study on the production of ethanol from mango peel

MINISTRY OF EDUCATION & TRAINING CAN THO UNIVERSITY BIOTECHNOLOGY RESEARCH & DEVELOPMENT INSTITUTE SUMMARY BACHELOR OF SCIENCE THESIS THE ADVANCED PROGRAM IN BIOTECHNOLOGY STUDY ON T

MINISTRY OF EDUCATION & TRAINING

CAN THO UNIVERSITY

BIOTECHNOLOGY RESEARCH & DEVELOPMENT INSTITUTE

SUMMARY BACHELOR OF SCIENCE THESIS

THE ADVANCED PROGRAM IN BIOTECHNOLOGY

STUDY ON THE PRODUCTION OF ETHANOL

FROM MANGO PEEL

SUPERVISOR STUDENT

Dr NGO THI PHUONG DUNG DUONG THI THANH XUAN

Student code: 5085861 Session: 34 (2008 – 2013)

Can Tho, 2013

Abstract

The increasing interest in biotechnological processes employing lignocellulosic residues is justifiable because these materials are cheap, renewable and widespread sugar sources Biotechnological conversion of biomass into fuels and chemicals requires hydrolysis of the polysaccharide fraction into monomeric sugars that are converted into ethanol The objective of this study was to examine the feasibility for the production of ethanol from mango peel The experimental activities included: studying the effects of H 2 SO 4 concentration, time and temperature on the hydrolysis of mango peel, assessing the effects of yeast inoculum levels, pH levels to the fermentation capacity, determining the favourable time and temperature for ethanol fermentation The results showed that the hydrolysis of mango peel with

H 2 SO 4 of 3% at 121 o C for 1 hour released 8.49% reducing sugars The fermentation conditions including yeast inoculum levels at 10 5 cells/mL and pH 5.5 were found to be favourable for ethanol fermentation In the treatment of 7 incubation days at 30°, the ethanol concentration of 3.08% (v/v) was obtained

Keyword: ethanol, fermentation, hydrolysis, mango peel,

Saccharomyces cerevisiae

CONTENTS

3.1.1 The effect of the concentration of H2SO4 on hydrolysis

1 INTRODUCTION

Nowadays, ethanol is an important industrial chemical with emerging potential as a bio-fuel to replace non-renewable fossil fuels (Alfenore et al., 2002) Ethanol may be produced commercially by chemical synthesis or biosynthesis

To produce ethanol using substrates such as molasses, fruit pulps etc are well known The economics of ethanol production by fermentation is significantly influenced by the cost

of the raw materials It accounts for more than half of the production costs To achieve a lower production cost, the supply

of cheap raw material is thus a necessity Production of value added products from agro-industrial and food processing wastes is now an area of focus because it reduces pollution in the environment in addition to the energy generation The major part

of this kind of waste is mostly discarded and it is the main source for increasing the pollution in environment on occasions and also, the discarding process become a very expensive step due to high transportation costs Majority of fruit and vegetable wastes available from their processing industries are seasonal and do not decompose rapidly Moreover, carbohydrate is the main source for ethanol production which is easily found in various plant-

parts It was reported that natural resources along with S

cerevisiae are the highest bidders for the commercial production

of ethanol

Mango is native to the Indian subcontinent, belonging to family Anacardiaceae, is the most cultivated and favourite fruit of the tropics (Purseglove, 1972) The king of fruits is nutritionally very rich, unique in flavor and smell thus account for

approximately half of all tropical fruits produced globally Ripe mango flesh contains carbohydrate and fiber Carotene, thiamine, riboflavin, niacin, ascorbic acid, tryptophan, lysine and minerals are also present in the fruit (Goldsmith, 1976) Mango peel fibre

is a good source of dietary fibre and its chemical composition may be compared to that of citrus fibre The peel fibre also shows higher values of antioxidant activity, glucose retardation and its aroma and flavour are pleasant (Reyes and Vega, 1988) Mango is processed to a maximum extent, thereby producing high quantity

of solid and liquid wastes Solid wastes, stones, stalks, trimmings and fibrous materials are obtained during the preparation of raw material Liquid waste is the waste material that comes out of a factory after washing of fruits, packaging, blanching, cooling and plant and machinery clean up and so on Utilization of this mango waste is both a necessity and a challenge In mango peel, there is

a high amount of lignocellulose and reducing sugars Lignocellulosic materials, are constituted primarily of lignin, hemicellulose and cellulose The carbohydrate fraction (hemicellulose and cellulose) can be depolymerised into sugars which act as a primary carbon source for the microbial biocatalysts for the production of xylitol, ethanol, organic acids, industrial enzymes, etc (Carvalheiro et al 2008; Mussatto and Teixeira 2010; Chandel et al 2011a) The present study was carried out to produce ethanol using mango peel, an agro-industry waste

Objectives:

Production of ethanol from mango peel using

Saccharomyces cervisiae

Activities in the research:

- Study on the effect of the concentration of H2SO4 on hydrolysis

- Study on the effect of time and temperature on hydrolysis

- Study on the effect of yeast density on fermentation

- Study on the effect of pH on fermentation

- Study on the effects of temperature and time on fermentation

2 MATERIALS AND METHODS

2.1 Materials

- Mango peel collected from Tien Giang Vegetables and Fruits Joint Stock Company

- Saccharomyces cerivisiae strain from Biotechnology

Research & Develop Institute of Can Tho University (notated as 2.1)

- Bien Hoa sugar from Co op Mart

- Medium: YPG (Yeast extract 0.3%, potato 20%, and Glucose 20%)

- Chemicals, equipments in Food Biotechnology laboratory

2.2 Methods

2.2.1 Preparation of mango peel

After collected, mango peel was washed and then dried in the sun for 2-3 days, followed by being chopped into pieces of 2-3

cm The material was transferred into an oven to dry at 65oC in 24 hours until the weight was unchanged and ground it in mixer into

a fine powder

2.2.2 Hydrolysis of mango peel

2.2.2.1 Study on the effect of the concentration of acid

H 2 SO 4 on hydrolysis

- The activity was designed with one factor – the

concentration of H2SO4 with five levels: 0.5, 1, 2, 3 and 4% The total number of experimental units was 5 treatments x 3 replicates

= 15 units

- 10 g mango peel powder was mixed with 100 mL dilute acid of different concentrations and autoclaved at 121ºC for 15 minutes (Arumugam và Manikandan, 2011)

- The acid pretreated samples were cooled and filtered

- pH of the hydrolysate was adjusted to 6.0 with NaOH (20N)

- The reducing sugar content in the hydrolysates was determined by DNS method (Walia et al, 2013) and the amount of soluble solids was measured by Brix meter

2.2.2.2 Study on the effect of time and temperature on hydrolysis

- The experiment was carried out with two factors: time

with five levels (15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours) and temperature at 30, 60, 90, 100 and 121oC Total number of treatments was 5 x 5 = 25 treatments The total number of experimental units was 25 treatments x 3 replicates = 75 units

- 10 g mango peel powder was mixed with 100 mL dilute acid with the concentration chosen from the previous activity The hydrolysis was carried out in the conditions of time and temperature as in the experimental arrangement

- The acid pretreated samples were cooled and filtered

- pH of the hydrolysate was adjusted to 6 with NaOH (20N)

- The reducing sugar content in the hydrolysates was determined by DNS method (Walia et al, 2013) and the amount of soluble solids was measured by Brix meter

2.2.3 Fermentation with Saccharomyces cerevisiae

2.2.3.1 Study on the effect of yeast density on fermentation

- The activity was designed with one factor: yeast density

at 104, 105 and 106 cells/mL The total number of experimental units was 3 treatments x 3 replicates = 9 units

- Transfered 100 mL hydrolyzed mango peel solution (pH 6) into flasks

- Sterilized the solution by NaHSO3 140 mg/L in 30 minutes

- Inoculated yeast suspension with experimental levels Cover flasks by water-locks and ferment for 7 days at room temperature

- Monitored Brix level, pH and ethanol content after fermentation Analyze the data with statistics program STATGRAPHICS Plus v3.0

2.2.3.2 Study on the effect of pH on fermentation

- The effect of pH was assessed with five levels: 4, 4.5, 5, 5.5 and 6 The total number of experimental units was 5 treatments x 3 replicates = 15 units

- Adjusted pH of the hydrolysate to mentioned levels and transfer 100 mL into flasks

- Sterilized the solution with NaHSO3 140mg/l in 30 minutes

- Inoculated yeast suspension as the result of previous activity Cover flasks by water-locks and ferment in the conditions of time and temperature mentioned

- Monitored Brix level, pH and ethanol content after fermentation Analyzed the data with statistics program STATGRAPHICS Plus v3.0

2.2.3.3 Study on the effects of temperature and time

- Transferred 100 mL hydrolyzed mango peel solution into flasks with pH as the result of activity before

- Sterilized the solution with NaHSO3 140mg/l in 30 minutes Inoculate yeast suspension as the result of previous activity Cover flasks by water-locks and ferment in the conditions of time and temperature mentioned

- Monitored Brix level, pH and ethanol content after fermentation Analyze the data with statistics program STATGRAPHICS Plus v3.0

3 RESULTS AND DISCUSSION

3.1 Hydrolysis of mango peel

3.1.1 Effect of the concentration of acid H 2 SO 4 on hydrolysis

Among factors affecting the efficiency of acid hydrolysis, acid concentration is considered one of the most important factors regarding the release of sugars High concentrations of acid may decompose the hemicellulosic structure, producing inhibitors and also causing damage to the equipment used Therefore, an appropriate acid concentration is essential for acid hydrolysis of lignocellulose (Taherzadeh and Karimi, 2007)

The results in Table 3 showed that the concentration of the acid had a great impact on hydrolysis, especially the amount

of reducing sugars produced which increased with the growth of the concentration of acid The catalyst activity was proportional to

H+ concentration The more hydrogen ions were formed in the solution, the more rapid the hydrolysis process occurred (Mosier

et al, 2002) Therefore, the breaking of glucosidic bounds would increase, leading to the high conversion of hemicellulose fraction into xylose Under harsh conditions if high acid concentration was applied, the cellulose fraction would be disrupted and glucose would be generated After 15 minutes hydrolyzing, the maximum reducing sugars was observed in the treatment of H2SO4 4% (7.74%) and the minimum was observed in the case of H2SO4

0.5% (6.14%) Soluble solids were also greater when mango peel was hydrolyzed with higher concentration of acid Specifically, with 4% acid, hydrolysate had the highest Brix level (11.7oBrix)

while the lowest (6.5Brix) was seen in the treatment of 0.5% acid

Table 3: Effect of the concentration of acid H 2 SO 4

of acid used and acid neutralization later According to the statistical result, there was no significant difference between the percentages of reducing sugars produced in the treatment of

H2SO4 3% and that of H2SO4 4% (7.61% and 7.74%, respectively) Therefore, the concentration of 3% H2SO4 was

suitable for the hydrolysis of mango peel

3.1.2 Effect of time and temperature on hydrolysis The results were presented in Table 4 It was indicated

that the hydrolysis rate gradually increased with respect to temperature Kim (1999) showed that temperature plays an important role in the reaction rate of acid hydrolysis Based on Arrhenius equation, at high temperatures, reaction should be faster According to Xiang et al., breakage of hydrogen bonds into cellulose and hemicellulose fractions takes place steeply in response to temperature At low temperature (30 – 90oC) the hydrolysis occurred more slowly than at high temperature (100 –

121oC) Particularly, the amount of reducing sugars doubled (about 8%) at high temperature (100 – 121oC) in comparison with lower levels of temperature According to Gírio et al (2010), temperature for hemicellulose hydrolysis was between 121 and

160oC

The extension of hydrolysis time also contributed to the increase of reducing sugars As can be seen in Table 4, in all treatments, after 1 hours hydrolyzing, the amount of reducing sugars grew to the levels that were significantly different from that in 15 and 30 minutes Remarkably, the rate of reaction was high at 121oC in 1 hour, producing reducing sugars with 8.49% After that, it increased to insignificantly different levels (8.39% after 2 hours and 8.58% after 3 hours) Soluble solids also went

up according to temperature and time The highest Brix level (12.5%) was seen at 121oC in 3 hours while at the temperature lower than 100oC it ranged only between 5 – 9.17oBrix Therefore, the hydrolysis of lignocellulosic compounds in mango peel occurred fast at 121oC in 1 hour In order to get higher yield,

time can be extended However, it will be time-consuming, reducing the efficiency of production Meanwhile, according to Lenihan et al, (2011), hydrolysis at too high temperature and in too long time, monosaccharides can be degraded into undesirable chemicals inhibiting the fermentation later such as furfural, hydroxylmethylfurfural-HMF, acetic acid, levulinic acid, formic acid, uronic acid, vanillic acid, phenol, cinnamaldehyde, formaldehyde… Thereby, 1 hour was adequate time for efficient hydrolysis The proportion of crude fiber in mango peel is 9.33% (Ashoush, 2011), so with 8.49% reducing sugars from the treatment of 121oC and 1 hour the efficiency of hydrolysis was 79.21% This figure was high and logical since in crude fiber there is lignin, a component that cannot be hydrolyzed (Palmquist and Hahn-Hagerdal, 2000; Taherzadeh, 1999) In comparison with 38,6% in hydrolysis on the sugar cane bagasse at 100o

C, 2% phosphoric acid concentration for 300 minutes (Gamez et al., 2004) The amount of reducing sugars released from the hydrolysis of durian peel is 5.6% (w/v) at 121oC in 45 minutes with 2% sulphuric acid (Matura et al., 2012) The hydrolysis of red macroalgae (Dwi, 2012) gave the highest level of reducing sugars (3.28%, w/v) in the conditions of 121oC and 1 hour Therefore, it could be concluded that the hydrolysis of mango peel at 121oC in 1 hour was feasible and efficient because high reducing sugars were produced and the degradation of these sugars could be avoided In addition, this choice is particularly important in making the process more economic and less time-consuming