Bioethanol Fermentation

LAB 4A
Dr. Maria Ghani
 Objective
To prepare for the ethanol fermentation

URL for observing laboratory presentation

https://www.youtube.com/watch?v=hAQ61hCxF2g
 Requirements
• Balance • Carboy (4L) or a big
• A big bowl bottle
• Funnel • Bottle (1.5L)
• Beaker • Plastic tubing
• Glass rod • Sugar (660g)
• Digital thermometer • Yeast Saccharomyces
cerevisiae (commercial
• Watch glass
grade)
• Spoon
• Duct tape (black
electrical tape)
• Silicon glue gun
 Theory
• Alcoholic fermentation converts one mole of glucose into
two moles of ethanol and two moles of carbon dioxide,
producing two moles of ATP in the process.
• The overall chemical formula for alcoholic fermentation is:
C6H12O6 → 2 C2H5OH + 2 CO2

• Sucrose is a dimer of glucose and fructose molecules. In

the first step of alcoholic fermentation, the
enzyme invertase cleaves the glycosidic linkage between
the glucose and fructose molecules.
C12H22O11 + H2O + invertase → 2 C6H12O6
Cont….
• Next, each glucose molecule is broken down into two pyruvate molecules
in a process known as glycolysis.
Glycolysis is summarized by the equation:

C6H12O6 + 2 ADP + 2 Pi + 2 NAD+ → 2 CH3COCOO− + 2 ATP + 2 NADH + 2H2O + 2 H+

CH3COCOO− is pyruvate, and Pi is inorganic phosphate.

• Finally, pyruvate is converted to ethanol and CO2 in two steps,

regenerating oxidized NAD+ needed for glycolysis:

• 1. CH3COCOO− + H+ → CH3CHO + CO2 catalyzed by pyruvate decarboxylase

• 2. CH3CHO + NADH + H+ → C2H5OH + NAD+ This reaction is catalyzed
by alcohol dehydrogenase (ADH1 in baker's yeast).
Diagrammatic Representation
 Procedure
• Take carboy and pour 3L of tepid water (24°C) in it.
• Weigh 660g of sugar (220g per liter of water used) and pour it into carboy.
• Firmly shake carboy until the sugar dissolves.
• Fill the beaker halfway with tepid water (24°C), Weigh 9.0g of yeast (3.0g
per liter of water used) then carefully pour it into the beaker.
• Gently stir in beaker to activate yeast.
• With the help of funnel and glass rod pour the activated yeast into the
carboy and stir to mix content.
• Cover the carboy with the lid having hole in it.
• Attach one end of tubing with the lid of carboy and put the other end
sealed with glass rod in bottle (1.5L).
• Fill half of the bottle with water.
• Seal the ends of tubing and lids.
• Store both containers at 25°C for 1-2 weeks.
 Observation
• The release of bubbles of carbon dioxide can
be easily observed in bottle from the end of
tubing.
Bioethanol Fermentation
LAB 4B
 Objective
To filter the fermented liquid
 Requirements
• Assembly of fermented liquid
• Clean carboy
• Funnel
• Filter paper
 Procedure
• Cut through the glue and remove the seal to
open the assembly.
• Place the funnel in clean carboy.
• Fold the filter paper and place it on funnel.
• Pour some of the fermented blend into the
funnel and allow it to filter through the paper.
• Repeat the filtration process until its done
completely.
Bioethanol Fermentation
LAB 4C
 Objective
To distill the filtered sample by using distillation
assembly
 Theory
Distillation means taking the fermented ethanol
and water mixture and adding heat to separate
them, typically in a still assembly.
Since ethanol evaporates faster than water, the
ethanol rises through a tube, collects and
condenses into another container leaving the
water left behind.
 Procedure
• Assemble the distillation assembly carefully.
• Pour filtered sample in the assembly flask.
• Turn on the heating mantle and allow the filtered
sample to boil.
• Collect 3 different fractions from the assembly.
• Collect first fraction from the first drop till 87°C.
• The second sample is collected between 87°C and 97°C.
• Collect the third sample on boiling temperature till end.
 Observation
• Now we have three different distilled
fractions.
Bioethanol Fermentation
LAB 4D
 Objective
To test distilled fractions for confirming
bioethanol production.
 Requirements
• Pippete 2mL
• Watch glass
• Absolute ethanol (as control)
• Fractions
 Procedure
• Measure 2mL of each fraction into watch
glass.
• Lit the match stick and light the sample to test
their flammability
 Results
• Positive results showing samples that are
flammable.
• While sample that doesn’t catch fire and non-
flammable isn't the desired fraction means it
is not ethanol.
• Compare your results with the flamed
absolute ethanol.