Can you elaborate on what you measure at 600 nm? From the basic organic chemistry that I remember, when you oxidize ethanol you get first ethyl aldehyde and then acetic acid. The K2Cr2P7 + sulfuric acid is a strong oxidizer, so after applying it on your sample there is no more alcohol left, since it is converted to acetaldehyde, which in  turm becomes acetic acid. So what do you measure at 600 nm?

See what I found when I googled: "Alcoholic fermentation by yeast cells":

The ability of yeast cells to convert sugar into Carbon dioxide and Alcohol is down to enzymes. Several enzymes are involved each does its step in the process. The final step is Zymase reduction which takes the end product of the other enzymes (acetaldehyde/glycerol), and turns this into good old ethyl alcohol. Sadly high concentrations alcohol actually destroys enzymes and kills the yeast cell. Different strains of yeast can tolerate different concentrations of alcohol.. Brewers yeast cannot withstand much beyond 5 or 6% Alcohol by volume. Wine yeast is more tolerant at a range of 10-15%

Specially cultured strains of yeast with the correct environment can withstand alcohol levels up to 21% alcohol.

Thank you for your reply.But my question was about the authentication of the procedure which i followed. Due to laboratory limitation, I was not able to measure ethanol by HPLC or GC. So, I used spectrophotometric method to measure ethanol concentration. But using this method, I have some confusion about the reliability of the method. And my another question was, what is the maximum percentage of ethanol is produced by an average yeast strain in the culture media?

See my second answer above.

In this reaction,ethanol is oxidized by dichromate in the presence of sulfuric acid with the formation of acetic acid(as you said) .Dichromate (Cr2O7--, Cr(VI)) is yellowish in color (Blank) and the reduced chromic product (Cr+++, Cr(III)) is intensely green(after the reaction). This chromic compex is measured at 600 nm.

One way to validate parameters would be to quantify remaining sugar concentration in the fermentation broth as well! As you would know substrate (feed eg sugar) concentration at the beginning of fermentation process and you would like to know the residual substrate (sugars?) along with the product of your interest (in this case Ethanol).

You can run standard reducing sugar assay along with potassium dichromate reagent assay to analyze the overall reaction stoichiometrically!

Hi Tahmina,

the concentration of ethanol produced by yeast during growth depends on the amount of sugar in the culture  media. In YPD, where sugar concentration is 20 g/L, yeast produces very low ethanol concentration not only due to the 'low' sugar concentration but also due to respiration. As you increase sugar concentration in the media, respiration is repressed and more ethanol is formed. If you have 200 g/L of sugar in the culture media you should expect about 12% (v/v) ethanol concentration. If more sugar is present you will get more ethanol. Of course some yeast strains tolerate high ethanol concentration better than others and you might see some residual sugar in the case of sensitive strains.

Now to the method, first make sure you are expressing ethanol using the right units, v/v is usually used by the food industry, so volume of ethanol per 100 mL of media. I am not completely sure but the reagents in the method you describe could potentially react with glycerol, also present at relevant concentrations. It is possible then that your higher values are a result of measuring ethanol+glycerol. You could prepare standard solutions of glycerol and mixtures glycerol/ethanol to confirm if you have any cross reactions with your method.

I hope this helps.

Cheers,

Cristian

Thanks a lot Mr.Prachand Shrestha and Mr.Cristian Valera for your informative suggestions and clear my confusions.

Can anybody recommend an effective method to develop an ethanol standard curve to be used in yeast fermentations?