This 14 g Glc/L is based on HPLC in the hydrolysate. I obtained ethanol level of 10.5 g/L using S. cerevisiae. Can this be trusted experimental evidence?
The maximum theortical yield of ethanol from glucose is 0.511g/g of glucose. So the maximum ethanol that can be produced from 14g/L glucose is 7.15g/L ethanol. 1 mole of glucose is oxidised to produce 2 moles of ethanol. I think you need to repeat your experiment as the ethanol obtained is higher and normally with even 90-95% efficiency levels of fermenting yeast the ethanol titre cannot exceed 6.7g/L.
Quantification by HPLC requires rigorous calibration with standards and regular repeats as column properties change over time. Mistakes can happen at various levels (the standard itself can be erroneous). Measuring ethanol levels accurate is also harder than you may think when you have a complex mixture of unfermented carbohydrates in your fermented broth. You should also measure glucose left in the broth after fermentation (glucose assays are very sensitive and easy these days as they are derived from routine diabetes test kits). Some of the glucose is not fermented, but used as building block by the yeast to make other bio-molecules to sustain cellular functions and to allow cell budding and growth (in other words some of your sugars and nutrients become more yeasts). As Deepti pointed out correctly, you cannot trust your experimental evidence and you need to do some systematic troublehooting.
Thanks J Denecke, D Agrawal and J Ghosh for all your invaluable inputs. I will recalibrate the instruments and repeat the experiments as well. Thank you.
Clearly, you are getting more yield than the theoretical expectation. This means there is an error, either in the ethanol analysis or there may be another carbon source in your media.
If you have HPLC with RID, then use Aminex organic acid column from biorad on which you can analyse glucose (retention time 8.5min) and ethanol (retention time 20.8 min) in single injection. Also you will be able to see other acids like formic, acetic, succinic produced in the fermentation process on same column. Mobile phase is 0.05M H2SO4 with 0.6 ml/min flow.
Once you have standard curve then redo all calculations.
Also check your media components for other carbon source.
I am replying it very late you must have already corrected your analysis.
If the ethanol determination is correct then you need to check the glucose contents in your hydrolysate or vise versa as both the results of yields are not matching as per the standard theoretical yields. It will give you correct results if you repeat it along with the correct reference standards.
if your using fungus other than s.cervesiae expect very less yeild
I also agree with all points suggested. Ethanol yield can't be exceed more that theoretical yield, which is 0.511g/g of glucose, but sacchro. Cerv. may convert only 90- 95% of total sugar present. It clearly shows that there is some experimental error. I uses Agilent, Waters HPLC, with HPX 87 P column for sugar detection. Use of sigma sugar standard and with proper calibration can reduces the error in quantification and GC for ethanol quantification.
Questioning about experiment may reduce the confidence for your research. You must repeat your experiment in triplicate to confirm validity. There is some calculation error, which you need to rectify....
With a non genetically engineered strain of S.cerevisiae only C6 sugars are fermented to alcohol C5 sugars are not affected.
You can try your analysis of C6 sugars with simple chemical methods like DNSA to confirm your analytical results.
From 1 g glucose, maximum 0.51 g ethanol (Theoretical yield) can be produced. Anything higher than this is not correct. There must be some error either in glucose or ethanol measurement.
C6H12O6 = 2C2H5OH +H2O + 2CO2
180 2*46=92
92/180= 0.51
please guide me. please tell me about condition process and amount of raw material.
The substrate is thatch grass hydrolysate
Sunita Singh
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