Sciencemadness Discussion Board

n-butanol from fermentation

Magpie - 2-2-2006 at 19:41

I've been thinking that n-butanol would be a useful reagent. My old organic text says that it can be made along with acetone by bacterial fermentation of carbohydrates in a process developed by Chaim Weizmann (1874-1952). I read somewhere else that the yield is rather low, like around 2%.

This sounds like a fun synthesis. I wonder what enzyme and conditions are used. I have found nothing useful on this via Google. A search at a university library should be more fruitful.

Has anyone done this? Does anyone have an interest in trying it?

BromicAcid - 2-2-2006 at 19:57

Isn't it amazing how often nothing is found on google, but something is actually hanging around on sciencemadness ;)

1-Butanol Synthesis
"Industrial Fermentations" by Paul Allen, c. 1926 (available here, I think, from the ftp or on the net via emule) page 107 tells how to isolate the yeast that produces butyl alcohol. Basically it can survive 90 C for 1-2 minutes where as most other yeasts are killed.

Magpie - 2-2-2006 at 22:27

Thank you Bromic Acid. I searched sciencemadness but entered n-butanol instead of 1-butanol.

Yes, I quite agree, that sciencemadness is becoming the search engine for chemistry. Perhaps someday soon sciencemadness threads will be referenced in the scientific journals. :D

Magpie - 3-2-2006 at 19:20

I found more potentially useful leads in my own old textbook library. According to Shreeve (1956), pp 686-690, n-butanol was made by fermentation of corn according to Weizmann's process using the bacteria Clostridium acetobutylicum. This yielded 3 mols n-butanol/2 mols acetone/1 mol ethanol. A different Clostridium bacteria can be used to produce n-butanol using a molasses feed. This culture gave 3/1 n-butanol/acetone. The yield of solvents is 30% and up, carbohydrate basis. The solvents are separated by careful fractionation. Other references and patents are given.

I'm wondering if these bacteria are exotic and difficult to get? How does one buy bacteria? :o

neutrino - 4-2-2006 at 10:31

Biological suppliers usually sell bacteria. Check some of those. I would suggest looking at Carolina when their site comes back up.

Magpie - 4-2-2006 at 12:16

Yes, I looked at Carolina and they do have the C. acetobutylicum. Now whether or not they will sell it to me is another question. I will ask them on Monday.

Once you know the keywords all sorts of information pops up on Google. n-Butanol production via fermentation is currently a hot topic of research it seems. The best bacteria specie seems to be C. beijerinckii BA101. I have emailed one of the principal researchers to see if he will help me get the bacteria.

neutrino - 4-2-2006 at 13:38

Unless the item is listed as restricted you can order it from Carolina without any problem.

Magpie - 9-2-2006 at 20:01

The owner of C. beijerinckii BA101 has replied to my inquiry. He notes that this is an anaerobe and wonders if I have the expertise and equipment for its culturing.

The quick answer is "no, not at this time. But I can learn and get equipment." Where is Chemoleo when you need him? ;)

BromicAcid - 9-2-2006 at 20:26

On vacation if memory serves. :D

Esplosivo - 9-2-2006 at 23:40

Is there any real challange in culturing anaerobes? All one needs is to remove the oxygen from the closed system, such as purging the system with CO2. Anaerobic conditions have been achieved since God knows when during normal fermentations by facultative anaerobes. All you need to do is remove the oxygen from the nutrient solution (boiling removes most of the gases) and 'close' the system with a sort of a U- tube containing water (the one used by wine-makers that is fixed at the mouth of the container). The latter allows gasses going out but prevents the entry of oxygen. Remember to flush the remaining air in the container with CO2 to removed the toxic oxygen. Temp. control etc is fairly basic stuff which is easy to set up. I usually carry out anaerobic fermentations in 2.5Lt opaque glass containers with only one exit tube as a batch process.

[Edited on 10-2-2006 by Esplosivo]

Magpie - 10-2-2006 at 19:58

Thank you Explosivo. That doesn't sound so tough. I have some questions:

I presume you start with a sterilized fermentation container.

1. Other than glucose and water what other nutrients, vitamins, and/or minerals should be added to the broth?

2. Would a small CO2 "gun" using cartridges like those used for pressurizing beer dispensers be a reasonable way to purge the sealed broth. Or is there a better way?

3. When the broth is ready are the anaerobes introduced via hypodermic needle/septum? Or is there a better way?

As you can see I know nothing about this. ;)

Esplosivo - 10-2-2006 at 22:59

Chemoleo has a better knowledge of biochemistry so you might double check what I recommend with him.

Adequate sterilisation of the container can be easily achieved. I usually wash the container with an OTC NaOCl solution followed by 2/3 washings of near to absolute ethanol.

1. The nutrient solution for optimal growth of the bacterium frequently depends on the species of bacterium, which except the basic nutrients might require some 'special' nutrient at a higher conc., etc... There are though certain basic mixtures which are adequate for all types of bacteria, but this would be expensive since protein digest and yeast extracts are used. I would suggest a basic mixture of glucose (or some other carbohydrate which is adequate for the bacterium), some basic minerals and vitamins (probably a good source is multi-vitamin tablets, or something similar). Again the conc. of each of the components depends on the type of bacterium, and which might therefore need some further research.

2. I guess so. What I do is to bubble CO2 (from a CO2 fire extringuisher) through the nutrient solution for a couple of minutes. CO2 accumulates above the solution in the 'closed' container, and flushes the O2 out.

3. It looks very good for inocculation of the nutrient solution. I do not usually bother too much about sterility (especially if I know that the bacterial suspension I am using is not composed only of 1 species), therefore I simply place approx.10mL of the dilutish bacterial suspension into the 2.5Lt container quickly. This will not work for bacteria very sensitive to O2 but your septum idea will.

PS. I actually mentioned bacteria but if the inoculant is a fungus it's pretty much the same. The fungus is usually more resistant to extremes of osmolarity than the bacterium and some other exceptions but the procedure is fairly similar.

Maybe sparkgap can also help. He looks very informed in pharmacology, where these things are being used ever more often.

chemoleo - 13-2-2006 at 20:45

I'd order the bacterium (C. beijerinckii BA101, or C. acetobutylicum) and do as follows:

- Find out the optimum temperature of growth for either species.
- For a growth medium, use something rich - e.g. molasse was mentioned above, which is used in numerous biotech industrial processes, and is very rich in all nutrients. Here, a minimal medium of glucose, minerals and vitamins cannot be advised - even E. coli, the work horse of biochemists, grows at a doubling time of 40 min or more, as opposed to 20 min in optimal media (really, do not bother. That medium stinks for growth of anything other than E coli). The optimal E.coli media is 2 x tryptone extract (can be purchased), at 1.5 % w/v , and 1 % yeast extract (w/v), and 0.5 % (w/v) NaCl, which is autoclaved before use. This should contain most nutrients for sufficient growth. Again, this can be autoclaved; a simple pressure cooker, and oven-heated reagent vessels will do (jams are made that way). Once cooled down, inocculate the medium with a small amount of the culture. Remember microbiological techniques (or look it up), flame the flask neck containing the medium with a Bunsen, then very quickly insert a metal loop (that was previously flamed and cooled in a Bunsen) that was dipped into the originator culture back into the flask containing the growth medium, and put the lid back onto the flask (the lid being being heat resistant to oven heat at least 120 deg C). Then you have to shake it, gently, and continuously. Don't worry about oxygen being initially present - it is used up very quickly (e.g. cider is also made initally in O2, but it is used up quickly and anaerobic yeast growth can continue). Once the O2 is used up, anaerobic growth will continue, out-competing any aerobic growth that might be present.
Be aware of the fact that you add the target bacterium at a much greater conc than any of those floating around in the air and infecting your flask, so your chances are good that anaerobic growth of your target bacterium will continue (where competing organisms play little or no role).
Just make sure you understand the following:
Microbiological techniques (check google)
Check patents for the optimal growth medium, or better contact that guy who used it (I can do that for you if you want to)
Dont worry about air as long as the air was thoroughly sterlised by heat (heat all the air above the growth medium with a bunsen)
Find out at what point, in the optimal growth medium (and find out the identity of the optimal growth medium), the production of 1-butanol is maximal.

Do post if you have more questions. On this stuff I can sort of help, because I frequently work with bacteria.

Magpie - 14-2-2006 at 09:50

Thanks very much Chemoleo. You have given me a lot of valuable detail. I just hope I can convince the owner to go to the effort and expense of sending me some C. beijerinckii. It is supposed to give about twice the yield of n-butanol as the C. acetobutylicum available commercially.