Biosynthesis and Purification of Ethanol Using Simple and Fractional Distillation

Fabris Kevin Sebineza

Student I.D number: 3153377

Lab for Organic Chemistry I 2202-075

Instructor: Simran Sandhu

Date Of Experiment Performed: September 26, 2023

Date Of Experiment Submission: October 16, 2023

Page 1 of 23
 Abstract

Ethanol is produced from yeast enzymatic conversion of sugar into alcohol. Our

experiment focused on the purification of ethanol hence, comparing simple and fractional

distillation to determine which method offers efficient separation of ethanol from impurities to

achieve high ethanol purity and concentration. We hypothesized that fractional distillation would

produce a higher ethanol purity and concentration than simple distillation. Experimentally, a

sucrose solution was fermented for a week, then distillation was carried out for the remaining

two weeks. Ethanol was distilled by using both simple and fractional. The density and volume of

distillates, ethanol content, and quantity were measured in all distillates. The results obtained

confirmed the hypothesis. simple distillation yielded 40.8% ethanol purity, while fractional

distillation achieved an average of 93.3% ethanol purity. To achieve a near 95% ethanol purity,

fractional distillation is ideal due to its fractionating column that allows for multiple vaporization

and condensation cycles. The study stressed why it is necessary to control contamination of the

wort to prevent flocculation and the ability to genetically manipulate yeast cells to increase

yields. Despite that increasing sugar concentration can result in high yields, it can also reduce

yields. Other methods such as self-cycling fermentation can increase ethanol productivity.

Considering that ethanol is crucial in producing healthcare products, alcoholic beverages, fuel

additives, and perfumes, it is important to advance research in genetic engineering and

fermentation to increase ethanol production and solve challenges that reduce ethanol production.

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 Introduction

Ethanol fermentation is a process where sugars are broken

down by microorganisms in the absence of oxygen. It contributes to the production of various

food products and the preservation of food and improves food safety through the inhibition of

pathogens or the removal of toxic compounds. It involves the conversion of sugars into alcohol

by microorganisms such as yeasts. Yeasts, provide enzymes needed for fermentation during

biochemical reactions. Their common occurrence in foods and long historical use contributes to

their acceptance as safe for human consumption. Sucrose, an important source of sugar for

ethanol production, is the main organic compound produced by plants. It is produced

commercially from sugar cane and sugar beets.

During fermentation, Sucrose is broken down into glucose and

fructose. Glucose molecules are then broken down into pyruvate molecules, through glycolysis,

yielding NADH and ATP. Finally, each pyruvate molecule is converted into an acetaldehyde

molecule and gives off a molecule of carbon dioxide whilst generating NAD+(P). The

acetaldehyde molecules are then reduced to ethanol by NADH, converting NADH back into

NAD+ [19,25,35,43,44].

Fig 1 shows the fermentation reaction where sucrose is converted to aqueous ethanol and
gaseous carbon dioxide.

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 Simple distillation was used to initially purify ethanol by heating the

fermented mixture to extract ethanol then, fractional distillation was used to separate miscible

liquids to achieve higher purity levels. Simple distillation involves heating a mixture of

significantly varying boiling points of liquids to vaporize the liquid with a lower boiling point.

As vapor pressure increases, the vapor travels through the longitudinal column, reaches the

condenser, and then is collected in the receiving flask whilst another liquid stays in the

distillation flask. However, in fractional distillation vapors from a boiling solution pass along a

fractionating column, which allows for many successive distillations to occur at once, packed

with plastic beads to improve the separation by providing more surface area for condensation and

evaporation. The substance with a lower boiling point boils first and converts into vapors which

pass through the column and are collected at the top, whilst the substance with a high boiling

point condenses on the column and returns to the solution.

We used both distillation methods to produce concentrated ethanol with high

purity. Simple distillation was used first to remove most of the ethanol from the fermentation

mixture, then fractional distillation was used to further purify the ethanol by separating it from

remaining impurities. We expected the fractional distillation to yield a higher percent purity and

concentration of ethanol compared to the simple distillation. In our study, we dissolved about

50g of sucrose in 250mL of water, added 2g yeast, and allowed fermentation to occur for a week

then the filtrate was subjected to both distillation techniques (for another two weeks) to isolate

ethanol in pure form and determine its boiling point and density using a hydrometer, then in

comparison to a table of values in figure 2, we determined the percent and concentration of

ethanol by weight and volume of the distillate.

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 Materials & Methods

All methods were adapted from CHEM 2202 Laboratory Manual.

50.0840g of Sucrose and 2.0366g of Na2HPO4 were dissolved in 250 mL of water in a

500 RB flask to which 2g of yeast was added. The flask was sealed with a one-hole stopper and

connected to another glass tube immersed in lime water. The ferment was decanted into a beaker,

to which boiling chips and 2 drops of 1-octanol were added. A simple distillation apparatus was

assembled, and the ferment was distilled up to 96℃, and the distillate’s density and volume were

determined. A fractional distillation apparatus was then assembled, and distillates were collected

at 81℃ and 91℃. The volume of each portion was recorded, and their densities were

determined. If the volume was insufficient for density measurement, 95% ethanol was added.

Finally, the ethanol content and quantity for all samples was determined.

Data and Results

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Figure 2. Relationship of Boiling point, % ethanol, and density. Note how at each
temperature, there is a specific density for the specific % ethanol.

Figure 3. This graph inter-relates boiling point and density as a function of the ethanol
content. Variations in Boiling Point and density with ethanol content. It shows the changing
properties of a solution as the ethanol content varies. As the ethanol content increases,
observe how both the boiling point and density of the solution change, revealing the unique
characteristics of ethanol-water mixtures.

graph drawn which

Page 6 of 23
inter-relate boiling point
and density as
a function of the ethanol
content
graph drawn which
inter-relate boiling point
and density as
a function of the ethanol
content
graph drawn which
inter-relate boiling point
and density as
a function of the ethanol
content
graph drawn which
Page 7 of 23
inter-relate boiling point
and density as
a function of the ethanol
content
graph drawn which
inter-relate boiling point
and density as
a function of the ethanol
content
Figure 4. Volume and density of the original ferment and distillates collected. Note how the
volume collected, density, ethanol quantity, and content decline as distillation occurs further.

Volume Density Ethanol Ethanol

collected quantity Content
(mL) (g/ml)
(g) (%)

Original 250.00 0.979 27.00

ferment

Simple 58.10 0.942 22.30 40.8

distillate
St
Fractional 1 17.00 0.832 14.28 94.1
distillate

Page 8 of 23
 nd
2 7.00 0.817 5.97 92.5

Sample Calculations

Calculating the mass of ethanol produced during fermentation.

1. n sucrose= 50.0840g/342.3 g/mol=0.1463 moles

2. 1 mole of sucrose : 4 moles of ethanol

0.1463 moles : X moles of ethanol

n ethano l= 0.5853 moles

3. met h anol=n et h anol x M .W et h anol

=0.5853 mol x 46.1 g/mol

= 27.0 g

Calculating maximum ethanol production possible

Yeast viability = (27.0 gethanol /250 mL) x 100

= 10.8%

If Yeast viability >12% = (12 g ethanol/ 100mL) x 250 mL

= 30. 0 g

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 Calculating atom economy

Atom economy = (total mass of desired product/total mass of all reactants) x 100

= (46.0 g/mol / 360.3 g/mol) x 100

= 12.8%

Analysis of simple distillate

d distillate collected = 0.942 g/mL v distillated collected= 58.10mL

et h anol grap h generated = 38.3%

ethanol content = (38.3g /100 mL) x 58.10 mL

= 22.3 g of ethanol

1. Calculating the % ethanol content

mdistillate = (58.1 mL x 0.942 g/mL) = 54.7 g

% ethanol = (mass of ethanol / total mass of distillate) x 100%

= (22.3 g / 54.7 g) x 100%

= 40.8%

Calculating Ethanol quantity and content in fractional distillate

1. distillate 1 Volume = 17.00mL, density total =0.832g/mL, v total=21.00 mL

mdistillate +m95 %= mtotal

(d distillate x v distillate) + (d 95 % x v 95% ) = d total x v total

= [(0.832g/mL x 21.00mL) - (0.800g/mL x 4.00mL)] / 17.00 mL

= 0.840 g/mL

met h anol quantity D=0.840 g/mL, m=? V=17 mL

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met h anol= 0.840 g/mL x 17.00 mL

=14.28 g¿distillate

% et h anol content h

met h anol = 17 mL x 0.789 g/mL et h anoldensity at S . T . P = 13.43 g

% ethanol content = (mass of ethanol / total mass of distillate) x 100%

= (13.43 g / 14.28 g) x 100%

= 94.1%

2. distillate 2 Volume = 7.00mL, density total =0.817 g/mL, V total=22.00 mL

d distillate 2 = [(0.817g/mL x 22.00mL) - (0.800g/mL x 15.00mL)] / 7.00 mL

= 0.853 g/mL

met h anol quantity E

d=0.853 g/mL, m=? V=7 mL

met h anol quantity = 0.853 g/mL x 7.00 mL

= 5.97 g ethanol in distillate

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 % et h anol content hhh

met h anol = 7 mL x 0.789 g/mL = 5.52 g

% ethanol = (mass of ethanol / total mass of distillate) x 100%

= (5.52 g / 5.97 g) x 100%

= 92.5%

Average % ethanol produced by fraction distillates.

Avg. = (92.5%+94.1%)/2

= 93.3%

Discussion

The experiment was performed over three weeks. In the first week, the fermenting wort

was prepared and left to ferment. Simple distillation was then carried out in the second week and

the distillate was stored in a corked flask. In the third week, distillate was distilled using

fractional distillation. Two fractional distillates were then collected at 81 ℃ and 91 ℃. Based on

calculations performed, distillate obtained from simple distillation yielded a 40.8% ethanol

purity, fractional distillate 1, and distillate 2 yielded 94.1% and 92.5% ethanol purity,

respectively. This confirms the hypothesis that fractional distillation yields a higher ethanol

Page 12 of 23
percent purity and concentration compared to simple distillation thus, is more effective at

separating ethanol into its component mixture because the fractionating column allows multiple

vaporization and condensation cycles to occur [12]. The glass beads in the column provide

“theoretical plates” on which the vapors can condense, re-evaporate, and re-condense, distilling

the compound many times over thus, the components in the mixture become more and more

separated as they move up the column [12]. This agrees with current literature that with

fractional distillation, high purity and high concentration of ethanol can be obtained beyond

which yeast can produce. Fermentation yields a solution that is only about ≤12% alcohol because

higher concentrations are toxic to the yeasts. However, with fractional distillation, ethanol

content can be raised to as high as 95% [36].

Notice how the volume collected in Figure 4 kept declining as distillation was carried

further. This is because as we kept distilling to obtain a pure ethanol content, we kept sifting

impurities out. This was reflected in the theoretical yield calculated. The wort yielded 27.0g of

ethanol before distillation. After simple distillation, the yield dropped down to 22.3g of ethanol,

then dropped down further to 14.28g of ethanol after the first fractional distillate was collected,

and finally to 5.97g of ethanol after second fractional distillation was collected giving us a

combined fractional distillate yield of 20.25g of ethanol. As we got more pure ethanol, we

reduced the impurity quantity hence, our yield kept on declining. In the initial theoretical yield of

27.0g ethanol, yeast viability was calculated at 10.8% showing that, yeast survived. Ethanol

production cannot exceed >12% ethanol content because yeasts die and the chemistry stops, only

water would be obtained and 30. 0 g ethanol would be the maximum ethanol produced as

demonstrated in the calculations. From Figure 1 and the graph in Figure 3, as the percent purity

Page 13 of 23
and concentration of ethanol increased, the density of ethanol obtained decreased. This was

consistent with our results in Figure 4. The density of our original ferment was 0.979g/mL, then

decreased to 0.942g/mL after simple distillation, and then decreased again to 0.832g/mL and

0.817g/mL after fractional distillate 1 and distillate 2 were collected, respectively. However, to

get a reading of the density of the fractional distillates collected using a hydrometer, 95% ethanol

was added (which Figure 4 reflects). The true density was calculated to be 0.840g/mL and

0.853g/mL, respectively. It should be noted that during the collection of the fractional distillate 2,

the receiving beaker fell and broke and the distillate collected was lost. However, we were still

able to collect 7mL. This might have led to contamination of the density value of the fractional

distillate 2.

In week1 of the experiment, lime water acted as a sealant to test for the presence of

CO2, a byproduct of fermentation, and precipitate it. the solution turned murky brown,

cloudiness, and specks of solids were observed in the beaker with limewater. In week2, the

fermented solution was carefully decanted into a large beaker, then transferred into the 500ml

RB flask with 2 boiling chips to prevent bumping and 2 drops of 1-octanal to disrupt foaming.

Then, the simple distillate was collected strictly at 97°C because the distillate above 97°C would

be alcohol-free. In week 3, the distillate was heated gently because rapid heating would result in

the liquid failing to equilibrate in the fractionating column. slow distillation results in better

separation of ethanol because it maximizes the number of vaporizations and condensations in the

column. Fast distillation causes the rising vapors to enter the receiving flask before condensing.

Ethanol has a lower boiling point than water, so it vaporizes first and condenses in the

column, while water vaporizes and condenses later. If impurities can lower the melting point of

Page 14 of 23
solids, they can also lower the boiling point of liquids. Impurities in Ethanol form an azeotrope

with ethanol which boils at a lower temperature than pure ethanol and water [12] and disrupts the

intermolecular forces between the ethanol molecules, making it easier for them to escape into the

vapor phase [37]. As a result, the impurities carry over into the distillate, reducing its purity

[12,26]. Adding ethanol to the mixture before fermentation is never a good idea. Ethanol

concentration above 12% kills yeast [19,38,39,40]. Ethanol added would already have

concentrations above 12%, thus, little or no fermentation would occur. Adding more sucrose to

the suspension would have resulted in more ethanol produced. The production rate of ethanol

increases at a high biomass ratio because larger quantities of biomass are available for

conversion into ethanol [29]. Despite that enhancing sugar concentrations leads to high ethanol

yields [31] other studies have found that high concentrations of sugars can decrease yields [33].

high levels of bacterial contaminations could cause the yeasts to flocculate. Yeast

flocculation is one of the biggest obstacles to industrial ethanol production [32]. This can be

overcome by metabolic engineering approaches and Manipulation of genes. Unfortunately, there

is still no economically viable process capable of thorough inhibition of wild Saccharomyces sp.

contaminations in the fermentation process[32].

The process of fermentation is not easy to be controlled [31]. Other methods [30] such

as Direct deletion of GPD and expressing GAPN genes in yeast genes could increase ethanol

production [32]. genetic engineering of microorganisms has been used to enhance ethanol

production [41]. Mixed cultures can also improve the efficiency of fermentation by reducing the

need for nutrient supplementation and increasing the tolerance to inhibitors [41]. applying self-

Page 15 of 23
cycling fermentation and reaction pathway engineering [35] could significantly increase

productivity [34]. Therefore, there is a need for further research into these domains. Ethanol

fermentation can be used to produce fuel additives to reduce emissions and improve engine

performance, perfumes, wine, spirits, and beer, and disinfectants and antiseptics in the healthcare

industry.

Conclusion

In this experiment, we investigated which method of distillation between fractional and

simple is more effective at separating ethanol from impurities thus, yielding high ethanol purity

and concentration. The results demonstrate that fractional distillation is more effective than

simple distillation because the column allows for many cycles of vaporization and condensation.

We dissolved sucrose in water, added yeast then allowed the wort to ferment for a week. In the

second week, we employed simple distillation to collect our first distillate. In the third week, we

used fractional distillation to collect two distillates at 81℃ and 91℃. Results show that we

Page 16 of 23
obtained 40.8% ethanol from simple distillation and an average of 93.3% ethanol from the

fractional distillates. While the experiment demonstrated the superior efficiency of fractional

distillation in ethanol purification, High levels of bacterial contamination could have led to yeast

flocculation which can be solved by genetic engineering. This is important because ethanol

produced through fermentation can used as a fuel additive to enhance engine performance, in

perfumes, alcoholic beverages, and as a disinfectant and antiseptic in the healthcare industry.

Continued research in the fields of genetic engineering and fermentation control is much

encouraged to enhance ethanol production.

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