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LABORATORY TECHNIQUES, SCIENCE

Alcohol Fermentation
Melissa Martinez
05/30/2022

Introduction
Alcoholic fermentation or ethanol fermentation is a biotechnological process in which sugars such
as glucose, sucrose, and fructose are converted into ethyl alcohol and carbon dioxide in the
presence of yeast (S. cerevisiae), some bacteria, or other microorganisms. 

Alcoholic fermentation is a type of fermentation process widely used in producing alcoholic

beverages such as beer and wine. Fermentation is a metabolic process where enzymes or
microorganisms induce the decomposition of organic substances. This metabolic process makes
beneficial changes in the food and beverages that maintain a healthy gut and increase the shelf
life of food. For instance, it makes the food more flavorful and nutritious and increases the shelf life. 

Ethanol fermentation is a complicated process that involves various chemical, biochemical, and
physicochemical processes. First, the sugar breaks down to form pyruvic acid, which is then
converted into ethanol and carbon dioxide. The regeneration of NAD+ provides the yeast with
energy to convert pyruvate molecules into ethanol and CO2. Typically, yeast has the capability to
function in the presence and absence of oxygen. However, alcoholic fermentation occurs in the
absence of oxygen (anaerobic condition). Under anaerobic conditions, the fermentation takes
place in the cytosol of yeast. (Lee, 1983)

Principle
The basic principle of alcohol fermentation is that it is carried out by living yeast cells under
anaerobic conditions. These cells absorb sugar molecules and break them in the presence of
oxidation and reduction enzymes; as a result, by-products such as ethanol, carbon dioxide, water,
and heat are produced. 

Alcoholic fermentation takes place in two steps, i.e., glycolysis and fermentation. Glycolysis involves
breaking down sugar to form pyruvate molecules in the presence of yeast. In this step, 2 pyruvic
acid molecules are produced. It is followed by fermentation in which the 2 pyruvate molecules are
converted into 2 ethanol molecules, 2CO2, and ATP. In the absence of oxygen, the pyruvate
molecule is first transformed into acetaldehyde and CO2 in the presence of the pyruvate
decarboxylase enzyme. At the same time, NADH regenerates NAD+ bypassing its electrons to
acetaldehyde in the presence of alcohol dehydrogenase enzyme, and as a result, ethanol is formed.
(Walker & Walker, 2018)

The overall chemical equation can be explained as follows:

C6H12O6 → 2 C2H5OH + 2 CO2

Apparatus 
Erlenmeyer flask 

Delivery tube 

Cork 

Glass tube 

Clamp stand

Stirring rods

Glucose powder

Yeast powder

Lime water

Paraffin oil

Procedure
– Prepare 5% glucose solutions by mixing 5 g of glucose powder in 10ml of lukewarm water in a
flask. 

– Prepare that 10% yeast suspension in another flask by adding 5g of yeast in water. 

– Take the prepared glucose solution in a larger test tube and add the yeast suspension in a 5:1
ratio. 

– Now add liquid paraffin drops along with the inner side of the test tube so that it covers the
surface of the glucose-yeast solution completely. 

– Cover the test tube with the cork containing the delivery tube. Ensure that the delivery tube’s end
does not touch the soil surface. 

– Seal the cork with glycerol 

– Set the larger test tube properly on the clamp stand and set a smaller test tube containing lime
water on the other end of the delivery tube. 

– Make sure that the tube is immersed well in the lime water.

– Allow this setup to stand for a few minutes until the air bubbles start to come out of the end of the
delivery tube and the lime water turns milky. 

– At the same time, a frothy layer will form on top of the oil layer in the larger test tube. 

– Disassemble the apparatus as fermentation has occurred. 

Results 
The contents in the larger test tube will give a strong ethanol smell, which suggests that alcohol
has been produced due to fermentation. 

The cloudiness of lime water indicates that the gas produced inside the test tube is carbon
dioxide, a by-product of fermentation.

Applications
Alcoholic fermentation is used in industries to produce alcoholic beverages, bread, and vinegar. For
instance, wine is synthesized by fermentation of natural sugars found in grapes. Similarly, rums are
produced by fermenting sugar cane product molasses followed by distillation.

(Boeira et al., 2021) conducted a study to mitigate nivalenol using alcoholic fermentation and
magnetic field application. The conditions set for this study were nivalenol (0.2 µg mL-1), magnetic
field application (35 mT) along with simultaneous use of mycotoxin. The results showed that
glutathione and enzyme peroxidase level was significantly increased during the experiment, and
nivalenol was mitigated by 56.6%. 

Soursop fruit is highly nutritious yet perishable, so (Ho et al., 2019) conducted a study to produce
soursop wine through alcoholic fermentation. Two cultures, i.e., mushroom (Pleurotus pulmonarius)
and yeast (Saccharomyces cerevisiae), were used together to determine fermentation effects on
physiochemical and antioxidant activities of soursop wine. Temperature, pH, time, and culture ratio
were optimized to maximize ethanol production. This alternative fermentation technique showed
increased ethanol production with higher antioxidant activities. 

Strengths and limitations

Strength 

Alcoholic fermentation is a simpler process that is carried out from renewable resources. It does not
require high amounts of energy for fermentation, due to which this is a low-cost process. The
average temperature required for fermentation is between 35 to 40°C. 

Limitations 

The major drawback of this alcoholic fermentation is that it slows down towards the end because of
the increased concentration of alcohol in the medium, which is toxic to yeast. So, fermentation
ceases even before the sugar is metabolized completely. This incomplete process has a high risk of
bacterial spoilage. 

The final product produced is impure, and it requires more steps to obtain the purified alcohol.
Moreover, it is a batch process that requires a large amount of time to produce sufficient alcohol. 

Precautions
Ensure to clean all the apparatus properly to avoid any bacterial contamination. 

Wear insulated gloves while handling hot materials

Use latex gloves while handling chemicals and samples

Wear proper clothes (long pants and closed-toe shoes), a lab coat, and safety glasses

Summary
– Alcoholic fermentation is a process in which glucose is converted into alcohol and carbon dioxide
in the presence of yeast or other microorganisms. 

– It is a batch process that involves two steps. 

– In the first step, the glucose breakdown to form pyruvate molecules by glycolysis. In the next step,
the pyruvate molecule gets converted into ethanol and carbon dioxide in the presence of NADH. 

– Fermented foods contain beneficial microorganisms and probiotics that maintain a healthy gut
and increase the shelf life of food.

– Alcoholic fermentation is widely used in industries for making bread, alcohol, and other products.

Reference 
 

Lee, F. A. (1983). Alcoholic Fermentation. Basic Food Chemistry, 323–341.

https://doi.org/10.1007/978-94-011-7376-6_14

Walker, G. M., & Walker, R. S. K. (2018). Enhancing Yeast Alcoholic Fermentations. Advances in

Applied Microbiology, 87–129. https://doi.org/10.1016/bs.aambs.2018.05.003

Boeira, C. Z., Silvello, M. A. de C., Remedi, R. D., Feltrin, A. C. P., Santos, L. O., & Garda Buffon, J. (2021).
Mitigation of nivalenol using alcoholic fermentation and magnetic field application. Food
Chemistry, 340, 127935. https://doi.org/10.1016/j.foodchem.2020.127935

Ho, C. W., Lazim, A., Fazry, S., Hussain Zaki, U. K. H., Massa, S., & Lim, S. J. (2019). Alcoholic
fermentation of soursop ( Annona muricata ) juice via an alternative fermentation
technique. Journal of the Science of Food and Agriculture, 100(3), 1012–1021.
https://doi.org/10.1002/jsfa.10103

Melissa Martinez
Melissa is responsible for developing and executing plans for the strategic direction and
operational growth of ConductScience. She provides guidance on product development,
strategy, and operations. She has extensive experience in project management and has a
deep understanding of the needs of the engineering and scientific communities. Melissa is a
strong advocate of efficiency and customer focus, and she enjoys leveraging her technical
background to solve problems and improve processes.

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