Sep 05, 2025
Version 3
Enzymatic Ethanol Assay V.3
- Sarah Hammer1,2,
- Dev Kapadia1,2,
- Shuen Hon1,2,
- Marybeth Maloney1,2,
- Daniel Olson1,2,
- Lee Lynd1,2
- 1Thayer School of Engineering, Dartmouth College, Hanover, NH, USA;
- 2Center for Bioenergy Innovation, Oak Ridge National Laboratory, Oak Ridge, TN, USA
- Lynd/Olson Lab
Protocol Citation: Sarah Hammer, Dev Kapadia, Shuen Hon, Marybeth Maloney, Daniel Olson, Lee Lynd 2025. Enzymatic Ethanol Assay . protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6b8n5gqe/v3Version created by Daniel Olson
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 05, 2025
Last Modified: September 05, 2025
Protocol Integer ID: 226504
Keywords: alcohol dehydrogenase, assay, ethanol, 96-well plate, rapid detection of ethanol concentration, ethanol concentrations in experimental sample, detecting ethanol evaporation, alcohol dehydrogenase, enzymatic assay, estimating ethanol concentration, ethanol concentration, determining relative ethanol production, ethanol evaporation, relative ethanol production from microbial fermentation, ethanol evaporation from media, ethanol, ul water to the assay plate, assay, assay plate, assay master mix, adding assay master mix, microbial fermentation, such as high performance liquid chromatography, high performance liquid chromatography, experimental sample, enzymatic ethanol assay, semicarbazide concentration
Funders Acknowledgements:
US Department of Energy - Center for Bioenergy Innovation
Grant ID: DE-AC05-00OR22725
US Department of Energy
Grant ID: ERKP886
Abstract
This protocol describes a 96-well-plate-based, enzymatic assay for reliably estimating ethanol concentrations in experimental samples in one hour. In the presence of excess NAD+, alcohol dehydrogenase (ADH) is employed to convert ethanol to acetaldehyde. The concomitant conversion of NAD+ to NADH is monitored via increased absorbance at 340 nm. When highly accurate analytical techniques (such as high performance liquid chromatography) are not necessary, or are too costly or low-throughput, this assay offers reliable, inexpensive, and rapid detection of ethanol concentrations. This assay is useful for applications such as determining relative ethanol production from microbial fermentations, and detecting ethanol evaporation from media.
Version notes:
10-27-2021. Add 50 ul water to the assay plate first, then add sample. This prevents ethanol evaporation and allows the assay to be started (by adding assay master mix) immediately before reading.
9-4-2025. The semicarbazide concentration was increased 10x, from 0.125 g to 1.25 g (i.e. 12.5 g/L). This results in a final concentration of 94 mM. This allows the reaction to proceed for longer before slowing down.
Materials
Reagents
Sodium pyrophosphate decahydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #221368
GlycineMerck MilliporeSigma (Sigma-Aldrich)Catalog #G7126
Semicarbazide hydrochlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #S2201
Hydrochloric Acid Solution, 1NFisher ScientificCatalog #SA48-1
β-Nicotinamide adenine dinucleotide hydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #N6522
Note
This specific vendor and catalog number are recommended for β-Nicotinamide adenine dinucleotide hydrate (NAD+) to avoid solubility issues.
Potassium phosphate dibasicFisher ScientificCatalog #P288
Potassium phosphate monobasicFisher ScientificCatalog #P380
Bovine serum albuminMerck MilliporeSigma (Sigma-Aldrich)Catalog #A3059
Alcohol dehydrogenase enzymeMerck MilliporeSigma (Sigma-Aldrich)Catalog #A3263
Ethyl alcohol, 200 proof, anhydrous, ≥99.5%Merck MilliporeSigma (Sigma-Aldrich)Catalog #459836
Clear 96-well flat-bottom microplateCorningCatalog #353072
ThermalSeal RTS sealing filmMerck MilliporeSigma (Sigma-Aldrich)Catalog #Z742256
Note
Several sealing films were tested during protocol optimization. The ThermalSeal RTS sealing film was the highest performing seal for this application, consistently preventing ethanol evaporation.
General Supplies and Equipment
- Laboratory balance
- P1000, P200, P20, and P10 pipettes and corresponding pipette tips
- pH meter
- 1.5-mL microcentrifuge tubes
- -80°C and -20°C Freezers
- Repeater pipette
- 5 mL repeater pipette tip
- Multi-channel pipette (8-channel) capable of transferring 10 μL
- Microplate spectrophotometer
Note
Protocol was developed with BioTek PowerWave XS microplate spectrophotometer.
Preparation
Create a microplate spectrophotometer program to read absorbance at 340 nm of each well in a 96-well plate.
- The program should take absorbance readings at 340 nm every 20 - 30 seconds (or at minimum interval), shaking for 10 seconds immediately before each reading.
- The program should be set to run for 1 hour at 30 °C .
Make nicotinamide adenine dinucleotide (NAD+) stock solution
| Component | Concentration | Amount | |
| NAD | 50 mM | 0.4976 g | |
| Water | --- | Up to 15 mL |
Vortex to ensure that NAD+ is completely dissolved.
Store aliquots in 1.5-mL microcentrifuge tubes at -80 °C .
Make 9.0 glycine buffer
| Component | Concentration | Amount | |
| Sodium pyrophosphate | 33.3 g/L | 3.333 g | |
| Glycine | 1.67 g/L | 0.167 g | |
| Semicarbazide hydrochloride | 1.25 g/L | 0.125 g | |
| Hydrochloric acid (1N) | --- | To pH 9.0 | |
| Water | --- | Up to 100 mL |
Store at 25 °C for up to 1 month.
Make alcohol dehydrogenase (ADH) enzyme stock solution
| Component | Concentration | Amount | |
| Potassium phosphate dibasic | 83 g/L | 1.66 g | |
| Potassium phosphate monobasic | 17 g/L | 0.34 g | |
| Bovine serum albumin | 1 g/L | 0.02 g | |
| Alcohol dehydrogenase enzyme (~300 U/mg) | 20 U/mL | 1.36 mg | |
| Water | --- | Up to 20 mL |
Store aliquots in 1.5-mL microcentrifuge tubes at -20 °C for up to 1 month.
Experimental Steps
Make ethanol standards at eight concentrations encompassing the range of concentrations expected from the experimental samples, including a 0 g/L ethanol standard. Dilute ethanol in the same media present in the experimental samples to make the standards.
Note
This protocol was optimized for detecting ethanol concentrations between 0.05 and 1.5 g/L in the assay solution, which corresponds concentrations between 1 and 30 g/L in the experimental samples. Measuring ethanol concentrations outside of this range will require preparing a different standard curve, and making different dilutions of samples (while maintaining 200 μL total volume in each well) to achieve final ethanol concentrations between 0.05 and 1.5 g/L in the assay solution.
Make master mix for enzyme assay on ice, adding components in the order listed in the table below. If NAD+ and ADH stock solution aliquots are frozen, defrost the necessary volume on ice before proceeding.
Note
The master mix in the table below is sufficient for one full 96-well plate (with each well containing 190 μL of assay master mix). If running more or less than one plate, scale the recipe accordingly.
| Component | Final Concentration | Volume | |
| NAD stock solution | 8 mM | 3.2 mL | |
| ADH stock solution | 0.1 U/mL | 111 uL | |
| Glycine buffer | --- | Up to 20 mL |
Keep the master mix on ice during and after preparation.
Turn on the microplate spectrophotometer and open the program (defined in Step 1) to begin heating to 30 °C , the temperature at which the assay will be run.
Fill all wells of a 96-well plate with 50 μL of water. The purpose of the water is to minimize ethanol evaporation during subsequent sample pipetting steps.
Note
Work as quickly as possible through the next three steps (Steps 9 -12) in order to minimize ethanol evaporation and substantial progression of the enzymatic reaction before the microplate program has started.
Designate two of the 12 columns in the 96-well plate for standards, and add 10 μL of each standard with a P10 pipette to the 190 μL of assay master mix.
In the remaining wells, add 10 μL of each sample. It is recommended to use a multi-channel (8-channel) pipette for this step to fill the plate as rapidly as possible.
Start the assay by adding 190 μL of the assay master mix using a repeater pipette and a 5 mL repeater pipette tip.
Note
At this point, the assay has started and the plate should be read as quickly as possible.
Seal the plate with a ThermalSeal RTS sealing film. Use a sealing film roller or a roll of tape to ensure that the film is adhered closely to the rim of each well, taking care to avoid any large wrinkles or gaps.
Place the sealed 96-well plate (without a lid) in the microplate spectrophotometer (preheated to 30 °C ) and start the program to read the absorbance at 340 nm.
Note
As alcohol dehydrogenase converts NAD+ to NADH, absorbance at 340 nm will increase with time. If after 30 minutes, the absorbance at 340 nm plateaus (is no longer increasing) for all of the samples and standards, the program can be terminated. If not, let the program run for the full hour.
1h
Data Analysis
Use the 340 nm absorbance data to calculate Vmax (change in absorbance per unit time) for each well in the plate, including wells containing experimental samples and standards. Use at least 30 data points in a range where absorbance is linearly increasing with time to calculate Vmax.
Use the standard wells with known ethanol concentrations to generate a standard curve, as specified in the sub-steps below. Sample data is attached.
Sample Data_2021_02_09.xlsx
Sample Data_2021_02_09.xlsx User-defined standard concentrations will serve as x-axis data inputs. Vmax data at 340 nm will serve as y-axis data inputs.
Use a four-parameter nonlinear regression curve fit to generate a standard curve with the formula Y = (A-D)/(1+(X/C)^B)+D.
A representative standard curve, relating Vmax (mOD/min) at 340 nm, denoted here as Max V [340], and ethanol concentrations (g/L).
Use the standard curve to calculate ethanol concentrations in the experimental samples.