Enzymatically Liquefaction Sweet Potato (Ipomea batatas L) to Bioethanol Production Using Saccharomyces cerevisiae.
Enzymatically Liquefaction Sweet Potato (Ipomea batatas L) to
Bioethanol Production Using Saccharomyces cerevisiae.
I Wayan Arnata, Bambang Admadi H., and Esselon Pardede
Agroindustrial Technology, Udayana University
Abstract
Sweet Potato (Ipomea batatas L) were hydrolyzed by enzymatically liquefaction process
and fermented by Saccharomyces cerevisiae to produce bioethanol. The objective of this
research was to determine (1) the optimum conditions of enzymatically liquefaction
process, (2) the optimum conditions of fermentation process using Saccharomyces
cerevisiae. These studies was designed by factorial randomized block design with two
factors. The liquefaction process consists of 2 factors, namely: the concentration of αamylase enzyme and temperature. The concentration of α-amylase enzyme consists of 3
levels, namely 0.8; 1 and 1.2 ml/kg of starch. The temperature of hydrolysis consists of 3
levels, namely : 90oC, 95oC and 100oC. In the fermentation process consists of 2 factors,
namely the pH of the substrate and temperature. The pH of the substrate consists of 3 level:
4.5; 5.0 and 5.5, while, the temperature consists of 3 level, namely: 30oC, 35oC, 40oC. The
best results on liquefaction process was the enzyme concentration 1.2 ml/kg of starch with
a temperature of 100oC. Liquefaction process was produced glucose with a concentration of
57.56 g/L. The best fermentation process carried out at a pH of 4.5 with a temperature of 35
o
C and produce bioethanol with a concentration of 5.32 %(v/v) and fermentation efficiency
of 70.16 % of theoretical ethanol production.
Keywords : Liquefaction, sweet potato, bioethanol, Saccharomyces cerevisiae
INTRODUCTION
An efficient method for conversion of biomass into fuel is by bioethanol production
because bioethanol is an economical as well as environmentally friendly fuel. Bioethanol
has the advantages of being renewable, cleaner burning and produces no greenhouse gases
(Altıntas et al., 2002). Sweet potato is one source of starch which is widely used in
industry, one of them was for the production of bioethanol. Bioethanol production from
starch raw materials requires the hydrolysis process, because of yeast are unable to
consume raw starch and hence, the starch must first be hydrolysis into simple sugars (Birol
et al., 1998). Hydrolysis process can be carried out enzymatically or chemically
(Taherzadeh and Karimi, 2007). The hydrolysis of starch to products with low molecular
weight, catalyzed by α-amylases is one of the most important commercial enzyme
processes. Amylase enzyme used to break down the polymer to smaller sugar units, which
is eventually converted to the individual basic glucose units (Delphine et al., 2000; Baskar
et al., 2008).
Yeast cells Saccharomyces cerevisiae are facultative anaerobes and under anaerobic
conditions can ferment glucose to bioethanol. S. cerevisiae is ideal for bioethanol
production due to several properties including fast growth rates, efficient glucose
repression, efficient ethanol production and a tolerance for environmental stresses, such as
high ethanol concentration and low oxygen levels.
In this work, the conversion of starchy materials into bioethanol have two steps,
enzymatically liquefaction of starch, and fermentation of sugar to etanol. Batch
fermentation runs were performed to produce bioethanol using strains of Saccharomyces
cerevisiae.
MATERIALS AND METHODS
Raw material. Sweet potato was collected from Tabanan. Saccharomyyces cerevisiae
was collected from microbiology laboratorium Bogor University. Sweet potato was
analysed to determine chemical compotition before it is used in liquefaction and
fermentation process.
Inoculum culture S. cerevisiae. The strain S. cerevisiae were maintained on potato
dextrose agar (PDA). The medium composition for cultivation was as follow: yeast extract
5 g/L, malt 5 g/L, glocose 10 g/L and peptone 5 g/L. Cultivation was conducted in 250 ml
Erlenmeyer flask for 24 hours at 30 oC and agitation 125 rpm.
Liquefaction. Sweet potato slurry was hydrolyzed enzymatically. The suspension sweet
potatoes made with a concentration of 30 % (w/v), pH was adjusted to 6.5 with NaOH.
Gelatinization process carried out at a temperature of 90 °C. The enzyme concentration and
temperature liquefaction process was conducted in accordance with the treatment for 1
hour.
Fermentation. Batch fermentation process carried out for 4 days in 250 ml erlenmeyer
with 200 ml work volume. Substrate was sterilized at 121 ° C for 15 minutes and cooled,
then cultured S. cerevisiae with a concentration of 10% (v/v) was added aseptically to the
substrate. The pH and temperature of the process carried out in accordance with the
treatment.
Experimental design. These studies was designed by factorial randomized block design
with two factors. The liquefaction process consists of 2 factors, namely: the concentration
of α-amylase enzyme and temperature. The concentration of α-amylase enzyme consists of
3 levels, namely 0.8; 1 and 1.2 ml/kg of starch. The temperature of hydrolysis consists of 3
levels, namely : 90oC, 95oC and 100oC. In the fermentation process consists of 2 factors,
namely the pH of the substrate and temperature. The pH of the substrate consists of 3 level:
4.5; 5.0 and 5.5, while, the temperature consists of 3 level, namely: 30oC, 35oC, 40oC.
Analysis methods. Glucose concebntration was assays by DNS reagent (Miller, 1959).
The pH of substrate assays by pH-meter. Ethanol concentration was analysed by gas
chromatography (GC) The ethanol concentration was analised at the end of time
fermentation process. Fermentation efficiency was measured according to formulation :
Fermentation efficiency (%) = (ethanol actual/ethanol teoritic) x 100% (Caylak and Fazilet,
1998).
RESULTS AND DISCUSSION
Proximate analyse showed that the contents of sweet potato flour was 67,94 ±
1,07% moisture, 0,91 ± 0,03 % ash, 16,07 ± 1,15 % fat, 0,04 ± 0,03 % proteine, 2.69 ±
0.04 % crude fiber and 15,05 ± 2,25% carbohydrate. The moisture content of the substrate
has a major impact on how long it can keep in the storage and still remains nutritions.
In liquefaction proses, the analysis variance of the concentration of glucose showed
that the difference of treatment liquefaction enzyme concentration and temperature
significantly affect the concentration of glucose. The highest concentration of glucose that
is 57.56 ± 4.81 g/L generated from the process liquefaction using an enzyme concentration
1.2 ml/g with a temperature of 100 °C and this treatment was significantly different from
other treatments. The lowest concentration of glucose that is 22.96 ± 11.20 g/L resulted
from treatment using enzyme concentrations of 0.8 ml/g with a temperature of 100 °C and
this treatment was also significantly different from other treatments. The average value of
the glucose concentration in the liquefaction process showed in the Table 1.
Table1. The average value of the glucose concentration in the liquefaction process
Temperature (T oC)
T1 (90 oC)
T2 (95 oC)
T3 (100 oC)
ab
ab
Concentration
C1 (0,8 ml/g)
28,99
32,64
22,96 c
b
ab
(α-amilase)
C2 (1 ml/g)
40,10
26,21
24,87 ab
ab
ab
C3(1,2 ml/g)
29,87
24,15
57,56 a
** Notation with different letters behind the averages indicate a significant difference between
treatments (p
Bioethanol Production Using Saccharomyces cerevisiae.
I Wayan Arnata, Bambang Admadi H., and Esselon Pardede
Agroindustrial Technology, Udayana University
Abstract
Sweet Potato (Ipomea batatas L) were hydrolyzed by enzymatically liquefaction process
and fermented by Saccharomyces cerevisiae to produce bioethanol. The objective of this
research was to determine (1) the optimum conditions of enzymatically liquefaction
process, (2) the optimum conditions of fermentation process using Saccharomyces
cerevisiae. These studies was designed by factorial randomized block design with two
factors. The liquefaction process consists of 2 factors, namely: the concentration of αamylase enzyme and temperature. The concentration of α-amylase enzyme consists of 3
levels, namely 0.8; 1 and 1.2 ml/kg of starch. The temperature of hydrolysis consists of 3
levels, namely : 90oC, 95oC and 100oC. In the fermentation process consists of 2 factors,
namely the pH of the substrate and temperature. The pH of the substrate consists of 3 level:
4.5; 5.0 and 5.5, while, the temperature consists of 3 level, namely: 30oC, 35oC, 40oC. The
best results on liquefaction process was the enzyme concentration 1.2 ml/kg of starch with
a temperature of 100oC. Liquefaction process was produced glucose with a concentration of
57.56 g/L. The best fermentation process carried out at a pH of 4.5 with a temperature of 35
o
C and produce bioethanol with a concentration of 5.32 %(v/v) and fermentation efficiency
of 70.16 % of theoretical ethanol production.
Keywords : Liquefaction, sweet potato, bioethanol, Saccharomyces cerevisiae
INTRODUCTION
An efficient method for conversion of biomass into fuel is by bioethanol production
because bioethanol is an economical as well as environmentally friendly fuel. Bioethanol
has the advantages of being renewable, cleaner burning and produces no greenhouse gases
(Altıntas et al., 2002). Sweet potato is one source of starch which is widely used in
industry, one of them was for the production of bioethanol. Bioethanol production from
starch raw materials requires the hydrolysis process, because of yeast are unable to
consume raw starch and hence, the starch must first be hydrolysis into simple sugars (Birol
et al., 1998). Hydrolysis process can be carried out enzymatically or chemically
(Taherzadeh and Karimi, 2007). The hydrolysis of starch to products with low molecular
weight, catalyzed by α-amylases is one of the most important commercial enzyme
processes. Amylase enzyme used to break down the polymer to smaller sugar units, which
is eventually converted to the individual basic glucose units (Delphine et al., 2000; Baskar
et al., 2008).
Yeast cells Saccharomyces cerevisiae are facultative anaerobes and under anaerobic
conditions can ferment glucose to bioethanol. S. cerevisiae is ideal for bioethanol
production due to several properties including fast growth rates, efficient glucose
repression, efficient ethanol production and a tolerance for environmental stresses, such as
high ethanol concentration and low oxygen levels.
In this work, the conversion of starchy materials into bioethanol have two steps,
enzymatically liquefaction of starch, and fermentation of sugar to etanol. Batch
fermentation runs were performed to produce bioethanol using strains of Saccharomyces
cerevisiae.
MATERIALS AND METHODS
Raw material. Sweet potato was collected from Tabanan. Saccharomyyces cerevisiae
was collected from microbiology laboratorium Bogor University. Sweet potato was
analysed to determine chemical compotition before it is used in liquefaction and
fermentation process.
Inoculum culture S. cerevisiae. The strain S. cerevisiae were maintained on potato
dextrose agar (PDA). The medium composition for cultivation was as follow: yeast extract
5 g/L, malt 5 g/L, glocose 10 g/L and peptone 5 g/L. Cultivation was conducted in 250 ml
Erlenmeyer flask for 24 hours at 30 oC and agitation 125 rpm.
Liquefaction. Sweet potato slurry was hydrolyzed enzymatically. The suspension sweet
potatoes made with a concentration of 30 % (w/v), pH was adjusted to 6.5 with NaOH.
Gelatinization process carried out at a temperature of 90 °C. The enzyme concentration and
temperature liquefaction process was conducted in accordance with the treatment for 1
hour.
Fermentation. Batch fermentation process carried out for 4 days in 250 ml erlenmeyer
with 200 ml work volume. Substrate was sterilized at 121 ° C for 15 minutes and cooled,
then cultured S. cerevisiae with a concentration of 10% (v/v) was added aseptically to the
substrate. The pH and temperature of the process carried out in accordance with the
treatment.
Experimental design. These studies was designed by factorial randomized block design
with two factors. The liquefaction process consists of 2 factors, namely: the concentration
of α-amylase enzyme and temperature. The concentration of α-amylase enzyme consists of
3 levels, namely 0.8; 1 and 1.2 ml/kg of starch. The temperature of hydrolysis consists of 3
levels, namely : 90oC, 95oC and 100oC. In the fermentation process consists of 2 factors,
namely the pH of the substrate and temperature. The pH of the substrate consists of 3 level:
4.5; 5.0 and 5.5, while, the temperature consists of 3 level, namely: 30oC, 35oC, 40oC.
Analysis methods. Glucose concebntration was assays by DNS reagent (Miller, 1959).
The pH of substrate assays by pH-meter. Ethanol concentration was analysed by gas
chromatography (GC) The ethanol concentration was analised at the end of time
fermentation process. Fermentation efficiency was measured according to formulation :
Fermentation efficiency (%) = (ethanol actual/ethanol teoritic) x 100% (Caylak and Fazilet,
1998).
RESULTS AND DISCUSSION
Proximate analyse showed that the contents of sweet potato flour was 67,94 ±
1,07% moisture, 0,91 ± 0,03 % ash, 16,07 ± 1,15 % fat, 0,04 ± 0,03 % proteine, 2.69 ±
0.04 % crude fiber and 15,05 ± 2,25% carbohydrate. The moisture content of the substrate
has a major impact on how long it can keep in the storage and still remains nutritions.
In liquefaction proses, the analysis variance of the concentration of glucose showed
that the difference of treatment liquefaction enzyme concentration and temperature
significantly affect the concentration of glucose. The highest concentration of glucose that
is 57.56 ± 4.81 g/L generated from the process liquefaction using an enzyme concentration
1.2 ml/g with a temperature of 100 °C and this treatment was significantly different from
other treatments. The lowest concentration of glucose that is 22.96 ± 11.20 g/L resulted
from treatment using enzyme concentrations of 0.8 ml/g with a temperature of 100 °C and
this treatment was also significantly different from other treatments. The average value of
the glucose concentration in the liquefaction process showed in the Table 1.
Table1. The average value of the glucose concentration in the liquefaction process
Temperature (T oC)
T1 (90 oC)
T2 (95 oC)
T3 (100 oC)
ab
ab
Concentration
C1 (0,8 ml/g)
28,99
32,64
22,96 c
b
ab
(α-amilase)
C2 (1 ml/g)
40,10
26,21
24,87 ab
ab
ab
C3(1,2 ml/g)
29,87
24,15
57,56 a
** Notation with different letters behind the averages indicate a significant difference between
treatments (p