Paper No. T.5-1.3, pp. 1-5
The 6th PSU-UNS International Conference on Engineering and Techn ol ology ogy (ICET (ICET-201 -2013), 3), Novi Sad, Serbi a, May May 15-17, 15-17, 2013 2013 University o f Novi Sad, Faculty Faculty o f Technical Sciences
BIO-ETHANOL FROM PREBIOTIC EXTRACTED JACKFRUIT SEEDS 1*
Sininart Chongkhong , Bancha Lolharat
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Prince of Songkla University, Faculty of Engineering, Thailand *Authors to correspondence should be addressed via email:
[email protected] [email protected]
Abstract: 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 petroleum based 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
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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 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.
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. 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. 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 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). 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
seeds remained proportionally essential components for ethanol production. Table 1. Compositions of the fresh jackfruit seeds and prebiotic extracted jackfruit seeds Compositions Protein Crude Fat
Moisture Ash Crude Fiber
Analytical methods AOAC (Kjeldahl Method) AOAC (Soxhlet Extraction Method) AOAC (Loss on Drying at 95-100°C) AOAC AOAC (Fritted Glass Crucible Method)
Total Carbohydrate
Calculation
Energy
Calculation
Total Sugar
Lane & Eynon Modified dinitrosalicylic acid method
Reducing Sugar
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
Fig. 1. Effect of heating time on ethanol content in the product for the pretreatment using 3% starter at 30 C 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. 3.2.2 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.
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Fig. 2. Effect of heating temperature on ethanol content in the product for the pretreatment using 3% starter at 30 C 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 30 C 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 30 C 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 30 C for 120 h with pH 5.0
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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
Fig. 6. Effect of pH on ethanol content in the product using 3% starter at 30 C 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.
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 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
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]. 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. 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].
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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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