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Life Science Archives (LSA)

ISSN: 2454-1354
Volume 1; Issue - 1; Year 2015; Page: 53 - 58

Research Article
PRETREATMENT HYDROLYSIS OF AQUATIC WEED AS RESOURCES IN
BIOETHANOL PRODUCTION USING Saccharomyces cerevisiae
M. Saranya, P. Udhayaraja* and G. Muniraj,
Department of Microbiology, Selvamm Arts and Science (Autonomous), Namakkal, Tamil Nadu. India.
Abstract
In the present study, the bioethanol production was optimized by Saccharomyces cerevisiae. Ethanol
fermentation from enzymes hydrolyzed was analyzed in the present research and the reducing sugar content
was recorded. Inoculums level 6 per cent, 8 per cent and 10 per cent. Bioethanol production was important
for environmental production. Because of the weed wastes was created the environmental pollution and also
affected other aquatic species. So, the two weed wastes were used for bioethanol production like Eichhronia
crassipes and Ipomoea carnea. Saccharomyces is most popular because of its high efficiency in ethanol
production fast growth rates and unique tolerance to environmental stresses such as high ethanol
concentration toxic fermentation inhibitors and low oxygen levels. The low lignin content means that
cellulose and hemi cellulose could be easily converted to fermentable sugars resulting in enormous amount
of utilizable biomass for the bio fuel industry.
Article History
Received : 25.02.2015
Revised : 05.03.2015
Accepted : 17.03.2015

Key words: Bioethanol, S. cerevisiae, Eichhronia

crassipes, Ipomoea carnea and Pretreatment.

1. Introduction

Bioethanol in its purest form is a

colourless clear liquid with mild characteristic. In
the first generation production starch and sugar
derived from sugar cane and maize are employed
as feedstock but in the second generation process,
Lignocellulosic materials, which are cheapest
abundant and renewable are used (Kootstra et al.,
2009). Besides lignocellulosic materials do not
negatively affect the human food in favor of
bioethanol production (Alvira et al., 2010).
Nowadays, research is going on all over world for
production of bioethanol from biomass, such as
* Corresponding author: P. Udhayaraja
Tel.: +91-9715636363
E-mail: rise.raja@rediffmail.com

cellulosic wastes, animal wastes, agriculture

wastes, and etc. Water hyacinth is very difficult to
eradicate by physical, chemical and biological
means and a substantial amount is spent on their
control annually throughout the world. Eichhornia
crassipesisa monocotyledonous freshwater aquatic
plant belonging to the family pontederiaceae,
related to the Liliaceae and is a native of Brazil
and Equador region. It is a well know cellulosic
plants found in water gardens and aquariums bears
beautiful blue to lilac coloured flowers along with
their round to oblong curved leaves and waxy
coated petioles. The world bioethanol production
in 2001 was 31 billion litres (Berg et al., 2001). It
has grown to 39 billion litres in 2006 and is
expected to reach 100 billion litres in 2015
(Karimi et al., 2007).). Enzymatic hydrolysis for
conversion is one main aim of pre treatment. In

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P. Udhayaraja /Life Science Archives (LSA), Volume 1, Issue 1, Page 53 to 58, 2015

the words pre treatment is the crucial and costly

unit process in converting lignocelluloses
materials into fuels (Mosier et al., 2005).
Water hyacinth is low in lignin content 10
per cent and contains high amounts of cellulose 20
per cent and hemi cellulose 33 per cent (Bolenz et
al., 1990). Ipomoea carnea Jacq. and Vitex
negundo Linn. Leaves have been used for
medicinal and agricultural purposes by the human
kind. Ipomoea carnea was reported for wound
healing activity. Preliminary pharmacological
study on the glycosides from the leaves of
Ipomoea carnea and antimicrobial activities of
metal complexes prepared from leaves proteins of
Ipomoea carnea have been reported (Agarwal and
Upadhyaya, 1978). Preliminary qualitative
phytochemical screening of Ipomoea carnea
revealed the presence of phenolic compounds,
terpenoids, flavonoids and steroids. New
alternative and renewable energy sources, such as
solar, wind, geothermal, hydrogen and biomass,
are the new sources for supply energy demand for
the whole world.
2. Materials and Methods
2.1. Weed wastes materials
The weed wastes such as Eichhornia
crassipes and Ipomoea carnea were used for the
study. The materials were collected from Erode
district of Tamil Nadu, India and they were cut
into small bits and were sun dried for a weak.
Further, the dried substrates were finally
powdered using electric grinders and sieved
through 56 m mesh sieved. Then, these samples
were used for all the studies are this research.
2.2. Cultures used
The cultures used for fermentation process
were Saccharomyces cerevisiae isolated from
fermented graph juice and maintained in AWE
CARE Research institute Erode. This culture was
maintained in (YPD) medium slant at 302 C
with monthly transferred.
2.3. Inoculum preparation and Fermentation
The yeast Saccharomyces cerevisiae were
inoculated in conical flask grow on yeast broth.

54

Inoculated media was kept on room temperature

for 48 hours. The yeast Saccharomyces cerevisiae
were inoculated into the slurry and fermented by
incubating for 2 weeks at room temperature. The
fermented medium was aliquoted after 3 days, 6
days, 9 days and 12 days interval and distilled to
ethanol content.
2.4. Estimation of reducing sugar in acid
treatment using DNS Method (Mamta Awasthi
et al., 2013)
Pre-treatment was carried out in Erlenmeyer
flasks (250 ml). The dried Eichhorniya crassipes
and Ipomoea carnea samples were treated in
different acids with different concentrations. The
dried Eichhornia crassipes and Ipomoea carnea
powder was pre-treated with different acids, like
H2SO4 (Sulphuric acid), HCL (Hydrochloric acid),
and HCOOH (Formic acid) and different alkalis,
Like NaOH (Sodium hydroxide), KOH (Potassium
Hydroxide) are also different concentration 6 per
cent, 8 per cent, 10 per cent. Different percentage of
acids and alkalis was made upto 100 mg/l of
distilled water and 3 gram of dried plant was mixed
together. The mixer was autoclaved at 121 C for 15
minutes and further cooled down to room
temperature. The hydrolysate was filtered using
filtered paper to remove the un-hydrolysed
materials. The filtrated was collected and analyzed
for the reducing sugar content using DNS method
(Di-Nitro Salicylic acid method).
2.5. Estimation of reducing sugar using DiNitro Salicylic acid method (Miller, 1972)
A convenient and sensitive method for the
estimation of reducing sugar, particularly when
large numbers of samples are to be analyzed, were
developed by Miller (1972) and involves the use
of dinitrosalicyclic acid method. Twenty l of
sample was pipette out into test tube. In the test
tube 0.5 mg/l of DNS reagent was added and
mixed well. Tubes were kept in boiling water bath
for 15 minutes. After the colour developed, 0.5
mg/l of 40 per cent potassium sodium tartarate
(Rochells salt) was added to tube are still warm
and the tubes were cooled under running tap
water. The OD value was measured at 575 nm
using colorimeter. The amount of reducing sugar

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P. Udhayaraja /Life Science Archives (LSA), Volume 1, Issue 1, Page 53 to 58, 2015

was calculated using standard prepared from

glucose.
2.6. Hydrolysate detoxification (Muhammad
MuktarNamadi, et al., 2013)
The hydrolysate of each sample was heated
to 60C (for dissolution) then adding NaOH by
starting with 0.5 gram at interval and measured
with a pH paper till it reaches pH 9.0 - 9.5. One
gram of Ca(OH)2 was added to the solutions to
detoxify harmful materials present in the
hydrolysate and filtered to remove insoluble
residues. The filtered was used as fermentable
sugars. Two gram of peptone was added to the
previously detoxified hydrolysate and the pH was
adjusted to 5.6 by adding 10 per cent sulphuric
acid (H2SO4). The medium was sterilized by
autoclaving at 121C for 15 minutes.
2.7. Estimation of Ethanol using potassium
dichromate method (Nitesh Kumar et al., 2013)
Amount of ethanol produced by
fermentation was estimated by potassium
dichromate method. Initially, 10 mg/l of liquor
was centrifuged at 10,000 rpm for 30 minutes.
One gram of potassium dichromate was dissolved
in 100 mg/l of 5M sulphuric acid solution,
prepared chromic acid reagent was added to
standard as well as test sample by 10 minutes of
water bath at 90C and addition of 40 per cent
Rochelle salt solution.
3. Result and Discussion
Ethanol was produced in the Ipomoea
carnea and Eichhornia crassipes hydrolysates
with fermentation of Saccharomyces cerevisiae in
different acids (Sulphuric acid, Hydrochloric acid,
Formic acid) and alkali (Sodium hydroxide,
Potassium hydroxide) with different concentration
like 6%, and 8%, 10%. The quantity of ethanol
produced from Ipomoea carnea and Eichhornia
crassipes after fermentation with Saccharomyces
cerevisiae aliquoted for 3, 6, 9 and 12 days
interval with the calculated.
In Ipomoea carnea and Eichhorniya
crassipes, 6 per cent of sulphuric acid sample was
produced 1.40 mg/l and 1.80 mg/l of ethanol on
3rd day fermentation. On 6th day 2.20 mg/l of
ethanol was produced in Ipomoea carnea and 2.50

55

mg/l of ethanol was produced in Eichhorniya

crassipes. In 9th day fermentation, the Ipomoea
carnea produced 2.70 mg/l of ethanol and
Eichhorniya crassipes produced 3.90 mg/l of
ethanol. End of the 12thdayfermentation, 2.40 and
3.10mg/l of ethanol was produced from Ipomoea
carnea and Eichhorniya crassipes. Ipomoea
carnea and Eichhornia crassipes hydrolysates
fermented with Saccharomyces cerevisiae have
higher quantity of ethanol, yielding 2.70 mg/l and
3.90 mg/l at 9th day. Similarly, Ipomoea carne and
Eichhorniya crassipes yielded the same quantity
of ethanol on 12th day and there is a slight
variation on 6th day. Eichhorniya crassipes
fermented with only Saccharomyces cerevisiae
has the highest quantity of ethanol of 3.90 mg/l
on the 6rd day and lowest quantity of 1.40 mg/l of
ethanol was found in Ipomoea carnea. Overall in
the 4 days, the highest quantity of ethanol was
produced in 9th day of fermentation in Ipomoea
carnea and Eichhorniya crassipes.
In Ipomoea carnea and Eichhorniya
crassipes, 8 per cent of sulphuric acid sample was
produced 2.10 mg/l and 2.80 mg/l of ethanol on
3rd day fermentation. On 6th day 2.80 mg/l of
ethanol was produced in Ipomoea carnea and 3.60
mg/l of ethanol was produced in Eichhorniya
crassipes. In 9th day fermentation, the Ipomoea
carnea produced 3.20 mg/l of ethanol and
Eichhorniya crassipes produced 4.10 mg/l of
ethanol. End of the 12thday fermentation, 2.50 and
3.80 mg/l of ethanol was produced from Ipomoea
carnea and Eichhorniya crassipes. The higher
quantity of ethanol, yielding 3.20 mg/l and 4.10
mg/l at 9th day. Similarly, Ipomoea carnea and
Eichhorniya crassipes yielded the same quantity
of ethanol on 12th day and there is a slight
variation on 6th day. Eichhorniya crassipes
fermented with only Saccharomyces cerevisiae
has the highest quantity of ethanol of 4.10 mg/l on
the 6rd day and lowest quantity of 2.10 mg/l of
ethanol was found in Ipomoea carnea. After 4
days, the highest quantity of ethanol was produced
in 9th day of fermentation in Ipomoea carnea and
Eichhorniya crassipes.
In Ipomoea carnea and Eichhorniya
crassipes, 10 per cent of sulphuric acid sample
was produced 2.50 mg/l and 3.50 mg/l of ethanol

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P. Udhayaraja /Life Science Archives (LSA), Volume 1, Issue 1, Page 53 to 58, 2015

on 3rd day fermentation. On 6th day, 2.90 mg/l of

ethanol was produced in Ipomoea carnea and 3.90
mg/l of ethanol was produced in Eichhorniya
crassipes. In 9th day fermentation, the Ipomoea
carnea produced 3.10 mg/l of ethanol and
Eichhorniya crassipes produced 4.30 mg/l of
ethanol. During the end of 12thday fermentation,
2.60 mg/l and 3.60 mg/l of ethanol was produced
from Ipomoea carnea and Eichhorniya crassipes.
Ipomoea carnea and Eichhornia crassipes
hydrolysates fermented with Saccharomyces
cerevisiae have higher ethanol yielded 3.10 mg/l
and 4.30 mg/l at 9th day. Similarly, Ipomoea carne
and Eichhorniya crassipes yielded the same
quantity of ethanol on 12th day and there is a slight
variation on 6th day. Eichhorniya crassipes
fermented with only Saccharomyces cerevisiae
has the highest quantity of ethanol of 4.30 mg/l on
the 6rd day and lowest quantity of 2.50 mg/l of
ethanol was found in Ipomoea carnea. Overall in
the 4th day, the highest quantity of ethanol was
produced in 9th day of fermentation in Ipomoea
carnea and Eichhorniya crassipes.
In Ipomoea carnea and Eichhorniya
crassipes, 6 per cent of Hydrochloric acid sample
was produced 1.60 mg/l and 1.40 mg/l of ethanol
on 3rd day fermentation. On 6th day, 2.90 mg/l of
ethanol was produced in Ipomoea carnea and 1.70
mg/l of ethanol was produced in Eichhorniya
crassipes. In 9th day fermentation, the Ipomoea
carnea produced 3.20 mg/l of ethanol and
Eichhorniya crassipes produced 2.20 mg/l of
ethanol. During the end of 12thday fermentation,
2.50 and 1.90 mg/l of ethanol was produced from
Ipomoea carnea and Eichhorniya crassipes.
Ipomoea carnea and Eichhornia crassipes
hydrolysates fermented with Saccharomyces
cerevisiae have higher quantity of ethanol,
yielding 3.20 mg/l and 2.20 mg/l at 9th day.
Similarly, Ipomoea carne and Eichhorniya
crassipes yielded the same quantity of ethanol on
12th day and there is a slight variation on 6 th day.
Ipomoea
carnea
fermented
with
only
Saccharomyces cerevisiae has the highest quantity
of ethanol of 3.20 mg/l on the 9rd day and lowest
quantity of 1.40 mg/l of ethanol was found in
Eichhorniya crassipes overall in the 4 days, the
highest quantity of ethanol was produced in 9 th

56

day of fermentation in Ipomoea carnea and

Eichhorniya crassipes.
In Ipomoea carnea and Eichhorniya
crassipes, 10 per cent of Hydrochloric acid sample
was produced 3.50 mg/l and 1.90 mg/l of ethanol
on 3rd day fermentation. On 6th day 4.60 mg/l of
ethanol was produced in Ipomoea carnea and 2.10
mg/l of ethanol was produced in Eichhorniya
crassipes. In 9th day fermentation, the Ipomoea
carnea produced 5.80 mg/l of ethanol and
Eichhorniya crassipes produced 3.40 mg/l of
ethanol. During the end of 12th day fermentation,
4.20 and 2.20 mg/l of ethanol was produced from
Ipomoea carnea and Eichhorniya crassipes.
Ipomoea carnea and Eichhornia crassipes
hydrolysates fermented with Saccharomyces
cerevisiae have higher quantity of ethanol,
yielding 5.80 mg/l and 3.40 mg/l at 9th day.
Similarly, Ipomoea carne and Eichhorniya
crassipes yielded the same quantity of ethanol on
12th day and there is a slight variation on 6 th day.
Ipomoea
carnea
fermented
with
only
Saccharomyces cerevisiae has the highest quantity
of ethanol of 5.80 mg/l on the 9rd day and lowest
quantity of 1.90 mg/l of ethanol was found in
Eichhorniya crassipes Overall in the 4th day, the
highest quantity of ethanol was produced in 9 th
day of fermentation in Ipomoea carnea and
Eichhorniya crassipes.
In Ipomoea carnea and Eichhorniya
crassipes, 6 per cent of Formic acid sample was
produced 1.20 mg/l and 1.00 mg/l of ethanol on
3rd day fermentation. On 6th day 1.90 mg/l of
ethanol was produced in Ipomoea carnea and 1.80
mg/l of ethanol was produced in Eichhorniya
crassipes. In 9th day fermentation, the Ipomoea
carnea produced 2.20 mg/l of ethanol and
Eichhorniya crassipes produced 2.60 mg/l of
ethanol. At the end of 12th day fermentation, 1.50
and 1.80 mg/l of ethanol was produced from
Ipomoea carnea and Eichhorniya crassipes. The
higher quantity of ethanol yielding 2.20 mg/l and
2.60 mg/l at 9th day in Ipomoea carnea and
Eichhorniya crassipes.

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P. Udhayaraja /Life Science Archives (LSA), Volume 1, Issue 1, Page 53 to 58, 2015

57

Concentration of ethanol in ml

ETHANOL CONCENTRATION FROM I.carnea, AND

E.crassipes IN HCOOH
25
10 per cent

20

8 per cent

15

6 per cent

10

Ipomoea

10 per cent

8 per cent
3days

6days

9days

12days

6 per cent

Ethanol concentration in different days

Sugar Concentration (g)

SUGAR CONCENTRATION IN PRE TREATED

SAMPLE (HCOOH)
100
80

I.carnya

60
40
20
0
6 PER CENT

8 PER CENT

10 PER CENT

Different concentration in acid

Similarly, Ipomoea carne and Eichhorniya

crassipes yielded the same quantity of ethanol on
12th day and there is a slight variation on 6 th day.
Ipomoea
carnea
fermented
with
only
Saccharomyces cerevisiae has the highest quantity
of ethanol of 5.20 mg/l on the 9rd day and lowest
quantity of 1.70 mg/l of ethanol was found in
Ipomoea carnea. Overall in the 4th day, the highest
quantity of ethanol was produced in 9th day of
fermentation in Ipomoea carnea and Eichhorniya
crassipes.
4. Conclusion
Eichhronia crassipes and Ipomoea carnea
were evaluated in this study as substrates for
ethanol production. Biomass energy could be
considered as one the cheapest energy types in the
world. The raw materials can be obtained using
simple, basic systems. Bioethanol is becoming
more popular than gasoline because bio ethanol is

a clean biofuel and can be used safely in

transportation vehicles. Eichhronia crassipes is
one of the worst weeds causing the major problem
to the aquatic ecosystem particularly in the tropics.
The technique described above helps lowering the
plant population while providing a simple and
low-cost process that is suitable to the developing
countries where the cellulose portions of the herbs
are hydrolyzed by enzymes into glucose sugar that
are fermented to bioethanol. Its an important role
for environmental production. Because of the
weed wastes was created the environmental
pollution and also affected other aquatic species.
weed in many parts of the world as it grow very
fast and depletes nutrient and oxygen rapidly from
water bodies, adversely affect ting flora and fauna
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P. Udhayaraja /Life Science Archives (LSA), Volume 1, Issue 1, Page 53 to 58, 2015

2)

3)

4)

5)

6)

7)

8)

9)

58

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