International Journal of Pharma and Bio Sciences

RESEARCH ARTICLE BIO TECHENOLOGY

ARTICALTICLE
ETHANOL FUEL PRODUCTION THROUGH MICROBIAL EXTRACELLULAR
ENZYMATIC HYDROLYSIS AND FERMENTATION FROM RENEWABLE
AGROBASED CELLULOSIC WASTES

MIR NAIMAN ALI*1, MAZHARUDDIN KHAN MOHD2 AND MAJID

MOHIUDDIN3
1
Department of Microbiology, Mumtaz Degree & P.G College, Hyderabad, Andhra Pradesh (India)
2
Prof. & Head Dept. of Microbiology & Biotechnology Mumtaz Degree & P.G College, Hyderabad,
Andhra Pradesh (India)
3
Department of Microbiology, Mumtaz Degree & P.G College, Hyderabad, Andhra Pradesh (India)

MIR NAIMAN ALI

Department of Microbiology, Mumtaz Degree & P.G College, Hyderabad, Andhra Pradesh
(India)

*Corresponding author

ABSTRACT
In the present work renewable agricultural cellulosic wastes groundnut hulls and rice husks
were selected for production of bioethanol fuel. Two locally isolated microorganisms
cellulase producing fungus Aspergillus niger and ethanol producing Saccharomyces
cerevisiae were employed for saccharification and fermentation respectively. Substrates
were enzymatically saccharified by using A. niger followed by addition of S. cerevisiae for
fermentative production of bioethanol. Two methods of fermentation i.e. stationary and
shaking were adopted. High yield of ethanol was obtained from groundnut hulls in stationary
fermentation method.

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KEY WORDS
Ethanol, cellulose, saccharification, Aspergillus niger, Saccharomyces cerevisiae.

INTRODCUTION
In 1925, Henry Ford quoted ethyl alcohol, Ethanol represents closed carbon
ethanol as the fuel of the future. He furthermore dioxide cycle because after burning of ethanol,
stated, The fuel of the future is going to come the released CO2 is recycled back into plant
from apples, weeds, saw-dust almost anything. material because plants use CO2 to synthesize
There is fuel in every bit of vegetable matter that cellulose during photosynthesis cycle1,3.
can be fermented. Today Henry Fords futuristic Ethanol production process only uses energy
vision significance can be easily understood 1. from renewable energy sources; no net CO2 is
The increasing demand for ethanol for added to the atmosphere, making ethanol an
various industrial purposes such as alternative environmentally beneficial energy source. In
source of energy, industrial solvents, cleansing addition, the toxicity of the exhaust emissions
agents and preservatives has necessitated from ethanol is lower than that of petroleum
increased production of this alcohol. Ethanol sources11. Ethanol derived from biomass is the
production is usually accomplished by chemical only liquid transportation fuel that does not
synthesis of petrochemical substrates and contribute to the green house gas effect 12.
microbial conversion of carbohydrates present in
agricultural products. Owing to depleting As energy demand increases the
reserves and competing industrial needs of global supply of fossil fuels, it causes harm to
petrochemical feedstocks, there is global human health and contributes to the green
emphasis in ethanol production by microbial house gas (GHG) emission. Hahn-Hagerdal 13
fermentation process. Increased yield of ethanol alarmed to the society by seeing the security of
production by microbial fermentation depends on oil supply and the negative impact of the fossil
the use of ideal microbial strain, appropriate fuel on the environment, particularly on GHG
fermentation substrate and suitable process emissions. The reduction of GHG pollution is
technology 2. the main advantage of utilizing biomass
conversion into ethanol14. Ethanol contains
In the current time, the importance of 35% oxygen that helps complete combustion of
alternative energy source has become even fuel and thus reduces particulate emission that
more necessary not only due to the continuous pose health hazard to living beings. The
depletion of limited fossil fuel stock but also for present study was therefore undertaken to
safe and better environment. With an inevitable utilize lignocellulosic biomass for bioethanol
depletion of the worlds energy supply, there has (biofuel) production. The objective of the
been an increasing worldwide interest in present study was to produce ethanol as a fuel
alternative sources of energy 1, 3, 4, 5, 6, 7, 8, 9 & 10. from renewable agricultural cellulosic wastes
Keeping in view all the above said advantages, through microbial extracellular enzymatic
biomass based fuel development technologies hydrolysis and fermentation. The process was
should rapidly gain momentum and the barriers carried out in two steps saccharification and
imposed earlier should be removed for fermentation, with saccharification at 300C by
successfully attempting the production of Aspergillus niger and fermentation by
bioethanol at the commercial level. Saccharomyces cerevisiae at 300C. For
comparative studies two different methods of

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fermentation were adopted: stationary and incubated at 300C for 72 hrs until the mycelium
shaking. sporulates black conidia. Inoculum was
produced in 250 ml Erlenmeyer flasks
MATERIALS & METHODS containing 100 ml potato dextrose broth by
transferring 2 discs from the PDA plates. The
Raw materials: In present study lignocellulosic flasks were incubated for another 72 hrs at
agricultural wastes- groundnut hulls and rice 300C till the mycelial mat develops. This
husks were utilized as substrates. The solid mycelial mat was used as inoculum in further
particles of substrates with particle sizes of 2.4 x saccharification experiments.
1.0cm for groundnut hulls and 1.0 x 0.3cm for
rice husks were used. Saccharomyces cerevisiae:
The yeast Saccharomyces cerevisiae was
Pre-treatment of substrates: The substrates isolated from soil samples collected from
were treated chemically with 1%NaOH for a vineyards rich in waste materials which include
period of 2 hrs 15, 16. fallen and discarded grapes. Samples were
collected in sterile containers and transferred
Chemical analysis of Substrates: The to laboratory. The soil samples were
substrates were subjected to the estimation of suspended in sterile distilled water and allowed
total sugars 17, reducing sugars 18 and cellulose to settle, then the supernatant was diluted by
content 19. serial-10 fold dilutions and the samples were
inoculated on to sterile Yeast-extract, Peptone
Microorganisms and culture: and Dextrose (YEPD) plates. The plates were
Aspergillus niger: incubated at 300C for 48 hrs. The grown yeast
The fungal culture Aspergillus niger was isolates were identified as Saccharomyces
screened from different soil samples of local cerevisiae by studying some of the
paddy and groundnut fields and identified with morphological, biochemical and physiological
the help of manuals like Dermataceous fungi by characteristics 23.
Barnett20, Text book of Mycology by Alexopolus
21
and Handbook of soil fungi by A. Nagamani, Setting up of fermentation:
I K Kunwar and C. Manoharachary 22. The The fermentative production of bioethanol was
fungus was cultured and maintained on Potato carried out in two steps- a) saccharification and
Dextrose agar medium at 300C. After optimum b) fermentation. Two methods i.e. stationary
growth the culture was stored at 40C in and shaking were adopted. The chemically
refrigerator for further use. pre-treated substrates were used for all the
experiments. In order to optimize bioethanol
Preparation of Inoculum: production the substrates were taken in three
For the preparation of inoculum the culture was different variations in the following manner
plated on PD agar plates. The plates were (Table-1).

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 Table1
Design of fermentation experiments
EXP No RICE HUSKS GROUNDNUT HULLS

1. 5g substrate 5g substrate
+ +
100ml distilled water 100ml distilled water
2. 5g substrate 5g substrate
+ +
100ml distilled water 100ml distilled water
+ 0.5% Lactose + 0.5% Lactose
3. 5g substrate 5g substrate
+ +
100ml chemically defined 100ml chemically defined
media media

Chemically defined medium (Table-2) was used in experiment-3. All the flasks were autoclaved at
15lbs for 15 minutes.
Table 2
Composition of chemically defined medium (%)
Component Percentage (%)
L-Glutamic acid 0.03
NH4No3 0.14
KH2PO4 0.2
CaCl2 0.03
MgSO4 0.03
Proteose peptone 0.75
FeSO4 0.5
MnSO4 0.16
ZnSO4 0.14
Tween 80 2%

Saccharification and fermentation studies were an orbital shaking incubator was employed and
performed in 250 ml Erlenmeyer flasks in which shaking was performed at 100 rpm at 300C
5 grams of substrate was taken in each flask (as temperature.
presented in Table-1) and fermentation The A. niger was selected for
experiments were carried out. saccharification as it is cellulolytic in nature and
can hydrolyze cellulose present in the
Saccharification of substrates by Aspergillus substrates to simple sugars. Generally this
niger: step is carried out by commercially available
For saccharification of substrates locally isolated cellulase enzyme which is very expensive. In
fungal culture Aspergillus niger was employed. our study an attempt was made to design an
The chemically treated substrates were economical process by the use of intact fungal
autoclaved and inoculated with sporulating organism as a source of cellulase enzyme
mycelial mat of Aspergillus niger. instead of commercially available enzyme. As
Saccharification was carried out in stationary and A. niger grows on the cellulosic substrates it
shaking methods for a period of six days at 300C hydrolyzes cellulose of the substrate and
and the process was monitored every 24 hrs for release simple sugars which can be fermented
total sugars released 24. For the shaking method to produce bioethanol.

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Fermentative production of bioethanol by RESULTS
Saccharomyces cerevisiae:
For fermentative production of bioethanol In the present study fuel ethanol was produced
(stationary and shaking) yeast Saccharomyces by saccharification and fermentation of
cerevisiae was employed. After six days of cellulosic substrates. The abundantly and
saccharification mycelial mat of Aspergillus niger cheaply available renewable cellulosic
was removed under aseptic conditions and 10% substrates- groundnut hulls and rice husks
of Saccharomyces cerevisiae culture was added were utilized. Before starting the
to all the flasks. The process was carried out for saccharification and fermentation experiments
a period of six days at 300C. During the total sugars, reducing sugars and cellulose
fermentation process every 24 hours samples content were estimated (Table-3).
were taken for the estimation of bioethanol 25.

Table - 3
Chemical composition of Cellulosic substrates (% w/w)
Substrates Rice Husks Groundnut Analytical Method
Hulls
Total Sugars 20% 25% Spectrophotometric method of
Hedge et al., 1962
Reducing 3.2% 4.5% Spectrophotometric method of
Sugars Krishnaveni et al., 1984
Cellulose 45% 65% Spectrophotometric method of
Sadasivam et al., 1992

Stationary Fermentation day of saccharification from all the substrates.

Highest amount of sugar was released from
Saccharification: In stationary fermentation the groundnut hulls followed by rice husks (Table-4
total sugars released increased from day 1 to and Figure-1).
day 6, highest amount of sugars released on 6th

Table 4
Sugar released in saccharification (Stationary method)(g/100g)

Substrate Variation Day1 Day2 Day3 Day4 Day5 Day6

Distilled Water 7.5 8.2 9 10.4 11.2 12
Groundnut Lactose 12 13.2 14.8 16 17.2 18.5
Hulls Chemically defined
medium 13 14.2 15 16.5 1.8 20
Distilled Water 5.2 6.8 7.4 8 9.2 10
Rice Husks Lactose 11 12.4 13.9 14.4 15.2 16
Chemically defined
medium 11.5 13.8 15 16.4 17.2 18

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 Fig-1: Sugar released in saccharification (Stationary
method) (g/100g)

Total sugar released in

25
20

g/100g
15
10 Day1
5 Day2
0
Day3

Distilled

Lactose

Distilled

Lactose
Chemically

Chemically
Water

Water
medium

medium
defined

defined
Day4
Day5
Day6
Groundnut Hulls Rice Husks
Number of days

Fermentation: In stationary fermentation In the case of groundnut hulls high

increasing trend in ethanol production was amount of ethanol was obtained from
observed from day 1 to day 6. The results of chemically defined medium + groundnut hulls
ethanol produced are presented in table-5 and combination (6.2g/100g), followed by
trend is shown in figure2. Highest amount of groundnut hulls + lactose combination
ethanol was produced on 6th day which is in (5.8g/100g) and least ethanol was recorded
accordance with release of total sugars. Among from groundnut hulls alone (4.0g/100g).
two substrates highest amount of ethanol was Similar results were also obtained in
obtained from groundnut hulls, followed by rice saccharification and fermentation of rice husks.
husks indicating that the efficiency of
saccharifying and fermenting enzymes on these
two substrates shows variations in performance.

Table 5
Ethanol produced in stationary fermentation (g/100g)

Substrate Variation Day1 Day2 Day3 Day4 Day5 Day6

Distilled Water 1 1.6 2 2.9 3.4 4
Groundnut Lactose 1.8 2.5 3.2 4.6 5 5.8
Hulls Chemically defined
medium 2 3.2 4 4.8 5.4 6.2
Distilled Water 0.8 1 1.6 2 2.4 3
Lactose 1 1.8 2.4 3 4.2 5
Rice Husks
Chemically defined
medium 1.4 2 2.6 3.2 4.8 5.5

Fig-2: Ethanol produced in stationary fermentation

(g/100g)
Ethanol produced g/100g

7
6
5
4
3 Day1
2
1 Day2
0
Day3
Distilled

Lactose

Distilled

Lactose
Chemically

Chemically
Water

Water
medium

medium
defined

defined

Day4
Day5
Day6
Groundnut Hulls Rice Husks
Number of days

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Shaking Fermentation: released on 5th day. The highest amount of
Saccharification: In shaking fermentation from sugar released from groundnut hulls, followed
day 1 to day 5 steady increases in release of by rice husks. The results of total sugars
total sugars was observed during released in shaking fermentation are shown in
saccharification with highest amount of sugars table-6 and figure-3.

Table 6
Sugar released in saccharification (Shaking method) (g/100g)

Substrate Variation Day1 Day2 Day3 Day4 Day5 Day6

Distilled Water 5.8 6.5 7 8.2 9 8.5

Groundnut
Hulls Lactose 8.5 9.2 10 10.5 11.2 10.8
Chemically defined
medium 11.5 12.2 13 13.5 14.2 13.9
Distilled Water 4.6 5 5.9 6.5 7.2 6.8
Rice Husks Lactose 4.5 5.8 6.2 7.7 8.2 7.5
Chemically defined
medium 8.2 9 9.5 10.2 10.8 10

Fig-3: Sugar released in saccharification (Shaking

method) (g/100g)
Total sugars released

16
14
12
g/100g

10
8 Day1
6
4 Day2
2
0
Day3
Distilled

Lactose

Distilled

Lactose
Chemically

Chemically
Water

Water
medium

medium
defined

defined

Day4
Day5
Day6
Groundnut Hulls Rice Husks
Number of days

Fermentation: In shaking fermentation consumed by 5th day. The results are shown in
comparatively less yield of ethanol was obtained table-7 and figure-4. The trend of ethanol
than the stationary method. The time course of production in shaking fermentation followed
ethanol produced and total sugar released was similar pattern to stationary fermentation-
similar to stationary fermentation with a minor highest amount of ethanol was obtained from
fluctuation. With increase in time of fermentation, groundnut hulls, followed by rice husks.
ethanol production increased up to 5th day. On In shaking fermentation also high yield
6th day the yield of ethanol decreased indicating of ethanol was obtained from groundnut hulls
that the maximum amount of sugar was with chemically defined medium combination

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(4.0g/100g), followed by groundnut hulls + Similar results were also obtained in
lactose combination (3.2g/100g) and least from saccharification and fermentation of rice husks.
groundnut hulls alone (2.4g/100g).

Table 7
Ethanol produced in shaking fermentation (g/100g)

Substrate Variation Day1 Day2 Day3 Day4 Day5 Day6

Distilled Water 0.8 1.2 1.8 2.4 2.8 2.4
Groundnut
Lactose 1.2 1.8 2.2 2.8 3.5 3.2
Hulls
Chemically
defined medium 1.8 2.6 3.2 4 4.5 4
Distilled Water 0.5 0.8 1.2 1.8 2.2 2
Rice Husks Lactose 0.8 1.2 1.8 2 2.5 2.2
Chemically
defined medium 1 1.6 2 2.5 3.2 2.8

Fig-4: Ethanol produced in shaking fermentation

(g/100g)
Ethanol produced g/100g

5
4
3
2 Day1
1 Day2
0
Day3
Distilled

Lactose

Distilled

Lactose
Chemically

Chemically
Water

Water
medium

medium
defined

defined

Day4
Day5
Day6
Groundnut Hulls Rice Husks
Number of days

DISCUSSION pretreatment methods have been designed 26,

27, 28
. Vaccarino et al., 29 studied the
In the present work bioethanol production effects of SO2, Na2CO3 and NaOH
process was studied with a saccharification pretreatments on the enzymatic digestibility of
process by A. niger and fermentative production grape marc. Silverstein et al., 30 studied the
of ethanol by S. cerevisiae. Both the substrates effectiveness of sodium hydroxide, hydrogen
were chemically treated with 1%NaOH for a peroxide and ozone pretreatments on cotton
period of 2 hrs before enzyme hydrolysis to stalks. Further hydrolysis using enzyme
improve enzyme amenability. Normally cellulosic increased the release of glucose (sugar). It
materials can be hydrolyzed chemically or was found that about 95% of cellulose in
enzymatically. It was found that native biomass pretreated bagasse pulp residue was
is extremely recalcitrant to enzyme converted to glucose by cellulase enzyme 31.
saccharification, to improve enzyme amenability Enzymatic saccharification is the
to holocellulose fraction a number of main step in biomass to bioethanol conversion.

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In our study an attempt was made to use the The results obtained in our study are in
fungal culture A. niger as a source of cellulase correspondence with these reports.
enzyme in saccharification step which
hydrolyzes complex cellulosic substrates by the The overall results showed that cellulolytic
release of extracellular cellulase enzyme and activity and ethanol yields are low in the flasks
release simple sugars. The work of Chandel et where substrate alone is available. Whereas in
al., 1 clearly demonstrates that cost of cellulases flasks where lactose and chemically defined
and recovery of fermentable sugars after media are present along with the substrates
enzymatic saccharification are the important showed increase in cellulolytic activity and
factors which will decide the tangible cost of ethanol production. Maximum cellulose
biomass to ethanol process. As all these utilization was observed in the flasks where
methods of saccharification adds extra cost in chemically defined media is present along with
the production process and to the final product in the substrate. The results indicate that
our study an attempt was made to use the fungal additions of chemically defined media or
culture A. niger as a source of cellulase enzyme lactose are enhancing the cellulolytic activity,
in saccharification step which hydrolyzes the amount of cellulose metabolized and the
complex cellulosic substrates by the release of total ethanol yield. These combinations gave
extracellular cellulase enzyme and produce higher ethanol yield than the substrate alone.
simple sugars.
Bioethanol production is a widely CONCLUSION
studied process for biofuel production. Different
workers have studied various raw materials and Finally we can conclude that cellulosic agro
different methods for bioethanol production but, wastes particularly groundnut hulls and rice
recently it has been observed that lignocellulosic husks are potential substrates which can be
materials are focused for bioethanol production. exploited in industries in future for bioethanol
Hence, we have selected cheaply and (biofuel) production as they are cheap,
abundantly available agrobased wastes for abundant and more importantly renewable.
bioethanol production. Cellulosic substrates were Based on the results obtained it can be
also used by Arthe et al., 32 for bioethanol concluded that when crude cellulosic wastes
production by microbial extracellular enzymatic are used as raw materials, intact
hydrolysis and fermentation where a yield of microorganism such as A.niger can be used for
8.9g/l was recorded. In another report of saccharification of cellulose as a substitute to
Vaithanomsat et al., 33 bioethanol was produced pure cellulase enzyme, lactose can be added
from enzymatically saccharified sunflower stalks as an enzyme inducer to enhance the cellulase
where the yield of 0.02g/100g was obtained. activity, groundnut hulls can be utilized and
Enzymatically pretreated agricultural residues stationary fermentation is ideal method.
were also used by Seema Patel et al., 34 for
ethanol production by different fungal cultures.

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