I am using a fermentation media of composition mentioned below and yeast (S. cerevisiae) culture
•3g/L yeast extract
•43g/L reducing sugars(glucose),
•3g/L peptone,
•1g/L KH2PO4
•0.5g/L (NH4)2SO4
•And 0.5g/L MgSO4·7H2O
•10%v/v inoculum
Can anyone suggest me the best way to recover and quantify ethanol (without HPLC/ GC)
How much liquid you have ? If you have enough you may try the traditional method in the wine industry: distil a known volume until no more ethanol is distilled (from the boiling point). The distillate contains all the ethanol. Adjust to the initial volume and measure the density. There are tables which give the % alcohol as a function of density ans T.
Glucose fermentation converts one mole of glucose into two moles of ethanol and two moles of carbon dioxide, producing two moles of ATP in the process. The overall chemical formula for alcoholic fermentation is: C6H12O6 → 2 C2H5OH + 2 CO.
You can use an ethanol meter to measure the ethanol percentage, but the accuracy is a little bit doubtable. However, it is possible to make an alternative method to detect ethanol production, such as the Durham tube test while fermentation. The inverted Durham tubes trap carbon dioxide gas that indicates sugar fermentation. But it cannot use to quantify the ethanol content.
refer Article Isolation and Identification of Cellulase Producing and Suga...
for the Durham tube test
I don't know what is an "ethanol meter". I proposed the historical density measurement.
You may check the introduction of the attached paper as a mini-review of the various standard methods. The density method is called pycnometry.
@ Jean-François Gal
The fermentation volume used is 500 mL and live yeast culture has been used.
I am afraid that we do not have the specific gravity bottle too for measurement of ethanol density.
The other process which I found is titration method using dichromate. Is it a reliable method? I have never tried it.
Further, to obtain ethanol from fermentation media, should we directly use the fermentation media with live yeast cells for distillation or should we first settle down the yeast cells (by adjusting the pH, followed by addition ZnSo4 for settling) and filter the fermentation media, after that use for distillation?
The titration method is reliable, and widely used as demonstration for student labs. You can find a lot of tutorials. I used it myself long time ago in student lab. It works on relatively dilute solutions so you may have to dilute your samples. It is possible that distillation is not required, a simple filtration removing organic residues could be sufficient. I have found an example (attached) in which wines and beers are directly titrated after dilution.
Jean-François Gal
Thank you very much for your reply.
Should NaoH (for adjusting the pH) and ZnSo4 be used for settling the microbial biomass or other organic residues ? After which the fermentation media can be filtered, or we can directly filter the fermentation media? I am not getting the exact procedure of uses of chemicals for settling out the microbial biomass, followed by recovery of ethanol. Please suggest me the procedure to follow.
If we want to store the ethanol sample then should we follow the distillation? Then should we do distillation at 70 degree centigrade only, without adding any other chemicals (As water and ethanol are azeotropic mixture, chances of water contamination is there in ethanol distillate)?
Waiting for your response
As recall many alcohol and wine manufacturers use an specific gravity hygrometer to measure %ethanol. But they have a known matrix and have developed a linearity chart with 'official' results from a GC. The AOAC has a graph in their latest edition (I think, its been 50 years).
I don't know if the treatment you mention (to stop the fermentation I suppose) is necessary before filtration. The broth will be heated at >100° anyway.
In fact, the distillation will occur at about 100°C or above in the liquid phase. The temperature at the head of the column will be about 78°C, corresponding to the azeotrope (96% ethanol) and will increases progressively to 100°C, when all ethanol will be distilled. You will have to adjust the volume of the distillate before any titration or measurement because you have to refer to the initial volume.
There might br an ELISSA kit for the analysis of Ethanol. It would be marketed to food manufacturers. Check the Boehringer Ingelheim website.
I checked this one
Ethanol Assay Kit Buy for the measurement of ethanol | Megazyme
It is for very diluted samples. It needs a spectrophotometer. Working on so small volumes cannot be highly accurate.
There are other spectrophotometric methods based on absorbance of oxidants (bichromate or permanganate as far as I remember), also used in student labs.
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