Universities Research Journal 2018, Vol. 11, No. 3

Preparation of Bioethanol from Waste Banana 1 2 3 War War Thin , Khin Swe Oo & Soe Soe Than

Abstract In this research, bioethanol was prepared from waste banana (Musa Accuminata Colla sub-sp) through saccharification and fermentation. Saccharification of waste banana (pulp and peel) was conducted using glucosidase enzyme. The maximum saccharification rate (Vmax) and substrate concentration (Km) were determined by using Lineweaver- Burk plot. Fermentable sugar content was determined by Lane and Eynon method. Baker’s yeast (Saccharomyces cerevisiae) was used for fermentation. During fermentation, the effect of amount of yeast, pH and fermentation period on strength of ethanol were also studied. The strength of the resultant bioethanol was measured by specific gravity method and identified by gas chromatography (GC). Key words: Waste Banana, bioethanol, saccharification, fermentation

Introduction

The excessive consumption of non-renewable energy causes in environmental deterioration and public health problems (Kahiaet al., 2016). This in turn has resulted in the need to find a source of renewable energy. The cheapest, eco-friendly and easily available source for the production of bioethanol is waste fruits. Waste fruits can be converted to bioethanol through biochemical or thermochemical processes. Biochemical conversion uses biocatalysts such as enzymes and microbial cell to convert biomass first to an intermediate sugar stream and then to ethanol and co-product (Foust, 2007). Enzymatic hydrolysis is quite a new approach and requires less energy, mild environment conditions and low utility cost compared to acid or alkaline hydrolysis (Diasa, 2009).The influence factors to the

1 Assistant Lecturer, Department of Industrial Chemistry, East Yangon University 2 Associate Professor, Department of Industrial Chemistry, University of Yangon 3 Professor, Department of Industrial Chemistry, University of Yangon 60 Universities Research Journal 2018, Vol. 11, No. 3

saccharification of complex carbohydrates to fermentable sugars are composition and structure of the feedstock, pretreatment method, type and loading of enzyme, cellulose crystallinity and available surface area (Ruan, 2004).The critical step for bioethanol production is saccharification where complex carbohydrates are converted to simple monomers. In this research, waste bananas were chosen as substrate because it consists of useful sugars and monomers of sugars that could be fermented to produce ethanol and found suitable to be used as alternative energy source (Chandel, 2007). The present study was to investigate the effect of enzymatic saccharification and further ethanol fermentation of waste banana by Saccharomyces cerevisiae.

Materials and Methods Materials The rejected waste Banana was collected from ThiriMingalar Market, Hlaing Township. Chemicals Potassium sodium tartrate (Analar grade, BDH, England), anhydrous copper II sulphate (Analar grade, BDH, England) and Sodium hydroxide (Analar grade, BDH, England) were purchased from Golden Lady (Chemical Dealers), 28th street, Pabedan Township, Yangon Region. Saczyme Plus (approximate density = 1.15 g/ml) (Novozyme, Denmark) was purchased from MY Associates Co., Ltd., Sanchaung Township, Yangon Region.

Preparation of Bioethanol Waste bananas were thoroughly washed with water. Flesh and peels were sliced into small pieces and pulped using a household blender. Some pulps were directly fermented with baker’s yeast (Saccharomyces cerevisiae). Saccharification of some pulp were conducted by varying the amount of enzyme 1 % (v/w), 2 % (v/w), 3 % (v/w), 4 % (v/w), 5 % (v/w) and 6 % (v/w), saccharification time 30 (min.), 60 (min.), 90 (min.), 120 (min.), 150 (min.) and 180 (min.), saccharification temperature 45 ºC, 50 ºC, 55 ºC, 60 ºC, 65 ºC and 70 ºC and substrate concentration 25 % Universities Research Journal 2018, Vol. 11, No.3 61

(w/v), 50 % (w/v), 75 % (w/v), 100 % (w/v), 125 % (w/v) and 150 % (w/v). After saccharification, the hydrolysates were maintained at 90ºC for 5 min. to inactivate the enzyme. Fermentation of the resultant sugar was conducted by varying the amount of yeasts 0.2% (w/w), 0.4% (w/w), 0.6% (w/w), 0.8% (w/w), 1.0% (w/w) and 1.2 % (w/w), fermentation period 1 (day), 2 (day), 3 (day), 4 (day), 5 (day) and 6 (day) at various pH (3.5, 4.0, 4.5, 5.0, 5.5, 6.0).Ethanol was then separated from the fermented broth by simple distillation at 78 ± 1 ºC. The first distillate was further purified by fractional distillation at 78 ± 1ºC using fractionating column (50 cm in height, 2 cm in outside diameter, 1.75 cm in inside diameter). After 3 hr. fractionating time, the resulting second distillate was analyzed by gas chromatography (GC). Observation of Saccharification Rate The values of sccharification rate were determined at each concentration of the samples which contain identical amount of enzyme and increasing amount of substrate by using the following equation; Beer Lambert Law, A = ε CL, where A = absorbance (nm) C = concentration (mol/L) ε = molar absorptivity (L/mol-cm) L = length of solution the light passes through (cm) Rate of Reaction = A × 103/ε × T where T = time (min.) The results are shown in Table (5). Ref; https://www.chemguide.co.uk.uvvisible

Results and Discussion The yields of fermentable sugar obtained from saccharificaiton of waste banana are shown in Tables (1 to 4). According to the results, the 62 Universities Research Journal 2018, Vol. 11, No. 3

maximum amount of reducing sugar 890 (mg/g) was achieved by using 100 % (w/v) of substrate concentration and 4 % (v/w) of glucosidase enzyme at 60ºC for 90 (min). At very low temperature, glucosidase enzyme does not show its activity because this enzyme is optically active at 50-60ºC (Kearsley, 1995). Table 1. Effect of Glucosidase Enzyme Volume on the Yield of Fermentable Sugar during Saccharification Volume Substrate Yield of Sr. Temperature of fermentable Concentration pH No (ºC) Enzyme sugar %(w/v) % (v/w) (mg/g) 1 50 60 4.7 1 306 2 50 60 4.7 2 419 3 50 60 4.7 3 568 4 50 60 4.7 4 610 5 50 60 4.7 5 512 6 50 60 4.7 6 461

Table 2. Effect of Saccharification Time on the Yield of Fermentable Sugar Sr. Substrate Volume of pH Time Yield of No Concentration Enzyme (min.) fermentable %(w/v) % (v/w) sugar (mg/g) 1 100 4 4.7 30 380 2 100 4 4.7 60 610 3 100 4 4.7 90 823 4 100 4 4.7 120 581 5 100 4 4.7 150 493 6 100 4 4.7 180 449 Universities Research Journal 2018, Vol. 11, No.3 63

Table 3. Effect of Saccharification Temperature on the Yield of Fermentable Sugar Volume Substrate Sr. of Time Temperature Yield of Concentration% Enzyme fermentable sugar (mg/g) No (w/v) % (v/w) (min.) (ºC) 2 100 4 90 50 548 3 100 4 90 55 658 4 100 4 90 60 823 5 100 4 90 65 714 6 100 4 90 70 688

Table 4. Effect of Substrate Concentration of Waste Banana on the Yield of Fermentable Sugar during Saccharification Volume Substrate Sr. of Time Temperature Yield of Concentration Enzyme fermentable sugar (mg/g) No % (v/w) (min.) (ºC) % (w/v) 2 4 90 60 50 848 3 4 90 60 75 862 4 4 90 60 100 890 5 4 90 60 125 879 6 4 90 60 150 831

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Table 5. Saccharification Rate Observed Sr. Absorbance at Substrate Reaction Rate No 405 (nm) Concentration (V) (C) (mol/L-min.) (mol/L) 1 0.977 0.14 1.56 2 0.848 0.28 3.12 3 0.709 0.42 4.66 4 0.664 0.56 6.2 5 0.617 0.69 7.7 6 0.53 0.83 9.2

When the reciprocal of the substrate concentration is plotted versus the reciprocal of the reaction rate, linear plots are obtained which mean that glucosidase enzyme obey Michaelis-Menten Kinetics. If the correlation, R2, for waste banana were nearly 1, it indicates an excellent fit between the data points and the regression line (Cleland, 1963a).

Lineweaver-Burk Plot

0.70 y = 0.0896x + 0.0007 0.60 R2 = 1 0.50 0.40 0.30 0.20

1/Reaction Rate1/Reaction 0.10 0.00 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 1/Substrate Concentration

Figure 1. Lineweaver-Burk plot Ref; Kenneth, 2013 Universities Research Journal 2018, Vol. 11, No.3 65

Km and Vmax values were calculated by using the linear equation from the Lineweaver-Burk plot. If an enzyme has a high Km value, it means substrate bind weakly to the enzyme and the higher substrate concentration will be needed to reach the half maximum reaction rate (Schnell, 2000). From the results, waste banana was achieved maximum saccharificaiton rate of 1429 (mol/L) per min. at a low substrate concentration of 123 (mol/L).The microbial enzyme used in the present study exhibited a high efficiency in the conversion of starch from fruit peels, which was comparable with the results obtained by many other researchers (Mojovic, 2006). As shown in Table (6), the maximum strength of ethanol 11.17 % (v/v) and 9.92 % (v/v) were obtained from the fermentation of saccharification and without saccharification of waste banana by using the same amount of yeast 0.8% (w/w).

Table 6. Effect of the Amount of Yeast on the Strength of Ethanol during Fermentation Sr. Fermentation Amount Ethanol Strength% (v/v) No Temp. of Yeast Analyzed by Sp.gr (ºC) % (w/w) with without Saccharification Saccharification 1 32 0.2 6.21 3.96 2 32 0.4 7.11 6.68 3 32 0.6 8.16 7.24 4 32 0.8 11.17 9.92 5 32 1 9.26 8.02 6 32 1.2 8.16 7.35

From the result in Table (7), the strengths of ethanol 14.47 %(v/v) and 12.34 %(v/v) were obtained at 4 days of fermentation period for saccharification and 5 days of fermentation period for without saccharification of waste banana. The decline in the ethanol production beyond 4 days and 5 days of fermentation period might be probably due to 66 Universities Research Journal 2018, Vol. 11, No. 3

decrease in the number of viable yeast cells or because of the denaturation of enzyme by the ethanol produced during fermentation.

Table 7. Effect of Fermentation Period on the Strength of Ethanol

Sr. Fermentation Fermentation Ethanol Strength% (v/v) No Temp. Period Analyzed by Sp.gr (day) (ºC) with without Saccharification Saccharification 1 32 1 8.6 4.21 2 32 2 10.22 5.91 3 32 3 12.99 9.84 4 32 4 14.47 10.75 5 32 5 12.8 12.34 6 32 6 11.17 9.92

From the result as shown in Table (8), the maximum strength of ethanol 16.09 % (v/v) and 12.34 % (v/v) were obtained at pH 4 and pH 4.5 for saccharification and without saccharification of waste banana respectively because the favorable pH for the growth of yeast was in a slightly acidic environment with the pH between 4-6 (Janani, 2013).

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Table 8. Effect of pH on the Strength of Ethanol during Fermentation

Ethanol Strength% (v/v) Sr. Fermentation Analyzed by Sp.gr pH Temp (ºC) No with without Saccharification Saccharification 12.88 9.73

2 32 4 16.09 11.51 3 32 4.5 14.47 12.34 4 32 5 12.63 9.92 5 32 5.5 10.59 7.71 6 32 6 8.68 5.59

According to the GC analysis as shown in Figure (2), saccharification accelerated the fermentation process and increased the final ethanol strength from 48.671 % (v/v) to 62.498 % (v/v) although the obtained ethanol strength was comparably lower than other starchy, sugary and lignocellulosic feed stocks. Hammond et al., (1996) have reported an increased sugar recovery and ethanol production from bananas and banana wastes using commercial α-amylase and glucosidase.

(a) 68 Universities Research Journal 2018, Vol. 11, No. 3

(b)

Figure 2. Gas Chromatogram of Bioethanol from waste banana (a) without Saccharification (b) with Saccharification

Conclusion This present research studied the optimization of saccharificaiton and fermentation process for the determination of the most suitable amount of enzyme to obtain the highest ethanol strength. From the comparative study of bioethanol production from waste banana, it was clear that fermentation of saccharified waste banana with Saccharomyces cerevisiae can yield maximum ethanol strength than direct fermentation under anaerobic condition.

Acknowledgements I would like to express my greatest appreciation to Dr Thin Thin Naing, Professor and Head, Department of Industrial Chemistry, East Yangon University for her permission to submit this research paper. I am very grateful to Dr Soe Soe Than, Professor and Head , Department of Industrial Chemistry, University of Yangon and Dr Khin Swe Oo, Associate Professor, Department of Industrial Chemistry, University of Yangon, for providing research facilities, editing many parts of the manuscript, and also for their invaluable suggestions offered to me during research work. Universities Research Journal 2018, Vol. 11, No.3 69

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