC should be definitive as to what is actually in it.

ANYWAY first set of questions as I start this Tuesday

If anyone has a particular brand of yeast you want me to put in this test let me know now. I have 4 slots out of nine left.

If there is a particular sugar or additive you want me to add again say before Tuesday. I have 5 slots for sugar left

I will post the details of the first set ups tonight or in the morning, I waiting to find out if the reactor vessels are 250ml or 350ml. I have posted some paper in the reference section, I have more to post but having trouble uploading them (my internet is the problem and times out). Please though no more comments on the legal side. I have checked and double checked, I dont need a license for this as its research and under a certain limit, all Ethanol will be made unfit to drink or will be disposed of.




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[*] posted on 26-11-2014 at 19:32


I did not mean to imply you are breaking the law.
Just a general warning particularly for those in the US.
Someone may try to duplicate your work and find
themselves on the wrong end of revenuers as we call them.
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[*] posted on 28-11-2014 at 03:37


Yeah sorry its just I have had no end of u2u's saying how bad it is to distill etc etc.
I use two types of reactor one is 2 litre but most are 400ml.
In my case this is not for brewing or making drinks, I am working on making other products including clean ethanol, what I am interested in is the manipulation of the yeast to use a pathway that gives repeatable results, at this stage nothing I am doing would help anyone interested in making a drink, brewing is more about flavour etc and uses all kinds of pathways within the organism, this research is for a distillery but the approach is different.
non sterile worts and mashes are normally used, without going into detail this is totally different. Its also a long way from the bio ethanol people although the research would be more helpful to them in many ways at this stage.
Fermenting isnt illegal, 90% of my reactor products will not be distilled, now I have a GC and centrifuge I dont need to distill the fermentation product to assess what is in there etc,I can take a sample centrifuge so any cells or bacteria are separated and then take 2ul of the solution and run it through the GC.
I have some interesting results from 3 different turbo yeasts I brought, on doing a DNA sequence on them the three I tried are very very closely related to a common bakers yeast strain, most of the packet contents were actually just nutrients so the yeast favored a certain path. Genetically there were zero difference with the three I tested and bakers yeast.
What I mean by that is no genes had been switch off or removed, so if these strains had indeed been special you would expect missing genes in them.
When yeast is bred for high tolerance it is often a case of breeding yeast that has some of the genetic pathways missing.
A very good example is sake yeast strains and turborg yeast. So far and admittedly I have only tested three strains this time the yeast is a bit of a con.
If you want good fast yeast highly tolerant, then your better off breeding it yourself from haploid cells on plates. It isnt the rocket science some would have you believe.




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[*] posted on 28-11-2014 at 05:44


@Little_Ghost_again;

Nice that you did sequencing on your strains! But isn't it still like 500 euro's per genome to sequence?

Can't it be true that there is a difference between your strains but it has more to do with regulation of genes rather than the complete absence of them? Of course the absence of a gene is a real nice way to identify a mutant, but regulation is often much more important than the total sum of genes present. After all, we ourselves are 25% cucumber...
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[*] posted on 28-11-2014 at 06:46


Quote: Originally posted by Tsjerk  
@Little_Ghost_again;

Nice that you did sequencing on your strains! But isn't it still like 500 euro's per genome to sequence?

Can't it be true that there is a difference between your strains but it has more to do with regulation of genes rather than the complete absence of them? Of course the absence of a gene is a real nice way to identify a mutant, but regulation is often much more important than the total sum of genes present. After all, we ourselves are 25% cucumber...


Simple sequencing just cost me the price of the reagents, so around £6 a test.
Yeast is pretty special in many ways, I have posted stuff all over the forums with diagrams of the different pathways it can use for respiration, what yeast kicks out the other end is mostly down to which pathway it used to respire. So some strains have been bred so the yeast lack the genes to use certain pathways, this limits there ability to just go off and do there own thing. Or put another way if you take some the pathways away by breeding out or whatever then you get a more predictable result.
The real turbo yeasts are mostly strains that are deficient in certain genes or have those genes switched off (depends how you want to view it), But you dont need to mess genetically with yeast to get it to produce a whole range of stuff or to get it sugar tolerant.
The strains I had sequenced came from sources that claimed the yeast were 'special', in fact there was nothing special about them, in one packet all they had done was double the amount of yeast in the packet, chucked in a load of amino acids and nutrients and advised that the yeast be kept at a higher temperature.
You can take plain cheap old bakers yeast and do exactly the same thing, it will produce 18% alcohol. But run the alcohol through a GC and you find that out of that 18% around 8-11% is actually Ethanol.
If you want mainly Ethanol and dont want to spend a fortune then go for a champagne yeast, these are a mix of yeasts species. Most of then have been line bred over generations and produce a good product.
I only sequenced then to see if the so called turbo yeast were specially bred and different in some way to bakers yeast or a different species. The ones I tested were not.
So anyone buying those particular brands are paying over the top for bakers yeast with added nutrients.


No I am not naming the brands, but I have contacted the companies asking them to clarify there product on there website as its misleading. If they dont then as they are in the uk I will contact trading standards.
With a bit of effort you can often get high yielding wild yeast from fruit blooms, but it takes effort plating them out and isolating those that have tolerated the high dextrose [insert sugar of choice] agar plate.
Same with all the additives, I normally use weak beef broth plates for first plates, it dosnt matter if you get bacteria or other stuff growing, if you have plated out correctly you should still have isolated colonies of what your after, then you can isolate the pure yeast one you want.
There are so many techniques it would take a book! But generally if its a good culture I look for haploids and grow in broth 4 days.
The point with gene sequencing is similar to humans, some lack the gene to handle say wheat products. There are some good papers on yeast genes and there function take a look at some they explain way better than I do.

LG




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[*] posted on 28-11-2014 at 06:57


Quote: Originally posted by Little_Ghost_again  

You can take plain cheap old bakers yeast and do exactly the same thing, it will produce 18% alcohol. But run the alcohol through a GC and you find that out of that 18% around 8-11% is actually Ethanol.


Then what is the other 7-10% ?




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[*] posted on 28-11-2014 at 08:23


Might be byproducts like higher alcohols, esters, glycerol, 2,3-Butanediol and succinic acid, basically other byproducts of the citric acid cycle.

@Little_Ghost_again
Really interested to know how you did sequencing on the genome, did you use STR or RFLP analysis?
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[*] posted on 28-11-2014 at 09:39


I suspect that there is a bit of literature covering removal of fusel oil out there. A better plan than trying to find yeast that doesn't make it (which apparently never occurred to anyone ever before) would be to work out the best purification method. Treating the azeotrope with CaO should remove acids, aldehydes, and obviously water.

Quote: Originally posted by macckone  
Salting out does not work with ethanol


Potassium carbonate is a very old and known agent for this.
http://books.google.com/books?id=fMsgBQAAQBAJ&pg=PA160




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[*] posted on 28-11-2014 at 13:44


Quote: Originally posted by confused  
Might be byproducts like higher alcohols, esters, glycerol, 2,3-Butanediol and succinic acid, basically other byproducts of the citric acid cycle.

@Little_Ghost_again
Really interested to know how you did sequencing on the genome, did you use STR or RFLP analysis?


Correct as well as other undefined peaks! I cheated this time with the sequencing and had my dad do it in his lab, I just got the report with straight answers to the questions I asked (hence cheap).
I dont know what method was used, they have some pretty high tech stuff there. I woud take a punt on SNP, Dad has RFLP capability at his home lab, but most that equipment came from his lab at work, so I doubt they use that anymore. Is a fair bit of messing about as well with that, although obviously quantity of sample wouldnt have been a problem.
I dont know what they call the new method they use at the uni lab, but its much much quicker and sample size is tiny compared to RFLP.
You have now made me curious lol I will have to go and ask now lol. I didnt go into it much, if I am honest I was pretty unscientific and was just wanting a yes your correct type answer on the bottom of the page.
I will ask for the full report (Tuesday he is next in) and post the PDF, I expect the method used is in the first page. You might get a kick out the report anyway.


Regarding what else comes over I have had this argument on another forum, when you do a GC of ethanol from fermentation there is a way with the live view to get the temperature markers of the column to be displayed on the chromatagram, I am not that used to this software yet (total chrom from elmer perkins). So once the run is complete I dont know how to get it to show that on the final graph.
But anyway what you see is a horizontal line with a vertical line at each end, this denotes the the temperature the column is at the point the sample enters the FID. On the HP machine when you set a run up you make a method and one the things in that is setting what temperature you want the inlet at and for how long and the same for the column (over simplified explanation).
When Ethanol shows on the FID I get could separation of other peaks but they are within the temperature range of the ethanol.
Now the argument I have been having on a brewing forum goes something like this...............

Because they use a large column and only take the fraction at the exact temperature of ethanol and the ethanol/water azeotrope then they think they are only getting Ethanol/ethanol and water.
But run it through a GC and this just isnt true, so when turbo yeast claims 18% ethanol its rubbish (in most cases), but without running a GC and MS of a sample you cant really prove it.
Thats one reason the set of experiments I am doing with yeast will include both fractional distillation and GC/MS data. Even then there are plenty of people that wont except that the Ethanol content is a lot lower than 18%.
If you really want high ethanol content then the best way is to use a mixture of yeast and other microbes, the downside is the work up to clean the solution afterwards, hence why I am looking at other ways.
Yes there are loads of papers on this, but most are looking at bio ethanol production using different feed stocks etc. I posted a paper on brewing and the microbes involved, yeast is not the only player in brewing. and you cant compare brewing and bio ethanol production or what I am doing as the same thing, they all use different techniques and methods, as well as (in the case of brewing in particular~) more fermenting microbes than yeast.

[Edited on 28-11-2014 by Little_Ghost_again]




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[*] posted on 28-11-2014 at 14:26


To give an example look at this page
http://www.hambletonbard.com/how-to-make-wine-beer-moonshine...

There are so many basic errors on that site, and they are trying to promote turbo yeast and yet reading it makes you wonder how much they actually know.
For example and taken from that page

"The key to this is the amount of oxygen available. Enough oxygen there, and the yeast will only reproduce. The six carbon atoms joined up in a glucose molecule (this is Dextrose sugar - if you use white sugar, it will break down into Glucose first before yeast can start working with it)"

For a start white sugar is generally regarded as sucrose not Dextrose, Dextrose is not broken down into Glucose because Dextrose IS Glucose to start with!

The title of the page
Alcohol fermentation with turbo yeast - how it works

and right at the bottom of the page in tiny print you get this

Turbo yeast fermentation will produce alcohol, but also by-products such as fusel oils, carbon dioxide and some other volatiles

No wonder there is so much rehashed rubbish on turbo yeast. Dont get me wrong everybody makes errors but these people are pushing turbo yeast, so you would think they would get the very very basics right like the sugars involved

[Edited on 28-11-2014 by Little_Ghost_again]

EDIT
maybe I am reading that wrong!! reading back on here it reads slightly different to how I read it on there page, so it might just be poorly written. Maybe they mean if you use sucrose (white sugar)then its broken down blah blah blah????
Anyway even they admit at the bottom that you get alot more than ethanol, try separating it though ;), even if you succeed look at the time spent and cost of chemicals etc and the actual ethanol yield, in the end your worse off (IMHO)

[Edited on 28-11-2014 by Little_Ghost_again]




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[*] posted on 28-11-2014 at 14:35


Is Turbo claiming an 18% distillation product? That's what people drink, right? So if they are drinking 10% ethanol plus 8% adehydes, glycol, butanediol, succinic acid, other alcohols, etc, wouldn't they get sick and die?



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[*] posted on 28-11-2014 at 14:43


Anyway this thread was going to be about a series of experiments and its kind of going off in the wrong direction lol.

As no one put a yeast forward I have chosen them all myself, I will name and shame them Tuesday :D. Two are wild strains collected from fruit blooms at the super market, so until the plate has finished incubating I have not much idea what they are. I put a acid and a alkali indicator in with the agar mix (I will detail agar plates I am using in another post).
Reaction vessels are 400ml straight Schott Duran beakers with no pour lips, I chose these because they fit the jenway multi stir plate perfectly! and are slim but tall.
The caps are RTV silicon custom molded by myself (pictures of the full set up soon). The first round of experiments are all at the same temp, so the stir plate will just be put in the environment chamber, as the vessels are sealed one way I am not bothering at this point with humidity control, the agar plates currently are in the other chamber and both temperature/humidity and oxygen levels in the chamber are being controlled.
Full aseptic techniques used, I did this as I was interested in seeing if any the commercial strain had any kind of contamination in them.
I am moving over to a new pc next week so that will make posting pics much easier than this laptop!




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[*] posted on 28-11-2014 at 21:30


Quote: Originally posted by S.C. Wack  
I suspect that there is a bit of literature covering removal of fusel oil out there. A better plan than trying to find yeast that doesn't make it (which apparently never occurred to anyone ever before) would be to work out the best purification method. Treating the azeotrope with CaO should remove acids, aldehydes, and obviously water.

Quote: Originally posted by macckone  
Salting out does not work with ethanol


Potassium carbonate is a very old and known agent for this.
http://books.google.com/books?id=fMsgBQAAQBAJ&pg=PA160


That only works if you have around 30% to begin with.
At least according to the diagram.
That is close to double what you get from yeast.
I should have been more specific.
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[*] posted on 29-11-2014 at 02:32


Quote: Originally posted by Magpie  
Is Turbo claiming an 18% distillation product? That's what people drink, right? So if they are drinking 10% ethanol plus 8% adehydes, glycol, butanediol, succinic acid, other alcohols, etc, wouldn't they get sick and die?

I dont think so, most is small amounts and higher alcohols etc,Drinking beer isnt any good for you anyway, it kills nearly as many as smoking, it causes death by drunk killing people in fights.
If it did produce 18% ethanol then the beer would taste different wouldnt it?
I cant prove a word of the above until I get the GAS for the GC, But I have seen enough GC print outs of yeast fermentation to know that Ethanol is not the only alcohol or 'other' in there.
When dad did the yeast work they wernt looking at this, but it did require in part the use of turbo yeast, I have never seen a turbo yeast match a champagne yeast for amounts of ethanol.
But you need to define what you want the ethanol for, there is no point people using anything from my experiments for making beer, it will taste awful, I am not trying to make a drink.
The one thing that might help though is immobilized yeast,very early days but I set some last night using several different different methods. these should make your IS much better as you dont get heaps of dead cells etc on the bottom of the vessel ,there are other reasons as well, but no point going into that until I have some data and facts.
I read some things on a brewing forum about immobilized yeast, the one thing I would say with that is, much of the poor results I saw could well be down to there use of beads way way to large, I altered a micro pipette like those used for filling multiple vials in one go, the end of them is made from those tiny 5ul pipette glass tubes you can get, my yeast beads are around 0.7mm diameter.
I have high hopes from using this method with yeast to get IA in a purer form




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[*] posted on 29-11-2014 at 02:56


Just a note to above, after the initial set of experiments I intend to have a go using immobilized cells in a continuous batch process at reduced pressure. in a ideal world it would be good if you could extract the ethanol as its made under enough reduced pressure that the temperature would be around that of the incubation temperature.
Isolating the yeast and the using one way valves should make it possible, but until I know what the yeast are making I cant work out what pressure or temperature etc. This is for another day though as I dont have huge amounts of space in the environmental chamber




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[*] posted on 1-12-2014 at 07:00


Any motivation to develop your own strains via mutagensis? Your previous posts make me think you have both the understanding, and the equiptment, to do so. I vaguely recall Ni(II) salts were most effective for wild Saccharomyces Cervisiae, though it would be nearly a year since I read the relevant texts.

Edit: Alternatively, Candida Utilis could be subtituted for S. Cervisiae if you want to branch out from the generic ethanol producing species. The common name is Torula yeast.

If you are looking more towards viability/efficiency, you may want to look into cellulosic ethanol production from cassava starch.

Perhaps there is a way to use the pearl beads from the plant as an immobilized support too. They have been shown to be surprisingly effective dessicants capable of producing ethanol with less than 0.5% water content after some simple preparation(Freeze drying to create porous structure.) It should also function fairly well as a support given the same treatment.
[Edited on 1-12-2014 by Mesa]

[Edited on 1-12-2014 by Mesa]
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[*] posted on 2-12-2014 at 18:25


DAMN, I wish I had found this thread earlier.

About when you started (back in September) I was hot on the trail of making my own lab ethanol. It's difficult both to research and procure these days – there's an extremely large amount of mis-information out there, as you have uncovered just attempting to chronicle your experiment. So before I get too carried away, I would like you to consider

Fleishman's active dry yeast(I think that it's name)in your study.

In the lower 48 here, this is the stuff in the yellow and red envelopes on display in the grocery stores for the last 50 years in the three packet packaging.

Trying to keep it simple, I use simply water, sugar and this yeast. I bought the economy low actinic (LOL) jar. I have had an awfully fun time of it, albeit somewhat frustrating. And I have lots of commentary which I'm holding back right now.

A Couple of Things

1.) I can't believe all the hoopla over the idea that it would be illegal to distill a little EtOH in the home, although after having read all I've read, I would be surprised, if they might not be able to kick down my door and seize my lab here at the man cave. However, I would fight to defend my hobby and my pursuit thereof. After two good 3.5 L runs, I have maybe a little under 200 mL neat Ethyl and that will hold me for a while.

2.) Those boys up in the mountains of East Tennessee know what they're doing. And they swear by copper – surely the process is not catalytic, but why question the experts? The for-run, as we call it can contain all source of volatile no-no's; aldehydes, acetates, acids and other alcohols all contribute to a "flavor" (poison), can also come from contamination of the fermentation species'



I will try to put up some photos. This one is a, "what's wrong with this picture?"





Goofy fractional1.JPG - 2MB
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