ORIGINAL RESEARCH ARTICLE

ETHANOL PRODUCTION FROM WASTE MATERIALS

Muhammad Shahid Iqbal(1), Muhammad Saad Ahmed(2), Tijen Talas Ogras(3),
Javed Hayat Asif(4), Saleem Ullah(5), Rizwan Bashir(5)

1) Department of Environmental System Analysis, Wageningen

University, Netherlands
2) Department of Bioinformatics & Biotechnology, International
Islamic University Islamabad, Pakistan
3) TUBITAK Marmara Research Centre, Genetic Engineering &
Biotechnology Institute, Turkey
4) Department of Chemistry, Edwardes College Peshawar, Pakistan
5) Department of Agricultural Chemistry, KP Agricultural University
Peshawar, Pakistan

2012

Corresponding Author: [email protected]

 ETHANOL PRODUCTION FROM WASTE MATERIALS

Muhammad Shahid Iqbal1, Muhammad Saad Ahmed2, Tijen Talas Ogras3, Javed Hayat
Asif4, Saleem Ullah5, Rizwan Bashir5

1
Department of Environmental System Analysis, Wageningen University, Netherlands
2
Department of Bioinformatics & Biotechnology, International Islamic University, Islamabad,
Pakistan
3
TUBITAK Marmara Research Centre, Genetic Engineering & Biotechnology Institute, Turkey
4
Department of Chemistry, Edwards College Peshawar, Pakistan
5
Department of Agricultural Chemistry, KP Agricultural University, Peshawar, Pakistan

Abstract
Experiment was designed for ethanol production using corn and other organic waste material
containing starch contents and cellulosic material while barely used for diastase and acidic
digestion methods. The effect of temperature, yeast, barely diastase and various dilutions of acid
(sulfuric acids) were investigated on ethanol production. The result showed that corn yielded
high amount of ethanol (445ml) as compared to cellulosic material which produced 132ml of
ethanol from one kg of weight. It was also noted that with the increase of barely and yeast
amount in a proper manner can increase ethanol production from different starch sources. It was
also noted that acid dilutions affected cellulose digestion where high yield of reducing sugar was
noted at 0.75% of sulfuric acid dilution. It was concluded from the present experiment that
economical sources of starch and various dilutions of acids should be tried on cellulose digestion
for bio-fuel production to withstand in this energy crisis time.

Key words: Ethanol, Biomass, Cellulose, Fermentation, Municipal waste.

Corresponding Author: [email protected]

Introduction

Ethanol is also used as fuel in gasoline engine, and as preservative for biological specimens
(Altınta et al. 2002). Ethanol is very old chemical and has been made since old times due to the
sugar fermentation (Hughes et al. 2009). Majority of the beverage industry produce ethanol by
fermentation process (Patle et al. 2008).
Ethanol is produced from any fodder crop which contains simple sugar in abundance or their
polymers (Hughes et al. 2009). The polymers like starch and cellulose are broken down into
simple sugars through chemical hydrolysis or enzymatic hydrolysis (saccharification), and then
converted by fermentation process to ethanol and carbon dioxide (Jamai et al., 2007), in
saccharification, process starch is converted into simple sugar (monosaccharide) using
microorganism or enzymes such as glucoamylase and -amylase (Shapouri et al. 2004).
Barley is first germinated, dried (baked on pan) and crushed for the whole process (Shapouri et
al. 2002). The resulting sugars are then by help of yeast (Saccharomyces cerevisiae.) converted
into ethanol (Oner et al. 2005). Generally the yeast attack on hexose sugars, but carbohydrates
containing pentose subunits could also be digested by specific yeast into ethanol (Ohgren et al.,
2006).
Along with the production of industrial ethanol research studies are also focused upon the
upgrading the bioconversion process of ethanol production. Ethanol as a fuel has promising
prospects. For biofuel purpose 85-95% alcohol is needed; the distilled out product contains
approximately 95% ethanol. However further treatment may lead to 99% pure ethanol.
Dehydrating agents like CaCl2, MgCl2 etc can be used produce absolute ethanol (99%) (Shigechi
et al. 2008).

METHODOLOGY
Ethanol is a basic ingredient of hot drinks but now-a-days its production is preferably doing as
engine fuel. The present project was conducted keeping this objective in mind.

Carbon Raw Material:

Corn grain: Corn was obtained from the local markets of Peshawar city. Waste vegetable
(Municipal Waste): Waste vegetable was also collected from local vegetable shops and main
sabzi mandi

Germination of barley for diastase activation

Barley malt contains appreciable amount of diastase enzyme. For the hydrolysis starchy material
during the experiment two kg of barley was malted as per the procedure of Davis et al. (2005).
Two kg of barley was weighted and was taken in crates made of wood and that were watered so
that excess amount of water would leach down and only desired moister retained for the
germination of barley. This process was carried out in dark for 3 days and night and every day
the moisture content were noted regularly. When the seedling of barley becomes 1cm long the
process was stopped. After that barley seeds were dried and baked on using pan. The dried
material was ground in home grinder and used as diastase source for ethanol production.

Isolation and selection of yeast strains

Yeasts samples were isolated in sterilized condition from corn (grains) samples collected from
four regions of KPK, namely Swat, Peshawar, Hazara division and D I Khan, Pakistan. Yeast
isolation was done at approximately 36 °C by using the technique which is called enrichment
which contains sugar cane juice (five percent to eight percent total sugars) (Consuelo et al,
2010), 0.05% ammonium sulphate and 4% (volume by volume) ethanol and with about (pH 4.7)
and without pH adjustment of about (pH 3.9). Cultures of pure yeast were isolated and were kept
on agar slants (One percent yeast extract, two percent peptone, two percent glucose and two
percent agar) and stored at about 10 °C.

Saccharification:
Enzymatic hydrolysis:
Saccharification of the raw material was conducted according to Guo et al. (2008) with some
modifications. Two kg of corn grains were boiled in a steel pot of 8L size. The boiled material
was cooled to 60oC and blended with home blender. (Generally the material in this condition is
called mash. The dilution of this material is termed as wort). The pH of the material was made up
to 4.85 with 0.05 M citrate buffer, and 20 g of diastase enzyme in the form of barley malt was
added to 100 g of the raw material used for ethanol production (about 3%, dry basis) in a total
working volume of 500 milliliter in one liter conical flask. These flasks were kept in incubation
at 52-55oC on an orbital shaker operated at 140 rpm. The percentage of saccharification was
calculated as follows:

Reducing sugars x 0.9

% saccharification = x100
Carbohydrates in substrate

Acid hydrolysis:
Municipal waste was treated with different concentration (0.25%, 0.5%, 0.75%, 1%, 2%, 3%,
4%) of H2SO4. 2kg of the waste was boiled with 3 L of each concentration of the H 2SO4 in glass
pot of 8L size. The reducing sugar was calculated by Fehling test. Hydrolysis efficiency was
calculated as follows. (Guo et al. 2008).

Reducing sugars x 0.9

% Saccharification= x100
Carbohydrates in substrate

Fermentation:
Fermentation of the carbon raw material is carried out with the help of yeast according to the
method of Voca et al. (2009). First of all the sample was prepared and filtered, meanwhile the
yeast was dissolved make its solution, about 10 ml of the yeast solution was added in the sample
and was kept for fermentation in dark up to seven days. The process of ethanol fermentation was
done by using glass fermenter and mud pitcher as fermenter tank containing 650 milliliter of 0.05
M sodium citrate buffer (pH 4.9), this process was done by following the two-step of the first
pre-saccharification and then the simultaneous saccharification and fermentation (SSF).

Distillation:
The mixture after fermentation was distilled out with the reflux column and reflux condenser for
the maximum percentage of alcohol, the method of Vander Griend et al. (2007) with some
modification was followed for research work to get good results.

Results and Discussion:

The ethanol production from maize, the effect of barley and amylase enzyme, time effect on
saccharification, temperature effect on ethanol production and also the effect of various yeast
strains isolated from grain belonging to different ecological regions were studied and results
were presented in the form of tables and figures

The minimum saccharification %age was noted at 48 hours intervals while analyzing reducing
sugar at this interval time. However the similar research was also conducted by Park et al. (2009)
strongly supported the present work. The present work is also in line with Hoyong Sohn et al.
(2008) which worked on the saccharification process.

Fermentation, Saccharification Vs Time

60
Fermentation & Saccharification % age

50

40

30

20

10

0 20 40 60 80 100 120 140

Time (Hours)
Time vs Saccharification
Time vs Fermentation

Fig. 1 Effects of time interval on Saccharification and Fermentation of maize through

amylase enzyme at 60 °C

%age Ethanol vs Temperature

8
%age Alcohol Production

2
20 25 30 35 40 45 50

Temperature in Centrigrade
% age Ethanol vs Temp

Fig.2 Ethanol Production on the basis of temperature

Reducing Sugar g/kg of raw material 300

250

200

150

100

50
0.25 0.5 0.75 1 2 3 4 Control

Sulphuric Acid Concentration

Reducing sugar g/kg vs Sulphuric Acid concentraions

Fig.3 Hydrolysis of Organic Material with different concentrations of sulphuric acid

 F(Vegitables)

E(Fruits)
Organic Material Used

D(Surgam)

C(Wheat)

B(Potato)

A(Corn)

0.0 0.2 0.4 0.6 0.8 1.0

Ethanol % age by Weight

Amaylase
Barley
Acid Hydrolysis

Fig.4 Effect of various agents (Amaylase, Barley, Acid Hydrolysis) on different

Carbohydrate sources
The saccharified maize sample, subjected to fermentation process by yeast (micro-organism) at
370C was presented in Fig.2. Here the maximum alcohol content was achieved at 37 0C on
further rise of temperature will reduce the alcoholic content up to great extent in Fig. 2. The
minimum concentration of alcohol was achieved at 250C than at 450C.

The maximum saccharified sample is subjected to the further process of fermentation as shown
in fig.1. The minimum fermentation in terms of sample alcohol content was observed at 24th
hour, where before 24th hour no alcohol was detected in the sample which indicates that
fermentation was not yet started. While the fermentation reached to its maximum at 120th hour
after that no appreciable change was observed. This means that fermentation required a great
span of time, which should be up to 120th hour at 50 0C in Fig. 1. Such type of experiment was
also conducted by Kyunghee et al. (2010).

Municipal waste was treated with different dilution of sulphuric acid (H2SO4). The hydrolysis of
the sample measured in the form of reducing sugars showed that dilution had profound effect on
hydrolysis. The minimum hydrolysis of the waste was obtained at 0.25% of the acid dilution
while maximum was got on 0.75% in Fig. 3. Although the higher dilution was efficiently
hydrolyzed the waste but that decreased with higher concentration of the acids (Zhao and Bai,
2009). For different waste the acid dilution should be adjusted. The present work was in line with
Patel et al. (2008). Although the study of Joseph Di Pardo (2000), was on the same subject, so
from the above graph we can say that for hydrolysis of municipal waste 0.75% dilution of acid is
quite well to get maximum results in Fig 3.

Various carbohydrates sources (corn, potato, wheat, fruits and vegetables (mixed waste)) treated
with three different hydrolyzing agents, been taken according to their equivalency action as in
Fig 4. The result showed that the best hydrolyzing agent was amylase showed in Fig 4, which
gave maximum hydrolyzing percentage in case of all sources followed by barley malt and acids.
Among the different sources corn, wheat and potato showed best hydrolyzing percentage while
the minimum percentage was noted in vegetables which might be due to some hindering
substances (e.g. corn) (Hashem and Darwish, 2010). This work is in line with Patel et al. (2008)
which support our study.

Similarly on various carbohydrate sources to get maximum reducing sugar content g/kg of the
raw material we use different enzymes alone and in combination. With amalyase enzyme we got
maximum reducing sugar content of 435 g/kg followed by the combination of enzymes 427.6
g/kg and minimum of 214.4 g/kg of reducing sugar by using cellulase enzyme as in Fig 5. This
work is in line with Hashem and Darwish 2010.

Conclusion:

Enzymatic hydrolysis of cellulosic material was best for obtaining maximum amount of reducing
sugar which further used for alcohol fermentation. Acid hydrolysis had posed various
hindrances. Best fermentation temperature was found at 37-50oC depending upon the conditions
and best fermentation time was 120hrs. Yeast strains had variability in their ethanol production.

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