Paper No. T.5-1.3, pp.

1-5

The 6th PSU-UNS International Conference on Engineering and

Technology (ICET-2013), Novi Sad, Serbia, May 15-17, 2013
University of Novi Sad, Faculty of Technical Sciences

BIO-ETHANOL FROM PREBIOTIC

EXTRACTED JACKFRUIT SEEDS
1

Sininart Chongkhong1*, Bancha Lolharat1

Prince of Songkla University, Faculty of Engineering, Thailand

*Authors to correspondence should be addressed via email: sininart.c@psu.ac.th

Abstract: Ethanol production from prebiotic extracted

jackfruit seeds using co-culture, rice cake starter, was
evaluated. The heating pretreatment step, and the
simultaneous hydrolysis and fermentation step were
investigated. The optimum pretreatment was attained at
90 C for 15 min. Then for the simultaneous hydrolysis
and fermentation, the optimum condition was using 4
%wt rice cake starter at 30 C for 144 h with initial pH
of 5.0 and 100 rpm oil bath shaking rate. This provided
the 15.3 %v ethanol product that could be commercially
reached at 95 %v using a rotary vacuum evaporator.
Key Words: Ethanol/ Prebiotic/ Jackfruit seeds/ Rice
cake starter

1. INTRODUCTION
Biomass is of interest in valuable alternative
resources as feedstock for bio-fuel production.
Renewable energies, inclusive of ethanol and biodiesel,
are sought for to be used as substitutes for petroleumbased fuels (respectively gasoline and diesel), especially
at times of energy crises [1]. Ethanol can be produced
economically from low cost starting materials such as
agricultural residues and wastes [2]. Three main types of
raw materials for ethanol production using biological
method are cellulose, carbohydrate and sugar [3-5]. The
majority type from agricultural materials is cellulose.
However, the other two types are easier to be
transformed into bio-ethanol fuel.
Ethanol production procedures from biomass
including corn, wheat, grain and seeds, which are rich in
carbohydrates, consist of feedstock preparation or
pretreatment, hydrolysis (transforming carbohydrate into
sugar), fermentation (converting the sugar into ethanol)
[3] and product purification (distillation or evaporation).
The essential hydrolysis step, which provides the
fermentable sugar, can be carried out by using enzyme or
microorganisms.
Loog Pang Kao Mhark (a rice cake starter) is an
inexpensive organism source which has been used
traditionally for fermented food and alcoholic drink
productions. Important organisms in the rice cake starter
that can transform cellulose and carbohydrate into
fermentable sugar are Aspergillus sp., Rhizopus sp., and

Mucor sp. and the starter contains also Saccharomyces

cerevisia sp. and Pichia sp. that can convert sugar into
ethanol [6-8]. In addition, Saccharomyces cerevisias
NP01, yeast, could be isolated from Loog Pang to
produce ethanol in a previous work by Laopaiboon et al.
[9].
Prebiotics are carbohydrates such as oligosaccharides
that are considered a functional food [10]. Following our
previous work studied on prebiotic extraction from
jackfruit seeds, in this work the prebiotic extracted
jackfruit seeds and their residues were evaluated for
ethanol production using microorganisms from Loog
Pang Kao Mhark (co-culture). Although the prebiotic
contents (culture media) that affected the growth of
organisms in a fermented agricultural product [11] are
low, prebiotic extracted jackfruit seeds still contain
several components that are of value for the ethanol
production. The study also focused on optimization of
operating conditions using Taguchi methods [12].
2. MATERIAL AND METHOD
2.1 Substrate and organisms
Prebiotic extracted jackfruit seeds, crushed down to 1
mm in size, were obtained from the DoE ChE (Discipline
of Excellent in Chemical Engineering), Faculty of
Engineering,
Prince
of
Songkla
University.
Compositions of the prebiotic extracted jackfruit seeds
are shown in Table 1.
Loog Pang Kao Mhark was purchased from a local
market in Songkhla province, Thailand.
2.2 Studies of the thermal pretreatment
The prebiotic extracted seed granules were processed
to eliminate impurities (from previous extraction
procedures). The seeds and clean water were mixed at a
weight ratio of 1 to 1 into 250 ml flasks. The flasks were
then immersed into an oil bath at a predetermined
temperature in the range of 70 95 C and a heating time
of 5 30 min with a constant shaking rate of 60 rpm.
After this heating pretreatment, the pretreated seeds were
cooled down to an ambient room temperature to be used
as substrates for simultaneous hydrolysis and
fermentation experiments.

seeds remained proportionally essential components for

ethanol production.

2.3 Studies of the simultaneous hydrolysis and

fermentation
Loog Pang, a rice cake starter and a co-culture, was
used to carry out hydrolysis process together with
fermentation process (transforming carbohydrate into
fermentable sugar and then converting sugar into ethanol
at the same time). The pretreated product was firstly
mixed with the rice cake starter followed by a nitrogen
gas feeding (for anaerobic process) in 250 ml air-locked
Erlenmeyer flasks. An oil bath was used to heat the
mixture in the flasks. Crucial factors, namely, rice cake
starter amount of 1 to 10 %wt, temperature from
ambience to 40 C, time of 24 to 240 h, initial pH of 3.0
to 6.0 and shaking rates of 40 to 120 rpm were examined.
After fermentation, the product mixture was separated
into liquid and solid phases using a fabric filter.
Thereafter the desired liquid phase was centrifuged at
5000 rpm for 5 min before being forced through a
syringe filter to obtain a clear liquid product. Its
compositions were then analyzed by gas chromatography
(GC). Taguchi method was employed to seek the
optimum condition for these laboratory-scaled tests.

Table 1. Compositions of the fresh jackfruit seeds and

prebiotic extracted jackfruit seeds
Compositions
Protein
Crude Fat
Moisture
Ash
Crude Fiber

AOAC
(Kjeldahl Method)
AOAC
(Soxhlet Extraction
Method)
AOAC
(Loss on Drying
at 95-100C)
AOAC
AOAC
(Fritted Glass Crucible
Method)

Total
Carbohydrate

Calculation

Energy

Calculation

Total Sugar

Lane & Eynon

Modified
dinitrosalicylic
acid method

Reducing
Sugar

2.4 Studies of the product purification

The fermented product could be purified to reach the
95 %v ethanol product (commercial grade) using an
evaporator to remove residual water and impurities.
Purification conditions were studied using a rotary
vacuum
evaporator
(S/N
100714835
model,
manufactured by Heidolph company) which was
operated at temperature of 40, 55, 65 and 78 C (normal
boiling point of ethanol) under a pressure of 0.175 bar
for 45 min. The purified ethanol product was then
analyzed by GC.

Analytical methods

Fresh
seeds

Prebiotic
extracted
seeds

5.48%

4.99%

0.21%

0.23%

56.51%

58.83%

1.42%

0.75%

1.27%

2.20%

36.38%

35.20%

169.33
kcal
0.60%
133.2
g/ml

162.83 kcal
0.40%
282.5
g/ml

3.2 Results of the thermal pretreatment

3.2.1 Effect of heating time

2.5 Analytical method

Gas chromatography with a flame ionization detector
(GC 6890 Hewlett Packard, USA) using an HP-FFAP
column (2.5 m length, 0.32 mm ID.) was used to analyze
components of the ethanol product. While the injection
ports were kept at 250 C, 30 L of the sample was
injected by an apparatus which used nitrogen carrier gas
with a flow rate of 20 ml/min. The column oven was
operated isothermally at 150 C. The combustion gas
was a mixture of hydrogen and air. Methanol was used as
the internal standard. Scanning was carried out to
produce a chromatogram which showed peak areas of
ethanol as volumetric percentages (%v).

Fig. 1. Effect of heating time on ethanol content in the

product for the pretreatment using 3% starter at 30C
for 120 h with pH 5.0 and 60 rpm shaking rate
Ethanol content of heating pretreatment versus
heating time at 80 C heating temperature before ethanol
fermentation using 3 %wt starter amount at 30 C for
120 h with 5.0 pH and 60 rpm shaking rate is shown in
Fig. 1. It was found that the best heating time was 15 min
as it yielded the maximum amount of ethanol.

3. RESULT AND DISCUSSION

3.1 Compositions of the prebiotic extracted jackfruit
seeds
Component comparisons of fresh jackfruit seeds and
prebiotic extracted jackfruit seeds are shown in Table 1.
After prebiotics extraction process, carbohydrate and
total sugar contents in the jackfruit seeds were decreased
because the prebiotics are carbohydrates or large
molecule sugars. However, the reducing sugar and
moisture contents that are significant preliminary
sustenance (inoculums) for organism subsistence were
increased from heating in the extraction process.
Moreover, the product residue and the prebiotic extracted

3.2.2 Effect of heating temperature

Fig. 2. illustrates the effect of heating temperature on
ethanol content for 15 min heating time before ethanol
fermentation using 3 %wt starter amount at 30 C for
120 h with 5.0 pH and 60 rpm shaking rate. For the
heating pretreatment, the highest ethanol content was
achieved at the heating temperature of 90 C.

Fig. 2. Effect of heating temperature on ethanol content

in the product for the pretreatment using 3% starter at
30C for 120 h with pH 5.0 and 60 rpm shaking rate

Fig. 3. Effect of rice cake starter amount on ethanol

content in the product operating at 30C for 120 h with
pH 5.0 and 60 rpm shaking rate

To sum up the optimum pretreatment condition,

another physical characteristic of the pretreated product
was scrutinized. The main objective was for the product
to reach gelatinization point. From the experiments,
viscosity of the heated jackfruit seeds increased with
increasing heating temperature. This increase in viscosity
was the gelatinization process in which the starch
molecules became expanded and active for the next
hydrolysis step. It was found that the gelatinization point
of the prebiotic extracted seeds was at 90 C for 15 min.
This gelatinization temperature of the prebiotic extracted
jackfruit seeds was higher than that of the fresh jackfruit
seeds, which should not be more than 73.3 C. This
could be because of the combination of gelatinization
and liquefaction that were readily set up for both
hydrolysis and fermentation because a gelatinization
temperature should be in the 63.1 to 73.3 C range and a
liquefaction temperature should be in the 79.6 to 90.2 C
range [13].

3.3.2 Effect of time

3.3 Results of the simultaneous hydrolysis and

fermentation
3.3.1 Effect of rice cake starter amount
The amount of rice cake starter used in our process
was varied from 1, 2, 3, 4, 5, to 6 %wt (weight of the rice
cake starter to weight of the seeds). The suitable amount
of the rice cake starter, a microorganism source, was
found to be 4 %wt as it produced the maximum ethanol
content (Fig. 3.). However, 3 and 4 %wt of the starter
were compared additionally for economy reason.

Fig. 4. Effect of time on ethanol content in the product

using 3% starter at 30C with pH 5.0 and 60 rpm
shaking rate
Fig. 4. shows the effect of resident time on ethanol
content at 30 C with 3 %wt rice cake starter and 60 rpm
shaking rate. Rapid formation of ethanol was observed
within the first 144 h, after that the conversion rate
declined. Resident times of 120 and 144 h, respectively,
were then adopted for further study.
3.3.3 Effect of shaking rate

Fig. 5. Effect of shaking rate of oil bath on ethanol

content in the product using 3% starter at 30C for 120 h
with pH 5.0

It was found from the experiments that the shaking

rate of 100 rpm was the most appropriate for the process
(Fig. 5.). There was no improvement of ethanol content
with higher speeds. Moreover, slower speeds down to 60
rpm still did not yield significant inappropriateness.

required experiments with a two-level-three-factor array,

L4 (23).
Table 2. Independent variables and Levels of L4(23) for
Taguchi method

3.3.4 Effect of pH
The ethanol contents in the experiments increased
with increasing pH from 3.0 to 5.0 (Fig. 6.).
Nevertheless, the conversion rate slowed down mildly in
the range of 5.0 to 5.5. Thereby, pH of 4.5 and 5.0 were
chosen for further study.
Table 3. Results of Taguchi experiments for determining
of the optimum condition of the simultaneous hydrolysis
and fermentation
Experimental
t
S
P Ethanol content
Number
(%v)
Level
1
1
1
1
11.3
2
1
2
2
13.0
3
2
1
2
14.6
4
2
2
1
15.3
Because 30 C and 100 rpm were already deemed
appropriate for the process, this rendered only the three
vital factors to be investigated by the Taguchi method. It
can be seen from Table 3. that Experimental Number 4
gave maximum ethanol content. Therefore, for
hydrolysis combined with fermentation the optimum
process was at a temperature of 30 C with 4 %wt starter,
5.0 pH for 144 h and 100 rpm.
This research showed that prebiotic extracted seeds
were a potential feedstock for ethanol production
comparable with other materials such as millet flour [14]
and sweet sorghum juice [15].

Fig. 6. Effect of pH on ethanol content in the product

using 3% starter at 30C for 120 h and 60 rpm shaking
rate
3.3.5 Effect of temperature
Fig. 7. depicts the effect of temperature on ethanol
content at 3 %wt of the starter for 120 h. The ethanol
content was found to decrease with increasing
temperature ranging from an ambient room temperature
(27-32 C) to 40 C. From the experiments, a process
could suitably be carried out at a constant temperature of
30 C.

3.5 Results of the product purification

Fig. 8. Effect of evaporation temperature on ethanol

content for purification under 0.175 bar for 45 min

Fig. 7. Effect of temperature on ethanol content in the

product using 3% starter for 120 h with pH 5.0 and 60
rpm shaking rate

Fig. 8. shows that the fermented product can be

purified with a low operating temperature of the
evaporator (A lower normal boiling point of pure
ethanol). To meet the standard requirement for
commercial ethanol product (95 %v ethanol), it is
suitable to operate at 40 C evaporating temperature
under 0.175 bar (almost a vacuum pressure) for 45 min,
and this is in good agreements with other works [16-18].

3.4 Determination of the optimum condition for the

simultaneous hydrolysis and fermentation by Taguchi
method
Rice cake starter amount (S), resident time (t) and pH
(P) were selected as independent variables. Variable
levels are laid out in Table 2. Table 3. shows the four

4. CONCLUSION
Results obtained from this work have demonstrated
that prebiotic extracted jackfruit seeds, the residual
products from prebiotic extraction, are a satisfactory
material for ethanol production. The main feature of the
process is a simple operation employing low cost
organisms (Loog Pang Kao Mhark) that could reduce
production steps and save costs. The final product can
meet a standard requirement for 95 %v commercial
ethanol product.

5. ACKNOWLEGDMENT
This research was financially supported by the
National Research Council of Thailand: NRCT 20112012, Contract No. ENG540091c-2, Faculty of
Engineering, Prince of Songkla University, Thailand.

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