study on the production of ethanol from mango peel

MINISTRY OF EDUCATION & TRAINING
CAN THO UNIVERSITY
BIOTECHNOLOGY RESEARCH & DEVELOPMENT INSTITUTE

SUMMARY
BACHELOR OF SCIENCE THESIS
THE ADVANCED PROGRAM IN BIOTECHNOLOGY

STUDY ON THE PRODUCTION OF ETHANOL
FROM MANGO PEEL

SUPERVISOR

STUDENT

Dr. NGO THI PHUONG DUNG

DUONG THI THANH XUAN
Student code: 5085861
Session: 34 (2008 – 2013)

Can Tho, 2013



APPROVAL

SUPERVISOR

Dr. NGO THI PHUONG DUNG

STUDENT

DUONG THI THANH XUAN

Can Tho, 2013
PRESIDENT OF EXAMINATION COMMITTEE


i


Abstract
The

increasing

interest

in

biotechnological

processes

employing lignocellulosic residues is justifiable because these materials
are cheap, renewable and widespread sugar sources. Biotechnological
conversion of biomass into fuels and chemicals requires hydrolysis of
the polysaccharide fraction into monomeric sugars that are converted
into ethanol. The objective of this study was to examine the feasibility
for the production of ethanol from mango peel. The experimental

activities included: studying the effects of H2SO4 concentration, time and
temperature on the hydrolysis of mango peel, assessing the effects of
yeast inoculum levels, pH levels to the fermentation capacity,
determining the favourable time and temperature for ethanol
fermentation. The results showed that the hydrolysis of mango peel with
H2SO4 of 3% at 121oC for 1 hour released 8.49% reducing sugars. The
fermentation conditions including yeast inoculum levels at 10 5cells/mL
and pH 5.5 were found to be favourable for ethanol fermentation. In the
treatment of 7 incubation days at 30°, the ethanol concentration of
3.08% (v/v) was obtained.
Keyword: ethanol, fermentation, hydrolysis, mango peel,
Saccharomyces cerevisiae.

i


CONTENTS
Abstract

i

Content

ii

1. Introduction

1

2. Materials and methods

4

3. Results and discussion

8

3.1. Hydrolysis of mango peel
3.1.1. The effect of the concentration of H2SO4 on

8
8

hydrolysis
3.1.2. The effect of time and temperature on

10

hydrolysis
3.2 Fermentation with Saccharomyces cerevisiae

13

3.2.1. The effect of yeast density on fermentation

13

3.2.2. The effect of pH on fermentation

14

3.2.3. The effects of temperature and time on

16

fermentation
4. Conclusion and Suggestions

20

4.1 Conclusion

20

4.2 Suggestions

20

References

21

ii


1. INTRODUCTION
Nowadays, ethanol is an important industrial chemical
with emerging potential as a bio-fuel to replace non-renewable
fossil fuels (Alfenore et al., 2002). Ethanol may be produced
commercially by chemical synthesis or biosynthesis.

To produce ethanol using substrates such as molasses,
fruit pulps etc. are well known. The economics of ethanol
production by fermentation is significantly influenced by the cost
of the raw materials. It accounts for more than half of the
production costs. To achieve a lower production cost, the supply
of cheap raw material is thus a necessity. Production of value
added products from agro-industrial and food processing wastes is
now an area of focus because it reduces pollution in the
environment in addition to the energy generation. The major part
of this kind of waste is mostly discarded and it is the main source
for increasing the pollution in environment on occasions and also,
the discarding process become a very expensive step due to high
transportation costs. Majority of fruit and vegetable wastes
available from their processing industries are seasonal and do not
decompose rapidly. Moreover, carbohydrate is the main source
for ethanol production which is easily found in various plantparts. It was reported that natural resources along with S.
cerevisiae are the highest bidders for the commercial production
of ethanol.
Mango is native to the Indian subcontinent, belonging to
family Anacardiaceae, is the most cultivated and favourite fruit of
the tropics (Purseglove, 1972). The king of fruits is nutritionally
very rich, unique in flavor and smell thus account for

1


approximately half of all tropical fruits produced globally. Ripe
mango flesh contains carbohydrate and fiber. Carotene, thiamine,
riboflavin, niacin, ascorbic acid, tryptophan, lysine and minerals
are also present in the fruit (Goldsmith, 1976). Mango peel fibre

is a good source of dietary fibre and its chemical composition
may be compared to that of citrus fibre. The peel fibre also shows
higher values of antioxidant activity, glucose retardation and its
aroma and flavour are pleasant (Reyes and Vega, 1988) Mango is
processed to a maximum extent, thereby producing high quantity
of solid and liquid wastes. Solid wastes, stones, stalks, trimmings
and fibrous materials are obtained during the preparation of raw
material. Liquid waste is the waste material that comes out of a
factory after washing of fruits, packaging, blanching, cooling and
plant and machinery clean up and so on. Utilization of this mango
waste is both a necessity and a challenge. In mango peel, there is
a

high amount

of

lignocellulose and

reducing sugars.

Lignocellulosic materials, are constituted primarily of lignin,
hemicellulose

and

cellulose.

The

carbohydrate

fraction

(hemicellulose and cellulose) can be depolymerised into sugars
which act as a primary carbon source for the microbial
biocatalysts for the production of xylitol, ethanol, organic acids,
industrial enzymes, etc. (Carvalheiro et al. 2008; Mussatto and
Teixeira 2010; Chandel et al. 2011a). The present study was
carried out to produce ethanol using mango peel, an agro-industry
waste.

2


Objectives:
Production

of

ethanol

from

mango

peel

using

Saccharomyces cervisiae
Activities in the research:
- Study on the effect of the concentration of H2SO4 on hydrolysis
- Study on the effect of time and temperature on hydrolysis
- Study on the effect of yeast density on fermentation
- Study on the effect of pH on fermentation
- Study on the effects of temperature and time on fermentation

3


2. MATERIALS AND METHODS
2.1 Materials
- Mango peel collected from Tien Giang Vegetables and
Fruits Joint Stock Company.
- Saccharomyces cerivisiae strain from Biotechnology
Research & Develop Institute of Can Tho University (notated as
2.1).
- Bien Hoa sugar from Co. op Mart

- Medium: YPG (Yeast extract 0.3%, potato 20%, and
Glucose 20%)
- Chemicals, equipments in Food Biotechnology laboratory.
2.2 Methods
2.2.1. Preparation of mango peel
After collected, mango peel was washed and then dried in the
sun for 2-3 days, followed by being chopped into pieces of 2-3
cm. The material was transferred into an oven to dry at 65oC in 24
hours until the weight was unchanged and ground it in mixer into
a fine powder.

2.2.2 Hydrolysis of mango peel
2.2.2.1. Study on the effect of the concentration of acid
H2SO4 on hydrolysis
- The activity was designed with one factor – the
concentration of H2SO4 with five levels: 0.5, 1, 2, 3 and 4%. The
total number of experimental units was 5 treatments x 3 replicates
= 15 units.
- 10 g mango peel powder was mixed with 100 mL dilute
acid of different concentrations and autoclaved at 121ºC for 15
minutes (Arumugam và Manikandan, 2011).

4


- The acid pretreated samples were cooled and filtered.
- pH of the hydrolysate was adjusted to 6.0 with NaOH
(20N).
- The reducing sugar content in the hydrolysates was
determined by DNS method (Walia et al, 2013) and the amount of
soluble solids was measured by Brix meter.
2.2.2.2. Study on the effect of time and temperature on
hydrolysis
- The experiment was carried out with two factors: time
with five levels (15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours)
and temperature at 30, 60, 90, 100 and 121oC. Total number of
treatments was 5 x 5 = 25 treatments. The total number of
experimental units was 25 treatments x 3 replicates = 75 units.
- 10 g mango peel powder was mixed with 100 mL dilute
acid with the concentration chosen from the previous activity. The
hydrolysis was carried out in the conditions of time and

temperature as in the experimental arrangement.
- The acid pretreated samples were cooled and filtered
- pH of the hydrolysate was adjusted to 6 with NaOH
(20N).
- The reducing sugar content in the hydrolysates was
determined by DNS method (Walia et al, 2013) and the amount of
soluble solids was measured by Brix meter.

5


2.2.3. Fermentation with Saccharomyces cerevisiae
2.2.3.1. Study on the effect of yeast density on
fermentation
- The activity was designed with one factor: yeast density
4

at 10 , 105 and 106 cells/mL. The total number of experimental
units was 3 treatments x 3 replicates = 9 units.
- Transfered 100 mL hydrolyzed mango peel solution (pH
6) into flasks.
- Sterilized the solution by NaHSO3 140 mg/L in 30
minutes.
- Inoculated yeast suspension with experimental levels.
Cover flasks by water-locks and ferment for 7 days at room
temperature.
- Monitored Brix level, pH and ethanol content after
fermentation.

Analyze

the

data

with

statistics

program

STATGRAPHICS Plus v3.0
2.2.3.2. Study on the effect of pH on fermentation
- The effect of pH was assessed with five levels: 4, 4.5, 5,
5.5 and 6. The total number of experimental units was 5
treatments x 3 replicates = 15 units.
- Adjusted pH of the hydrolysate to mentioned levels and
transfer 100 mL into flasks.
- Sterilized the solution with NaHSO3 140mg/l in 30
minutes.
- Inoculated yeast suspension as the result of previous
activity. Cover flasks by water-locks and ferment in the
conditions of time and temperature mentioned.

6


- Monitored Brix level, pH and ethanol content after
fermentation.

Analyzed the data with statistics program

STATGRAPHICS Plus v3.0.
2.2.3.3. Study on the effects of temperature and time
on fermentation
The activity was designed with two factors: fermentation
temperatures at: 25°C, 30°C and 35°C; fermentation time at: 3, 5,
7 and 9 days. Total number of treatments was 3 x 4 = 12
treatments. The total number of experimental units was 12
treatments x 3 replicates = 36 units.
- Transferred 100 mL hydrolyzed mango peel solution
into flasks with pH as the result of activity before.
- Sterilized the solution with NaHSO3 140mg/l in 30
minutes. Inoculate yeast suspension as the result of previous
activity. Cover flasks by water-locks and ferment in the
conditions of time and temperature mentioned.
- Monitored Brix level, pH and ethanol content after
fermentation.

Analyze

the

data

STATGRAPHICS Plus v3.0.

7

with

statistics

program


3. RESULTS AND DISCUSSION
3.1. Hydrolysis of mango peel
3.1.1. Effect of the concentration of acid H2SO4 on
hydrolysis
Among factors affecting the efficiency of acid hydrolysis,
acid concentration is considered one of the most important factors
regarding the release of sugars. High concentrations of acid may
decompose the hemicellulosic structure, producing inhibitors and
also causing damage to the equipment used. Therefore, an
appropriate acid concentration is essential for acid hydrolysis of
lignocellulose (Taherzadeh and Karimi, 2007).
The results in Table 3 showed that the concentration of
the acid had a great impact on hydrolysis, especially the amount
of reducing sugars produced which increased with the growth of
the concentration of acid. The catalyst activity was proportional to
H+ concentration. The more hydrogen ions were formed in the
solution, the more rapid the hydrolysis process occurred (Mosier
et al, 2002). Therefore, the breaking of glucosidic bounds would
increase, leading to the high conversion of hemicellulose fraction
into xylose. Under harsh conditions if high acid concentration was
applied, the cellulose fraction would be disrupted and glucose
would be generated. After 15 minutes hydrolyzing, the maximum
reducing sugars was observed in the treatment of H2SO4 4%
(7.74%) and the minimum was observed in the case of H2SO4

0.5% (6.14%). Soluble solids were also greater when mango peel
was hydrolyzed with higher concentration of acid. Specifically,
with 4% acid, hydrolysate had the highest Brix level (11.7 oBrix)

8


while the lowest (6.5oBrix) was seen in the treatment of 0.5%
acid.
Table 3: Effect of the concentration of acid H2SO4
on hydrolysis
Concentration of H2SO4

CV

0,5%

1%

2%

3%

4%

(%)

6.14c

6.21bc

6.47b

7.61a

7.74a

1.75

6.5

7.5

8.7

11.2

11.7

Amount of
reducing
sugars (%)
Brix level

Note: The difference was statistically significant mean only in rows. The
same characters show no difference statistically significant at 95%.

The determination of acid’s suitable concentration was very
important to the efficiency of ethanol production from mango
peel. The low level of acid (0.5%) led to low performance of

hydrolysis whereas the hydrolysis occurred faster with high
concentration of acid (4%) in spite of its high cost for the amount
of acid used and acid neutralization later. According to the
statistical result, there was no significant difference between the
percentages of reducing sugars produced in the treatment of
H2SO4 3% and that of H2SO4 4% (7.61% and 7.74%,
respectively). Therefore, the concentration of 3% H2SO4 was
suitable for the hydrolysis of mango peel.

9


3.1.2. Effect of time and temperature on hydrolysis
The results were presented in Table 4. It was indicated
that the hydrolysis rate gradually increased with respect to
temperature. Kim (1999) showed that temperature plays an
important role in the reaction rate of acid hydrolysis. Based on
Arrhenius equation, at high temperatures, reaction should be
faster. According to Xiang et al., breakage of hydrogen bonds into
cellulose and hemicellulose fractions takes place steeply in
response to temperature. At low temperature (30 – 90oC) the
hydrolysis occurred more slowly than at high temperature (100 –
121oC). Particularly, the amount of reducing sugars doubled
(about 8%) at high temperature (100 – 121oC) in comparison with
lower levels of temperature. According to Gírio et al. (2010),
temperature for hemicellulose hydrolysis was between 121 and
160oC.
The extension of hydrolysis time also contributed to the
increase of reducing sugars. As can be seen in Table 4, in all
treatments, after 1 hours hydrolyzing, the amount of reducing

sugars grew to the levels that were significantly different from
that in 15 and 30 minutes. Remarkably, the rate of reaction was
high at 121oC in 1 hour, producing reducing sugars with 8.49%.
After that, it increased to insignificantly different levels (8.39%
after 2 hours and 8.58% after 3 hours). Soluble solids also went
up according to temperature and time. The highest Brix level
(12.5%) was seen at 121oC in 3 hours while at the temperature
lower than 100oC it ranged only between 5 – 9.17oBrix.
Therefore, the hydrolysis of lignocellulosic compounds in mango
peel occurred fast at 121oC in 1 hour. In order to get higher yield,

10


time can be extended. However, it will be time-consuming,
reducing the efficiency of production. Meanwhile, according to
Lenihan et al, (2011), hydrolysis at too high temperature and in
too long time, monosaccharides can be degraded into undesirable
chemicals inhibiting the fermentation later such as furfural,
hydroxylmethylfurfural-HMF, acetic acid, levulinic acid, formic
acid, uronic acid, vanillic acid, phenol, cinnamaldehyde,
formaldehyde… Thereby, 1 hour was adequate time for efficient
hydrolysis. The proportion of crude fiber in mango peel is 9.33%
(Ashoush, 2011), so with 8.49% reducing sugars from the
treatment of 121oC and 1 hour the efficiency of hydrolysis was
79.21%. This figure was high and logical since in crude fiber
there is lignin, a component that cannot be hydrolyzed (Palmquist
and Hahn-Hagerdal, 2000; Taherzadeh, 1999). In comparison

with 38,6% in hydrolysis on the sugar cane bagasse at 100oC,

2% phosphoric acid concentration for 300 minutes (Gamez et al.,
2004). The amount of reducing sugars released from the
hydrolysis of durian peel is 5.6% (w/v) at 121oC in 45 minutes
with 2% sulphuric acid (Matura et al., 2012). The hydrolysis of
red macroalgae (Dwi, 2012) gave the highest level of reducing
sugars (3.28%, w/v) in the conditions of 121oC and 1 hour.
Therefore, it could be concluded that the hydrolysis of mango
peel at 121oC in 1 hour was feasible and efficient because high
reducing sugars were produced and the degradation of these
sugars could be avoided. In addition, this choice is particularly
important in making the process more economic and less timeconsuming.

11


Table 4: Effect of time and temperature on hydrolysis
Temperature
(oC)

30

60

90

100oC

121oC

Amount of

reducing
sugars(%
1.84l
3.54l
4.07k
4.10k
4.33k
3.29l
3.33l
4.02k
4.18k
4.42ik
4.83hi
5.07gh
6.17de
5.85def
5.50fg
5.76ef
6.22d
7.25c
7.67bc
8.03b
7.53c
7.58c
8.49a
8.53a
8.58a
3.69

Time

15 mins
30 mins
1 hr
2 hrs
3 hrs
15 mins
30 mins
1 hr
2 hrs
3 hrs
15 mins
30 mins
1 hr
2 hrs
3 hrs
15 mins
30 mins
1 hr
2 hrs
3 hrs
15 mins
30 mins
1 hr
2 hrs
3 hrs

CV (%)

Brix level
5

6.17
6.33
6.33
7.17
6.17
6.83
7
7.67
8.17
7.83
8.17
8.17
9
9.17
9.33
9.83
9.83
10.17
10.33
10.83
11.33
12.17
12.33
12.5

Note: The difference was statistically significant mean only in columns.
The same characters show no difference statistically significant at 95%.

12

3.2 Fermentation with Saccharomyces cerevisiae
3.2.1. Effect of yeast density on fermentation
The

initial

density

of

yeast

inoculated

effects

considerably the fermentation. With the sufficient level, the
fermentation takes place effectively, creating good-quality
product. The process will be slow if there are not enough yeast
cells added, whereas in the case of abundant yeast cells, it will be
wasteful and the product will have strange smell.
The result of the study on the effect of yeast density on
fermentation was shown in the Table 5.
Table 5: Effect of yeast density on fermentation
Yeast

pH after

Brix level after

density

fermentation

fermentation

Log 6
tb/mL
Log 5
tb/mL
Log 4
tb/mL

Ethanol
content
(% v/v ở 20°C)

5.25

9

3.47a

5.37

9.5

3.21a

5.91

11.5

0.37b

CV (%)

14.56

Note: The difference was statistically significant mean only in
columns. The same characters show no difference statistically significant at
95%.

After fermentation, pH of all fermented liquid was lower
than that before fermentation (initial pH was 6.0). pH of
hydrolysates decreased gradually through stages of fermentation,

13


ranging between 5.25 and 5.91. This decrease caused by sugars
oxidation into organic acids. During fermentation process, yeasts
converted sugars into alcohol and other intermediate products organic acids, reducing the pH of the fermenting liquid. Besides,
the results proved that the fermentation occurred in the good way,
with yeast overwhelming acidic bacteria that could cause pH to
decrease to the level 3.0 – 3.5.
Similarly, there was a difference in Brix level before and
after fermentation. The reason was the conversion of sugars into
ethanol by yeast. With the initial point of 12%, Brix level of the

treatment of log 6 cells/mL decreased to 9oBrix and 9.5oBrix in
log 5 cell/mL. There was little decrease in Brix level in the
treatment of log 4 cell/mL (11% after fermentation).
Statistical analysis with Statgraphics plus v3.0 releaved
that average ethanol of triplcates in the density log 5 cell/mL and
log 6 cells/mL (3.21% and 3.47%, respectively) was higher and
significantly different from that of the density log 4 cells/mL
(0.37%). Therefore, the density log 5 cells/mL (105 cells/mL) was
selected for fermentation in order to

produce high level of

ethanol as well as save a great amount of yeast in comparison
with the treatment of log 6 cells/mL.
3.2.2. Effect of pH on fermentation
pH has a big influence on the growth of yeast which
depends

on

different

fermentation

medium.

Therefore,

determining a suitable pH for the solution of hydrolyzed mango
peel is very important.

14


The hydrolysates were adjusted to pH 4, 4.5, 5, 5.5 and 6
and inoculated with the chosen level of yeast. The result was
presented in the Table 6.
Table 6: Effect of pH on fermentation
pH
before
fermentation

pH after
fermentation

Brix after

Ethanol content

fermentation (% v/v at 20°C)

4.0

3.92

11

0.43c

4.5

4.42

10.7

0.94b

5.0

4.67

10.2

1.13b

5.5

5.28

10

3.01a

6.0

5.25

9.2

3.14a

CV (%)

12.23

Note: The difference was statistically significant mean only in columns.
The same characters show no difference statistically significant at 95%.

The results of Brix level and pH in Table 6 proved that
ethanol fermentation occurred, changing Brix level (initial Brix
value was 12) and pH of fermented liquid.
The results of ethanol content indicated that in the
treatments pH 5.5 and pH 6.0 ethanol contents (3.01% and 3.14%,
respectively) were significantly higher than that of other ones.
This was because proteins have good activity in appropriate pH
(Berg, 2007). This result was consistent with the study published
by Arumugam and Manikandan (2011). Meanwhile, inhibitors of
fermentation are highly absorbed in basic pH (Fox, 1985).

15


According to statisticals analysis with the confidence of 95%,
ethanol contents in pH 5.5 and pH 6.0 were not significantly
different. Therefore, in the purpose of saving NaOH used in
neutralizing the hydrolysates, pH 5.5 was suitable for the
fermentation of ethanol from mango peel.
3.2.3. Effect of temperature and time on fermentation
Temperature has a great influence on metabolism as well
as the activities of yeast’s enzymes. If temperature is low and

time is long, the efficiency of the conversion of sugars into
ethanol will be high and the quality is good. Whereas, if the
temperature is high, the fermentation will be limited, leading to
low ethanol content, high residual sugars. Therefore, the
determination of suitable temperature is very important.
In this activity, hydrolyzed solution was fermented in 3,
5, 7 and 9 days at temperatures 25, 30 and 35oC. The values of
yeast density and pH initially were adjusted for optimum results
in the previous activity: yeast density 105 cells/mL, pH 5.5. After
fermentation, results were presented in Table 7.

16


Table 7: Effects of temperature and time on fermentation
Ethanol
Temperature
o

( C)

Time
(days)

pH

Brix

after

after

fermentation

fermentation

content
(% v/v at
20°C)

3

5.46

11.2

0.12e

5

5.36

10.7

1.86c

7

5.13

9.2

3.15a

9

5.12

8.8

3.21a

3

5.45

11

0.06e

5

5.34

10.2

1.61c

7

5.16

8.8

3.08ab

9

5.22

9.7

2.94ab

3

5.49

12

0e

5

5.43

10.8

0.73d

7

5.28

10.2

2.63b

9

5.27

9.7

2.87ab

25

30

35

CV (%)

16.96

Note: The difference was statistically significant mean only in columns.
The same characters show no difference statistically significant at 95%.

Like the results in the previous activities, Brix level and

pH after fermentation decreased (initial Brix level and pH were

17


12% and 5.5, respectively), which proved that the fermentation of
hydrolysate from mango peel occurred.
The results in Table 7 showed no difference of ethanol
content at 95% probability at two temperatures 25°C and 30°C
after 3, 5, 7 and 9 days of fermentation. That meant 25 and 30oC
were suitable temperature for fermentation because enzymes
acted efficiently at these ranges of temperature. The rate of
enzyme catalyzing reactions increased with the increase of
temperature until it reaches the temperature at which enzyme
started being denatured (Southerland, 1990). However, to lower
production costs, the temperature of 30°C was chosen because
this temperature is close to ambient temperatures, reducing
expense for cooling in the fermentation process. The temperature
of 35oC was not suitable for fermentation because ethanol content
was low (2.87% after 9 days incubating). The cause was that at
high temperature (35oC) some fermenting enzymes produced by
yeast activities might be inactivated, discouraging the conversion
of sugars into ethanol (Schandert, 1959). Meanwhile, fermenting
at this level of temperature comsumed more energy.
Similarly,

regarding

to

the

treatment

of

30oC,

fermentation duration was chosen at 7 days to get high ethanol
content (3.07%, v/v) which was insignificantly different from that
obtained after 9 days (2.94%, v/v). This choice was also to reduce
fermentation time and to avoid the risk of contamination from the
external environment. In summary, temperature 30°C and 7 days
of fermentation were favorable conditions for ethanol production.
The fermentation of mango peel hydrolysate produced
ethanol at 3.08% (v/v). In comparison with ethanol production

18


from starch which releases ethanol of 6 – 9.5% (v/v), the
efficiency of the production from mango peel was 50% as low as
that from starch. However, the results showed that mango peel
was a potential material for ethanol production since it is an
available and cheap agro-industry waste and has no effect on food
supply for human beings. At the same time, environmental
problems caused by this waste could be solved.

19