I am working on bioethanol production from lignocellulosic biomass. However, I require some clarification on the methods for calculating initial sugar concentration, sugar consumption rate, ethanol yield (in g/g and g/l). Could you please recommend simple calculations or protocols?
Sept. 21, 2023
Dear Merlin,
I will try to provide you with some information that hopefully will help you.
There are many methods for measuring sugar concentration. Some are very sophisticated, e.g., gas chromatography, HPLC, etc. If you don't have access to such instrumentation, you can use colorimetric methods. There are many of these. They use readily available chemicals and require only a bench-top spectrophotometer for analyses. One that I have used in the past was developed by Dubois et al. "Colorimetric method for determination of sugars and related substances". Analytical Chemistry, Vol. 28, p. 350-356. This is an OLD method (from 1956); however, it is still in use. It is very quick and easy to use. Requires phenol and sulfuric acid. Detects many different sugars, e.g., glucose, mannose, galactose, fructose, xylose, maltose, raffinose, etc. It is very sensitive: can detect as little as ~5 micrograms (even less) of carbohydrate (CHO). It can also detect the above sugars in polysaccharides (e.g., starch, cellulose, etc.) and oligosaccharides.
Reagents: 1) 5% phenol in water; 2) concentrated (18 molar) H2SO4.
Procedure: Add your test sample to DI-water to final volume of 1.0 mL. Then add 1 mL 5% phenol, followed by 5 mL H2SO4. (When the acid is added, the resulting solution gets VERY HOT, so hold the test tube by the top, not by the bottom.) Mix the tube contents (vortex mixer). An orange color develops very quickly. Wait until the solution is cool to the touch, then read the absorbance at 490 nm on a spectrophotometer. You will need to run a series of sugar standards for quantitation of the CHO in your samples. CAUTION!! Be sure to wear gloves, lab coat and especially safety goggles when doing the assay. If you're careful, you'll be OK.
For detection of sugar consumption rate, e.g., during a fermentation, you can take samples of the fermentation broth at various times, clarify the broth (centrifugation) and measure the sugar concentration (as above) and see how it decreases with time. You can also measure the corresponding increase in ethanol.
Calculating ethanol (EtOH) yield: Let's assume you are using yeast to ferment glucose to ethanol. Consider the reaction:
C6H12O6 ----------> 2 CH3CH2OH + 2 CO2
Glucose EtOH
If the yeast completely ferment 1 mole (180 g) of glucose, they will produce 2 moles of EtOH (2 x 46 g/mole) or 92 grams, plus 2 moles of CO2
(2 x 44 g/mole) or 88 grams. The EtOH yield is (92 g/180 g) x 100 = 51.1%. If the above fermentation is done in a 1-liter volume, you will have 92 g EtOH/liter or 92 mG/mL. If you divide the weight of ethanol by its specific gravity, that will give you the # grams of EtOH/L. If I recall, the Spec.Gravity of EtOH is ~0.79 g/mL.
From the equation and calculations above, you can see that if you start with 1 mole of hexose (e.g., glucose, galactose, etc.), the EtOH yield will be ~51.1% of the initial weight of the sugar. IMPORTANT: The 51.1% is only a THEORETICAL yield. You will NEVER get the theoretical yield. This is because during the fermentation, the yeast will use some of the sugar as a nutrient on which to grow and multiply in number, so a small amount of the sugar is never converted to EtOH . Of the total sugar, only ~93-94% of it is fermented to EtOH. This % will vary from one fermentation to another.
I hope this information helps you. If you have other questions, let me know. I will try to answer them for you. Good luck w/ your research.
Bill Colonna, Midwest Grape & Wine Industry Institute, Iowa State University, Ames, Iowa, USA. [email protected]
Initial sugar concentration: You need to Know total sugar concentration by phenol sulfuric acid method. Total sugar does not answer how much ethanol you will get. Most important what are the fermentable sugars present in it. So you need to know Reducing sugar by DNS method. As you are working on lignocelluloase biomass you need to know the method for hydrolysis method. You also need to know the what are the inhibitors.
Sugar consumption rate: Once you kow the sugar concentration you get idea about rate. Try to study growth kinetic of yeast.
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