BIO-ETHANOL FROM PREBIOTIC EXTRACTED JACKFRUIT SEEDS
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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
Sininart Chongkhong1*, Bancha Lolharat1
1
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 Mucor sp. and the starter contains also Saccharomyces
jackfruit seeds using co-culture, rice cake starter, was cerevisia sp. and Pichia sp. that can convert sugar into
evaluated. The heating pretreatment step, and the ethanol [6-8]. In addition, Saccharomyces cerevisias
simultaneous hydrolysis and fermentation step were NP01, yeast, could be isolated from Loog Pang to
investigated. The optimum pretreatment was attained at produce ethanol in a previous work by Laopaiboon et al.
90 C for 15 min. Then for the simultaneous hydrolysis [9].
and fermentation, the optimum condition was using 4 Prebiotics are carbohydrates such as oligosaccharides
%wt rice cake starter at 30 C for 144 h with initial pH that are considered a functional food [10]. Following our
of 5.0 and 100 rpm oil bath shaking rate. This provided previous work studied on prebiotic extraction from
the 15.3 %v ethanol product that could be commercially jackfruit seeds, in this work the prebiotic extracted
reached at 95 %v using a rotary vacuum evaporator. jackfruit seeds and their residues were evaluated for
Key Words: Ethanol/ Prebiotic/ Jackfruit seeds/ Rice ethanol production using microorganisms from Loog
cake starter Pang Kao Mhark (co-culture). Although the prebiotic
contents (culture media) that affected the growth of
organisms in a fermented agricultural product [11] are
1. INTRODUCTION low, prebiotic extracted jackfruit seeds still contain
several components that are of value for the ethanol
Biomass is of interest in valuable alternative
production. The study also focused on optimization of
resources as feedstock for bio-fuel production.
operating conditions using Taguchi methods [12].
Renewable energies, inclusive of ethanol and biodiesel,
are sought for to be used as substitutes for petroleum-
2. MATERIAL AND METHOD
based fuels (respectively gasoline and diesel), especially
at times of energy crises [1]. Ethanol can be produced 2.1 Substrate and organisms
economically from low cost starting materials such as Prebiotic extracted jackfruit seeds, crushed down to 1
agricultural residues and wastes [2]. Three main types of mm in size, were obtained from the DoE ChE (Discipline
raw materials for ethanol production using biological of Excellent in Chemical Engineering), Faculty of
method are cellulose, carbohydrate and sugar [3-5]. The Engineering, Prince of Songkla University.
majority type from agricultural materials is cellulose. Compositions of the prebiotic extracted jackfruit seeds
However, the other two types are easier to be are shown in Table 1.
transformed into bio-ethanol fuel. Loog Pang Kao Mhark was purchased from a local
Ethanol production procedures from biomass market in Songkhla province, Thailand.
including corn, wheat, grain and seeds, which are rich in
carbohydrates, consist of feedstock preparation or
2.2 Studies of the thermal pretreatment
pretreatment, hydrolysis (transforming carbohydrate into
The prebiotic extracted seed granules were processed
sugar), fermentation (converting the sugar into ethanol)
to eliminate impurities (from previous extraction
[3] and product purification (distillation or evaporation).
procedures). The seeds and clean water were mixed at a
The essential hydrolysis step, which provides the
weight ratio of 1 to 1 into 250 ml flasks. The flasks were
fermentable sugar, can be carried out by using enzyme or
then immersed into an oil bath at a predetermined
microorganisms.
temperature in the range of 70 – 95 C and a heating time
Loog Pang Kao Mhark (a rice cake starter) is an
of 5 – 30 min with a constant shaking rate of 60 rpm.
inexpensive organism source which has been used
After this heating pretreatment, the pretreated seeds were
traditionally for fermented food and alcoholic drink
cooled down to an ambient room temperature to be used
productions. Important organisms in the rice cake starter
as substrates for simultaneous hydrolysis and
that can transform cellulose and carbohydrate into
fermentation experiments.
fermentable sugar are Aspergillus sp., Rhizopus sp., and
12.3 Studies of the simultaneous hydrolysis and seeds remained proportionally essential components for
fermentation ethanol production.
Loog Pang, a rice cake starter and a co-culture, was
used to carry out hydrolysis process together with Table 1. Compositions of the fresh jackfruit seeds and
fermentation process (transforming carbohydrate into prebiotic extracted jackfruit seeds
fermentable sugar and then converting sugar into ethanol Fresh
Prebiotic
at the same time). The pretreated product was firstly Compositions Analytical methods extracted
seeds
seeds
mixed with the rice cake starter followed by a nitrogen AOAC
gas feeding (for anaerobic process) in 250 ml air-locked Protein 5.48% 4.99%
(Kjeldahl Method)
Erlenmeyer flasks. An oil bath was used to heat the AOAC
mixture in the flasks. Crucial factors, namely, rice cake Crude Fat (Soxhlet Extraction 0.21% 0.23%
Method)
starter amount of 1 to 10 %wt, temperature from AOAC
ambience to 40 C, time of 24 to 240 h, initial pH of 3.0 Moisture (Loss on Drying 56.51% 58.83%
to 6.0 and shaking rates of 40 to 120 rpm were examined. at 95-100°C)
After fermentation, the product mixture was separated Ash AOAC 1.42% 0.75%
AOAC
into liquid and solid phases using a fabric filter.
Crude Fiber (Fritted Glass Crucible 1.27% 2.20%
Thereafter the desired liquid phase was centrifuged at Method)
5000 rpm for 5 min before being forced through a Total
Calculation 36.38% 35.20%
syringe filter to obtain a clear liquid product. Its Carbohydrate
compositions were then analyzed by gas chromatography 169.33
Energy Calculation 162.83 kcal
kcal
(GC). Taguchi method was employed to seek the Total Sugar Lane & Eynon 0.60% 0.40%
optimum condition for these laboratory-scaled tests. Modified
Reducing 133.2 282.5
dinitrosalicylic
Sugar g/ml g/ml
2.4 Studies of the product purification acid method
The fermented product could be purified to reach the
95 %v ethanol product (commercial grade) using an 3.2 Results of the thermal pretreatment
evaporator to remove residual water and impurities.
Purification conditions were studied using a rotary 3.2.1 Effect of heating time
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.
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 μL of the sample was
injected by an apparatus which used nitrogen carrier gas Fig. 1. Effect of heating time on ethanol content in the
with a flow rate of 20 ml/min. The column oven was product for the pretreatment using 3% starter at 30C
operated isothermally at 150 C. The combustion gas for 120 h with pH 5.0 and 60 rpm shaking rate
was a mixture of hydrogen and air. Methanol was used as
the internal standard. Scanning was carried out to Ethanol content of heating pretreatment versus
produce a chromatogram which showed peak areas of heating time at 80 C heating temperature before ethanol
ethanol as volumetric percentages (%v). fermentation using 3 %wt starter amount at 30 °C for
120 h with 5.0 pH and 60 rpm shaking rate is shown in
3. RESULT AND DISCUSSION Fig. 1. It was found that the best heating time was 15 min
3.1 Compositions of the prebiotic extracted jackfruit as it yielded the maximum amount of ethanol.
seeds
Component comparisons of fresh jackfruit seeds and 3.2.2 Effect of heating temperature
prebiotic extracted jackfruit seeds are shown in Table 1. Fig. 2. illustrates the effect of heating temperature on
After prebiotics extraction process, carbohydrate and ethanol content for 15 min heating time before ethanol
total sugar contents in the jackfruit seeds were decreased fermentation using 3 %wt starter amount at 30 °C for
because the prebiotics are carbohydrates or large 120 h with 5.0 pH and 60 rpm shaking rate. For the
molecule sugars. However, the reducing sugar and heating pretreatment, the highest ethanol content was
moisture contents that are significant preliminary achieved at the heating temperature of 90 C.
sustenance (inoculums) for organism subsistence were
increased from heating in the extraction process.
Moreover, the product residue and the prebiotic extracted
2Fig. 2. Effect of heating temperature on ethanol content Fig. 3. Effect of rice cake starter amount on ethanol
in the product for the pretreatment using 3% starter at content in the product operating at 30C for 120 h with
30C for 120 h with pH 5.0 and 60 rpm shaking rate pH 5.0 and 60 rpm shaking rate
To sum up the optimum pretreatment condition, 3.3.2 Effect of time
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 Fig. 4. Effect of time on ethanol content in the product
temperature should be in the 63.1 to 73.3 °C range and a using 3% starter at 30C with pH 5.0 and 60 rpm
liquefaction temperature should be in the 79.6 to 90.2 °C shaking rate
range [13].
Fig. 4. shows the effect of resident time on ethanol
3.3 Results of the simultaneous hydrolysis and content at 30 C with 3 %wt rice cake starter and 60 rpm
fermentation shaking rate. Rapid formation of ethanol was observed
within the first 144 h, after that the conversion rate
3.3.1 Effect of rice cake starter amount declined. Resident times of 120 and 144 h, respectively,
The amount of rice cake starter used in our process were then adopted for further study.
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 3.3.3 Effect of shaking rate
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. 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
3It was found from the experiments that the shaking required experiments with a two-level-three-factor array,
rate of 100 rpm was the most appropriate for the process L4 (23).
(Fig. 5.). There was no improvement of ethanol content
with higher speeds. Moreover, slower speeds down to 60 Table 2. Independent variables and Levels of L4(23) for
rpm still did not yield significant inappropriateness. 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 Level (%v)
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
Fig. 6. Effect of pH on ethanol content in the product can be seen from Table 3. that Experimental Number 4
using 3% starter at 30C for 120 h and 60 rpm shaking gave maximum ethanol content. Therefore, for
rate hydrolysis combined with fermentation the optimum
process was at a temperature of 30 C with 4 %wt starter,
3.3.5 Effect of temperature 5.0 pH for 144 h and 100 rpm.
Fig. 7. depicts the effect of temperature on ethanol This research showed that prebiotic extracted seeds
content at 3 %wt of the starter for 120 h. The ethanol were a potential feedstock for ethanol production
content was found to decrease with increasing comparable with other materials such as millet flour [14]
temperature ranging from an ambient room temperature and sweet sorghum juice [15].
(27-32 C) to 40 C. From the experiments, a process
could suitably be carried out at a constant temperature of 3.5 Results of the product purification
30 C.
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
Fig. 8. shows that the fermented product can be
rpm shaking rate
purified with a low operating temperature of the
evaporator (A lower normal boiling point of pure
3.4 Determination of the optimum condition for the
ethanol). To meet the standard requirement for
simultaneous hydrolysis and fermentation by Taguchi
commercial ethanol product (95 %v ethanol), it is
method
suitable to operate at 40 C evaporating temperature
under 0.175 bar (almost a vacuum pressure) for 45 min,
Rice cake starter amount (S), resident time (t) and pH
and this is in good agreements with other works [16-18].
(P) were selected as independent variables. Variable
levels are laid out in Table 2. Table 3. shows the four
44. CONCLUSION [10] G.R. Gibson and M.B. Roberfroid, “Dietary
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This research was financially supported by the extracted from jackfruit seeds (Artocarpus
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