College of Agricultural Biotechnology, · Web viewEthanol is produced in Brazil in large scale using sugarcane as raw material by fermentation of sugars and distillation. Although

BIOETHANOL PRODUCTION FROM DIFFERENT

BIOMASS BY USING STRAIN OF

SACCHAROMYCES CEREVISIAE

By

TAKALKAR VISHAL ASHOKRAO

MGM COLLEGE OF AGRICULTURAL BIOTECHNOLOGY

AURANGABAD

2013

1

Bioethanol production from different biomass by using strain of Saccharomyces cerevisiae

Dissertation Submitted to

The Principal and Chairman of Scientific Advisory Committee

in partial fulfillment of the requirement for the degree of

Bachelor of Science

in

Agricultural Biotechnology

By

Takalkar Vishal Ashokrao

MGM/CABT/09/49Semester: VIII (New), Course No. HOT 481 (Hands on Training)

MITCON Bio-Pharma, PuneDuration: November 2012 to April 2013

Project Guide

Dr. Pande A.K.

Assistant Professor Department of Crop Science

MGM College of Agricultural Biotechnology, Aurangabad.

2

“Once we accept our limits,

We go beyond them.”

- Albert Einstein

Dedicated To,

My Teachers & Parents,

Who Always Believed In

My Abilities Which Will

Be Always Encouraging

Me For Lifetime….

3

Declaration

"I hereby declare that this submission is my own work and that, to the best of my

knowledge and belief, it contains no material previously published or written by

another person nor material which has been accepted for the award of any other

degree or diploma of the university or other institute of higher learning, except where

due acknowledgment has been made in the text.

Place: Signature:

Date: Name:

Reg.No:

4

(A Division of MITCON Consultancy & Engineering Services Ltd.)

Certificate

5

Mahatma Gandhi MissionCollege of Agricultural Biotechnology, Aurangabad.

(Affiliated to Krishi Vidyapeeth, Parbhani)(ISO 9001:2008 Certified)

Certificate

This is to certify that Mr. Takalkar Vishal Ashokrao Reg. No. MGM/CABT/09/49 has successfully completed Hands on Training from MITCON Biotechnology & Pharmaceutical Center, Pune during 05.12.2012 to 30.04.2013.

Evaluation Committee

Sign.,Name & DesignationModule Incharge (If outside of theHost Institute)

Sign.,Name & DesignationHands on Training coordinator of the concerned College

Sign.,Name & DesignationMember Secretary from the

University

Sign.,Name & DesignationPrincipal of the concerned college

Sign.,Name & Designation

ChairmanAssociate Dean, Biotechnology College/ Incharge,Biotechnology Centre of the respective university

6

Seal of the college

ACKNOWLEDGEMENT

This thesis is the end of my journey in obtaining my degree. I have not

traveled in a vacuum in this journey. This thesis has been kept on track and been seen

through to completion with the support and encouragement of numerous people

including teachers, my parents, my friends, colleagues. At the end of my thesis I

would like to thank all those people who made this thesis possible and an

unforgettable experience for me. At the end of my thesis, it is a pleasant task to

express my thanks to all those who contributed in many ways to the success of this

study and made it an unforgettable experience for me.

             At this moment of accomplishment, first of all I pay homage to Dr. B. N.

Chavan, Principal and Chairman of Scientific Advisory Committee, MGM College of

Agricultural Biotechnology, Aurangabad and Dr. A. K. Pande, Assistant Professor,

Crop Science, MGM CABT, Aurangabad. This work would not have been possible

without their guidance, support and encouragement. Under their guidance I

successfully overcame many difficulties and learned a lot.

            Also my special thanks to Dr. A. B. Kshirsagar, Associate Professor, Plant

Biotechnology, Mr. A. V. Kharde, Assistant Professor of Animal  Biotechnology, Mr.

N. M. Maske, Assistant Professor, Agronomy, Mr. T. B. Chavan, Assistant Professor,

Plant Pathology, Mr. N.S. Chavan, Assistant Professor, Biochemistry & Molecular

Biology for their valuable suggestion and guidance.

I will forever be thankful to my college advisor, Mrs. K. G. Deshpande,

Assistant Professor, Post-Harvest and Food Biotechnology. She has been helpful in

providing advice many times during my graduation.

            Last but not least, many thanks go to the head of the project, Mr. Chaitanya

Velhal whose have given his full effort in guiding me in achieving the goal as well as

his encouragement to maintain our progress in track. I take this opportunity to

sincerely acknowledge the MITCON Biotechnology & Pharmaceutical Center, Pune

for providing me with an excellent atmosphere and facility for doing research.

.

Place:

Date: / / (TAKALKAR V.A.)

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CONTENTS

1. Introduction……………………………………………………………..14

2. Review of literature……………………………………………………...21

3. Materials and methods…………………………………………………..27

3.1 Experimental site……………………………………………….27

3.2 Collection of material…………………………………………..27

3.3 Microbial culture and maintenance…………………………….27

3.4 Preparation of substrate………………………………………...28

3.4.1 Corn…………………………………………………..29

3.4.2. Sugarcane molasses………………………………….30

3.4.3 Rice straws……………………………………………31

3.5 Fermentation…………………………………………………...33

3.6 Distillation and dehydration……………………………………34

3.7 Feed products from stillage processing…………………………35

3.8 Quantitative estimation……………………………………….....36

4. Result and discussion……………………………………………………..37

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5. Summary and conclusion…………………………………………………..41

5.1 Summary…………………………………………………………41

5.2 Conclusion……………………………………………………….42

5.3 Future line of work………………………………………………43

6. Bibliography……………………………………………………………….44

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LIST OF TABLES

Table no.1.1 Annual Fuel Ethanol Production by Country (2007–2011)…………....16

Table no.3.1YPD media (liquid)……………………………………………………..28

Table no.3.2 Enzymes used to convert polysaccharides into simple sugar and for

fermentation process………………………………………………………………….29

Table no.4.1 Amount of bioethanol production at 370 C…………………………….38

Table no.4.2 Percentage of bioethanol at 37°C............................................................40

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LIST OF FIGURES

Fig no. 1.1 Structure of ethanol molecule………………...………………………….18

Fig.no. 3.1 YPD growth medium..…...……………………………………………....28

Fig.no. 3.2 Culture of Saccharomyces cerevisiae strain on YPD medium…...….…..28

Fig.no. 3.3 Corns selected for the fermentation process ......…………………….…..30

Fig.no. 3.4 Sugarcane molasses for the fermentation process…………………….... 31

Fig.no. 3.5 Rice straws for the fermentation process…………..……………….……32

Fig.no. 3.6 Fermentation of bioethanol……………………….………………….…..34

Fig no. 3.7 Distillation of ethanol obtained from fermentation of substrates…...…...35

Fig.no. 3.8 Stillage from corn………………………………………………….…….36

Fig.no. 4.1 Microscopic view of Saccharomyces cerevisiae…………………………37

Fig.no. 4.2 Amount of bioethanol produced in ml…………………………………...39

Fig.no. 4.3 Ethanol obtained………………………………………………………....39

Fig.no. 4.4 Bioethanol %..............................................................................................40

Fig.no. 4.3 Measurement of distilled ethanol by volume fraction…………………...40

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ABBREVIATIONS

Abbreviations Full from

% Percent

/ Per

bg Billion gallons

0C Degree Celsius

CD Critical difference

Conc. Concentration

DAI Days after inoculation

et.al. and other

Fig. Figure

FAO Food and Agricultural Organization

H Hour

Lit Liter

Mg Milligram

Min Minute

Ml Milliliter

Psi Pounds per square inch

SE Standard error

SSF Simultaneous Saccharification and Fermentation

v/v Volume by volume

w/v Weight by volume

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ABSTRACT

A lab experiment was carried out during summer season of 2012-13 at,

MITCON Biotechnology & Pharmaceutical Center, Pune to study “Bioethanol

production from different biomass by using strain of Saccharomyces cerevisiae”

under in vitro condition. The experiment was laid out in completely randomized block

design with three treatments of agricultural biomass such as corn, sugarcane molasses

and rice straws. The aim of this study is to make an evaluation of the comparative

possibilities of ethanol production increase through the introduction of yeast cells for

fermentation process. Making ethanol from cellulosic feedstock i.e. rice straw was

more challenging than using corn and sugarcane molasses. On an average within a

period of fermentation process, the percentage yield of ethanol from corn, sugarcane

molasses, and rice straws were 33.33%, 11% and 5.1% respectively. Among corn

showed significantly higher ethanol percentage; hence can be chosen for higher yield

of Bioethanol.

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1. INTRODUCTION

Bioethanol is a form of renewable energy and a promising alternative fuel

that can be produced from agricultural feedstock. It is primarily produced from starch-

based crops, such as corn. Cellulosic ethanol production volumes are very small.

Several commercial cellulosic ethanol production plants are under construction, and

intensive research and development is rapidly advancing the state of cellulosic ethanol

technology. The production of ethanol from non-starch, lignocellulosic materials is,

however, a fairly recent development. There are many ways to produce ethanol from

lignocellulosic material. The ethanol can be blended with gasoline or used neat in

combustion engines. As a fuel, ethanol burns cleaner than gasoline, is completely

renewable, and relatively less toxic to the environment (Hayward, 1993).

The high price of oil, limited availability of liquid fuels, and growing energy

demands in transportation, industrial and other sectors and concerns over effect of

greenhouse gas emissions on environment has led to a search for alternative fuels.

Because of limited area under crop cultivation and growing population, cellulosic

ethanol could be a key alternative fuel to meet energy requirements of countries like

India. Cellulosic ethanol and ethanol produced from other biomass resources have the

potential to cut greenhouse gas emissions by 86%. The best alternative for handling

such a huge quantum of biomass is the production of commercially important value-

added products like enzymes, ethanol, and organic acids. (Oberoi et al, 2011)

Distillation was well known by the early Greeks and Arabs. Greek

alchemists working in Alexandria during the 1st century A.D. carried out distillation,

and the medieval Arabs learned from the Alexandrians. In cold parts of Central Asia,

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freeze distillation was discovered and the earliest evidence of it being used dates back

to the early middle Ages. We also know that alcohol was distilled in Schola Medica

Salernitana in southern Italy during the 12th century, and that fractional distillation

was invented by Tadeo Alderotti in the 13th century. The first to mention absolute

alcohol, in contrast with alcohol-water mixtures, was Raymond Lull. In 1796, Johann

Tobias Lowitz obtained pure ethanol by mixing partially purified ethanol (the alcohol-

water azeotrope) with an excess of anhydrous alkali and then distilling the mixture

over low heat. Antoine Lavoisier described ethanol as a compound of carbon,

hydrogen, and oxygen, and in 1807 Nicolas-Theodore de Saussure determined

ethanol's chemical formula. Fifty years later, Archibald Scott Couper published the

structural formula of ethanol.

Ethanol was first prepared synthetically in 1825 by Michael Faraday. He

found that sulfuric acid could absorb large volumes of coal gas. He gave the resulting

solution to Henry Hennell, a British chemist, who found in 1826 that it contained

"sulphovinic acid" (ethyl hydrogen sulfate). In 1828, Hennell and the French chemist

Georges-Simon Serullas independently discovered that sulphovinic acid could be

decomposed into ethanol. Thus, in 1825 Faraday had unwittingly discovered that

ethanol could be produced from ethylene (a component of coal gas) by acid-catalyzed

hydration, a process similar to current industrial ethanol synthesis.

Ethanol has been utilized as a fuel source in the United States since the turn

of the century. However, it has repeatedly faced significant commercial viability

obstacles relative to petroleum. Renewed interest exists in ethanol as a fuel source

today owing to its positive impact on rural America, the environment and United

States energy security. Current technologies allow for 2.5 gallons (wet mill process)

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to 2.8 gallons (dry grind process) of ethanol (1 gallon = 3.785 l) per bushel of corn.

Valuable co-products, distillers dried grains with soluble (dry grind) and corn gluten

meal and feed (wet mill), are also generated in the production of ethanol (Bothast &

Schlicher, 2003) The world's top ethanol fuel producers in 2011 were the United

States with 13.9 billion U.S. liquid gallons (bg) (52.6 billion liters) and Brazil with

5.6 bg (21.1 billion liters), accounting together for 87.1% of world production of

22.36 billion US gallons (84.6 billion liters). Strong incentives, coupled with other

industry development initiatives, are giving rise to fledgling ethanol industries in

countries such as Germany, Spain, France, Sweden, China, Thailand, Canada,

Colombia, India, Australia, and some Central American countries. (Lichts, 2010)

Table 1.1: Annual Fuel Ethanol Production by Country (2007–2011)

(Millions of U.S. liquid gallons per year)

Worldrank

Country/Region 2011 2010 2009 2008 2007

1 United States 13,900 13,231 10,938 9,235 6,4852  Brazil 5,573.24 6,921.54 6,577.89 6,472.2 5,019.23  European Union 1,199.31 1,176.88 1,039.52 733.60 570.304  China 554.76 541.55 541.55 501.90 486.005  Thailand 435.20 89.80 79.206  Canada 462.3 356.63 290.59 237.70 211.307  India 91.67 66.00 52.808  Colombia 83.21 79.30 74.909  Australia 87.2 66.04 56.80 26.40 26.4010 Other 247.27

World Total 22,356.09 22,946.87 19,534.993 17,335.20 13,101.7(Lichts, 2010)

Ethanol is produced in Brazil in large scale using sugarcane as

raw material by fermentation of sugars and distillation. Although Brazil produces the

most sugarcane, India is the world’s number one producer of sugar. 60% of India’s

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sugar is produced in two states: Maharashtra and Uttar Pradesh and almost all the rest

is produced in Tamil Nadu, Karnataka, Gujarat and Andhra Pradesh. Most of the

domestic sugar demand in India is met by domestic production but in deficit years it

imports sugar and exports when there is a surplus. The majority of sugarcane grown

in India is by sugar mills to produce sugar and its two main by-products, molasses and

bagasse. Currently 70-80% of harvested sugarcane is used by regulated mills to

produce sugar. The other 20-30% is used for the production of alternate sweeteners:

gur and khandsari and for seeds (Raju et al 2009). The only sugarcane based product

that produces molasses as a byproduct however is standard sugar. Sugar extraction

rates from sugarcane in India average around 10.5% compared to about 14% for

Brazil (Pursell 2007)

As per the latest FAO statistics, India alone accounts for about 22% of

the total rice produced in the world. The availability of rice straw in huge quantities

and unavailability of proper infrastructure to handle such a large quantity of biomass

leads to burning of rice straw in countries like India, resulting in biomass loss and

environmental pollution problems. (Oberoi et al, 2011)

All biomass goes through at least some of these steps: it needs to be grown,

collected, dried, fermented, and burned. All of these steps require resources and an

infrastructure. The total amount of energy input into the process compared to the

energy released by burning the resulting ethanol fuel is known as the energy balance

(or "energy returned on energy invested"). Figures compiled in a 2007 by National

Geographic Magazine” point to modest results for corn ethanol produced in the US:

one unit of fossil-fuel energy is required to create 1.3 energy units from the resulting

ethanol. The energy balance for sugarcane ethanol produced in Brazil is more

favorable, with one unit of fossil-fuel energy required to create 8 from the ethanol.

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Energy balance estimates are not easily produced, thus numerous such reports have

been generated that are contradictory. For instance, a separate survey reports that

production of ethanol from sugarcane, which requires a tropical climate to grow

productively, returns from 8 to 9 units of energy for each unit expended, as compared

to corn which only returns about 1.34 units of fuel energy for each unit of energy

expended. A 2006 University of California Berkley study, after analyzing six separate

studies, concluded that producing ethanol from corn uses much less petroleum than

producing gasoline. (Sanders and Robert, 2006)

The basic steps for large scale production of ethanol are: microbial

(yeast) fermentation of sugars, distillation and dehydration . Prior to fermentation,

some crops require saccharification or hydrolysis of carbohydrates such as cellulose

and starch into sugars. Saccharification of cellulose is called cellulolysis. Different

types of enzymes are used to convert starch into sugar. (Goettemoeller, 2007)

Fig.1.1: Structure of ethanol molecule.

Glucose (a simple sugar) is created in the plant by photosynthesis.

6 CO2 + 6 H2O + light → C6H12O6 + 6 O2

During ethanol fermentation, glucose is decomposed into ethanol and carbon dioxide.

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C6H12O6 → 2 C2H5OH+ 2 CO2 + heat

During combustion ethanol reacts with oxygen to produce carbon dioxide, water, and

heat:

C2H5OH + 3 O2 → 2 CO2 + 3 H2O + heat (Goettemoeller, 2007)

Formulae:

(1) Estimation of ethanol -

Resource amount (kg) × Glucose content ×Sugar recovery with 4 FPU

cellulase × Fermentation efficiency (0.85) × Theoretical ethanol

yield(0.51)×Process recovery(0.9)

Ethanol (L) = ______________________________________________________

Specific gravity of ethanol（0.79 kg / L）

(2) Reducing sugar-

(Glucose) + 1.053 (cellobiose)

% Reducing sugar = ____________________________ ˟ 100

1.111 f (Biomass)

Considering the above points an experiment entitled “Bioethanol production from

different biomass by using strain of Saccharomyces cerevisiae” was planned during

the November 2012 to April 2013 in MITCON Biotechnology & Pharmaceutical

Center, Pune with the following objectives.

1. To convert cellulosic or starch material of selected agricultural feedstock into

bioethanol.

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2. To check the higher ethanol percentage within cellulosic or non-cellulosic

plant material.

3. To produce nutritional cattle feed from the residues of fermentation process.

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2. REVIEW OF LITERATURE

The present experiment was conducted to find out the higher amount of

ethanol production from different agricultural materials. The relevant literature

pertaining to these aspects has been reviewed and presented below.

Hayward (1995) demonstrated that ethanol is a promising alternative fuel

which can be produced biologically from a variety of waste materials such as paper

products, corn fiber, sawmill waste, straw, and rice. Ethanol has been made from

grapes, barley and potatoes for thousands of years. The production of ethanol from

non-starch, lignocellulosic materials is, however, a fairly recent development. There

are many ways to produce ethanol from lignocellulosic material. Ethanol can be

blended with gasoline or used neat in combustion engines. As a fuel, ethanol burns

cleaner than gasoline, is completely renewable, and relatively less toxic to the

environment.

Kim and Dale (2002) investigated the system expansion approach to net

energy analysis for ethanol production from domestic corn grain. Production systems

included in their study were ethanol production from corn dry milling and corn wet

milling, corn grain production (the agricultural system), soybean products from

soybean milling (i.e. soybean oil and soybean meal) and urea production to determine

the net energy associated with ethanol derived from corn grain. These five product

systems are mutually interdependent. That is, all these systems generate products

which compete with or displace all other comparable products in the market place.

Using ethanol as a liquid transportation fuel could reduce domestic use of fossil fuels,

21

particularly petroleum. Sensitivity analyses show that the choice of allocation

procedures has the greatest impact on fuel ethanol net energy.

Alegre et al (2003) investigated the catalytic role of chrysotile support on

the acceleration of alcoholic fermentation under non-aseptic conditions by

Saccharomyces cerevisiae. The fermentation medium employed consisted only of

diluted sugar-cane molasses. In the batch fermentations process with immobilized

yeasts, the initial rate of CO2 production increased roughly 27 % during the first 30

minutes, compared to systems containing no chrysotile. A study of continuous

alcoholic fermentation with chrysotile in the reactor bed showed a higher ethanol

production rate at the different dilution rates investigated compared to similar

fermentations without chrysotile.

Ryohei (2003) selected seven strains of the yeasts from hot spring drain

were evaluated for their ethanol- producing abilities from sugarcane molasses at high

temperatures. Maximum ethanol yields were obtained at 400C for the five strains and

they reduced total organic carbon (TOC) in molasses during fermentation. In the early

stage of molasses fermentation, use of the hot spring yeasts resulted in a 1-7 fold

increase (at best) in the ethanol production rates compared to that observed in the

culture of an industrial strain S. cerevisiae K-1, whereas they did not excel K-1 in the

extent of ethanol yields. Therefore, use of the selected strains in the continuous

fermentation was suggested as a possible application of these yeasts. Among the hot

spring yeasts, phylogenetic position of the strain RND14 which fermented glucose at

550C, was inferred from 18S rDNA sequence alignment: the strain was closely related

to Pichia fermenters, though significant difference in the physiological characteristics

was found between them.

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Bothast and Schlicher (2004) has been documented that ethanol has been

utilized as a fuel source in the United States since the turn of the century. Today, most

fuel ethanol is produced by either the dry grind or the wet mill process. Current

technologies allow for 2.5 gallons (wet mill process) to 2.8 gallons (dry grind process)

of ethanol (1 gallon = 3.785 l) per bushel of corn. Valuable co-products, distillers

dried grains with soluble (dry grind) and corn gluten meal and feed (wet mill), are

also generated in the production of ethanol. While current supplies are generated from

both processes, the majority of the growth in the industry is from dry grind plant

construction in rural communities across the Corn Belt. While fuel ethanol production

is an energy-efficient process, additional research is occurring to improve its long-

term economic viability. Three of the most significant areas of research are in the

production of hybrids with higher starch content or a higher extractable starch

content, in the conversion of the corn kernel fiber fraction to ethanol, and in the

identification and development of new and higher-value co-products.

Stanley (2008) had given the data of Indian ethanol production according

to its demand and supply in billion liters.

Elena et al (2009) observed that in commercial ethanol production,

producers often use sugar cane molasses as raw material due to their abundance and

low costs. The most employed microorganisms used for fermentation is

Saccharomyces cerevisiae yeasts due to their ability to hydrolyze sucrose from cane

molasses into glucose and fructose, two easily assimilable hexoses. So that, to

evaluate the application of different strains of Saccharomyces cerevisiae for sugar

cane molasses in order to produce bioethanol. According to the obtained results the

strain Safdistil C-70 achieved higher values of the specific growth rate in comparison

23

with other strains used. The maximum ethanol productivity of 2.33 g/L/h was

achieved around 36 hours of fermentation by using the yeast Safdistil C-70.

Cucek (2010) studied the simultaneous integration of technologies,

feedstock and energy towards the sustainable production of ethanol from corn in the

form of grain and Stover. It was found that the most economical process is based on

thermo-chemical route while the most economical integrated process consists of the

thermo-chemical route and dry-grind process due to the large impact of the heat

integration that can be achieved. The availability of energy at high temperature at the

reactor is useful to reduce the energy consumption of the beer column for the

dehydration of the ethanol. While in the short term the production of ethanol from

grain and stover will co-exist, the lignocellulosic material will eventually displace the

use of grain due to its lower cost.

Mino (2010) suggested that if the Government of India hopes to

successfully reach their ethanol blend goal for 2017 with minimum negative side

effects on the rest of its economy, it will need to convert its ethanol industry from one

dependent solely on sugarcane molasses to one based primarily on sugarcane juice.

His results also suggest that negative impacts on the domestic sugar and alcohol

industry and agricultural markets are unavoidable. However these impacts can be

lessened through investment in crop production technology and agricultural

infrastructure to improve yields and efficiency of irrigation systems to increase

availability of water or reduce water requirements for irrigated crop production.

Oberoi et al (2011) had the study in which Pichia kudriavzevii was

used first time for ethanol production from any lignocellulosic material. His study

demonstrated that the treatment of native straw with 1% NaOH effectively decreased

lignin by about 47% and increased glucan by about 50%. Increasing alkali

24

concentration beyond 1% for pretreatment led to higher loss of biomass and relatively

higher solubilization of hemicellulose.

Yamada et al (2011) have developed diploid yeast strain optimized for

expression of cellulolytic enzymes, which is capable of directly fermenting from

cellulosic materials. It was the first report of ethanol production from agricultural

waste biomass using cellulolytic enzyme-expressing yeast without the addition of

exogenous enzymes. Their results suggest that combining multigene expression

optimization and diploidization in yeast is a promising approach for enhancing

ethanol production from various types of lignocellulosic biomass.

. Bereche et al (2012) accomplished an evaluation of the mass balance

and energy consumption for the ethanol production process by enzymatic hydrolysis.

Moreover, his study showed the potential ethanol production increase due to the

introduction of the bagasse enzymatic hydrolysis in the conventional ethanol

production process. These results showed that a higher ethanol production is obtained

for higher solids concentrations in the hydrolysis process and the study and

characterization of lignin cake is important in order to enable the ethanol production

by enzymatic hydrolysis process.

Macrelli et al (2012) worked on the Techno-economic evaluation of

2nd generation bioethanol production from sugar cane bagasse and leaves integrated

with the sugar-based ethanol process. His modelling showed that the MESP for 2G

ethanol was 0.97 US$/L, while in the future it could be reduced to 0.78 US$/L. In this

case the overall production cost of 1G + 2G ethanol would be about 0.40 US$/L with

an output of 102 L/ton dry sugar cane including 50% leaves. Sensitivity analysis of

25

the future scenario showed that a 50% decrease in the cost of enzymes, electricity or

leaves would lower the MESP-2G by about 20%, 10% and 4.5%, respectively.

26

3. MATERIALS AND METHODS

The details of various material used and experimental methods adopted

during the course of present investigation are narrated in this chapter under suitable

sub-heads.

3.1 Experimental site:

The experiment was conducted in Biotechnology laboratory of MITCON

Biotechnology & Pharmaceutical Center, Pune.

3.2 Collection of material:

Sugarcane molasses was obtained from Jarandeshwar sugar factory of Satara

and used as a fermentation medium. Rice (Oryza sativa) straw was procured from the

agricultural fields of Pune. While the Corn grains were obtained from the College of

Agriculture, Pune. The Baker’s yeast, largely used in industrial process, was also used

in this work. Commercial Baker’s yeast was bought from local market of Pune for the

preparation of Saccharomyces cerevisiae culture.

3.3 Microbial culture and maintenance:

Yeast Extract, Peptone, Dextrose (YPD) media is a common growth medium

for S. cerevisiae. It is rich in amino acids, vitamins, and minerals necessary for S.

cerevisiae growth and fermentation process. This complex medium was supplied in

excess, so that nutrients are not a limiting factor. Although this S. cerevisiae will

grow at other pH conditions but pH 5 is chosen because it is optimal compromise for

SSF of most substrates when using common cellulases. (Hayward, 1995) The broth

27

cultures were maintained by sub-culturing every 20 days and the test tubes were then

incubated at 30 °C for 36 h. (Alegre, 2003)

Table 3.1: YPD medium (liquid)

Yeast extract 10 g/lit

Peptone 20 g/lit

Dextrose (glucose) 20 g/lit

Fig. 3.1 YPD growth medium Fig. 3.2 Culture of Saccharomyces

cerevisiae strain on YPD medium

3.4 Preparation of Substrate:

The substrates we have chosen for the ethanol production process are of

different make up. Hence are treated separately. According to their texture i.e.

lignocellulosic or non-lignocellulosic, some crops require saccharification or

hydrolysis of carbohydrates such as cellulose and starch into sugars for

degradation of lignocellulosic biomass. Following are the enzymes used to

28

convert polysaccharides into simple sugar and for fermentation process;

(Goettemoeller, 2007)

Substrate Enzyme for

Polysaccharide

Conversion

Enzyme for Fermentation

Corn α-amylase Saccharomyces cerevisiae

Sugarcane molasses ___ Saccharomyces cerevisiae

Rice straws Cellulase Saccharomyces cerevisiae

Table3.2: Enzymes used to convert polysaccharides into simple sugar and for

fermentation process

3.4.1 Corn- In the dry grind method of ethanol production, nothing was done to

pre-separate the corn starch from the kernel. The entire corn kernel was

ground into coarse flour through a grinder, then slurried with water to form a

“mash” of volume 500ml. Starch exists as insoluble, partially crystalline

granules in the endosperm of the corn kernel. Starch cannot be metabolized

directly by yeast, but must first be broken down into simple six carbon sugars

prior to fermentation. To accomplish this conversion, the pH of the mash is

adjusted to pH 6.0, followed by the addition of a α-amylase. A thermostable α-

amylase enzyme is added to begin breaking down the starch polymer to

produce soluble dextrins. (Bothast and Schlicher, 2004) Mash was heated to

100˚C & put it for 40 minutes. The mash was allowed to fall to 80–90°C.

Additional α-amylase is added and the mash is liquefied for at least 30 min.

Liquefaction greatly reduces the size of the starch polymer. The dextrinized

mash was then cooled, adjusted to pH 4.5, and glucoamylase enzyme was

29

added. Glucoamylase converts liquefied starch into glucose. Enough

glucoamylase was added such that the saccharification of the starch to glucose,

which occurs continually through the fermentation and does not limit the rate

of ethanol production. (Bothast and Schlicher, 2004)

Fig. 3.3 Corn

3.4.1 Sugarcane molasses- Molasses is commonly used as a feedstock for bioethanol

production. Molasses is the non-crystallizable residue remaining after sucrose

purification, has some advantages: it is a relatively inexpensive raw material,

readily available, it does not require starch hydrolysis and already used for

ethanol production. It is the liquid residue left after condensing the sap of

sugar cane or sugar beets until sugar crystals precipitate. After processing,

molasses contains about 60% sucrose and 40% other components. The non-

sucrose substances include inorganic salts, raffinose, kestose, organic acids

and nitrogen containing compounds. Sugar cane molasses were diluted with

water to a resultant sugar content of 180-200 g/l. The medium was not sterilized

30

and the volume was adjusted to 500ml while pH to 4.5 with 10% v/v

Hydrochloric acid from 7.5. (Elena et al, 2009)

Fig. 3.4 Sugarcane Molasses

3.4.1 Rice straws-

The bioconversion of lignocellulosic biomass to ethanol is a

multi-step process consisting of pretreatment, enzymatic hydrolysis and

fermentation. Among these, pretreatment is particularly crucial, as significant

presence of lignin and crystalline nature of cellulose impede the enzymatic

hydrolysis of lignocellulosic biomass. The lignin component in lignocellulosic

biomass acts as a physical barrier and must be removed or structurally altered

by pretreatments to make the carbohydrates available for transformation

processes (Kadam, 2000). Pretreatment gives high reaction rates and

significantly improves cellulose hydrolysis. Alkali pretreatment utilizes lower

temperature, pressure and results in lesser sugar degradation, in comparison to

acid pretreatment (Merino and Cherry, 2007). During alkali pretreatment, the

first reactions taking place are salvation and saphonication. This causes a

swollen state of the biomass leading to an increase in internal surface area, a

31

decrease in the degree of polymerization, a decrease in crystallinity, separation

of structural linkage between lignin and carbohydrates, and disruption of

lignin structure making biomass more accessible to action by enzymes (Zhao,

2008)

The straw was oven dried at 80 0 C for 1 h, cut into small pieces,

milled with grinder. The milled rice straw was treated with 0.2 M/lit KOH for

24 hrs at room temperature in 500ml Erlenmeyer flask. The flask was

incubated in an incubator shaker at 150 rpm, 4000 C for 1 h and thereafter

autoclave-sterilized at 1210 C for 30 min. The pretreated biomass (solid

residue) was collected by filtration using Buchner funnel lined with Whatman

filter paper. The biomass was washed repeatedly with tap water to a pH of

about 5.2. The neutralized alkali-treated straw obtained after treatment with

different alkali concentrations was separately dried in a hot-air oven at 700 C

to a constant weight (Oberoi et al, 2011). Pretreated rice straws were then

introduced with cellulase enzyme and incubated at 55 C for 36 hrs. Samples

were collected and used for reducing sugar. (Pasha et al, 2012)

Fig. 3.5 Rice Straws

32

3.5 Fermentation:

When the mash gets cooled to 32°C, each liquefied substrate was transferred

to the fermenter with approximate volume of 500ml where S. cerevisiae was added.

Ammonium sulfate was added as a nitrogen source for the growth of yeast. The

fermentation required 48–72 h. The pH of the ethanol declines during the

fermentation to below pH 4, because of carbon dioxide formed during the ethanol

fermentation. The decrease in pH is important for inhibiting the growth of

contaminating bacteria. However, simultaneous saccharification and fermentation

(SSF) lowers the opportunity for microbial contamination. (Bothast and Schlicher,

2005)

In case of molasses an experimental set up was done such that air did not

pass through flask but it would escape to test tube containing Ca(OH)2 solution

(limewater) otherwise there were chances of formation of acetic acid instead of

ethanol. (Ryohei, 2003)

The carbon dioxide released during fermentation is often captured and sold

at industrial level. The carbon dioxide is used in carbonating soft drinks and

beverages, manufacturing dry ice, and in other industrial processes. (Mino, 2010)

33

Fig. 3.6 Fermentation of the substrate.

3.6 Distillation and dehydration-

Distillation is the process of separating the ethanol from the solids and

water in the mash. Alcohol vaporizes at 78°C and water at 100°C (at sea level). This

difference allows water to be separated from ethanol by heating in a distillation

column. So the distillation unit was set at temp 78˚C-80˚C. Alcohol gets separated

due to vaporization. (Bothast and Schlicher, 2005)

Conventional distillation methods can produce 95% pure (190 proof)

ethanol. At this point, the alcohol and water form an azeotrope, which means further

separation by heat cannot occur. In order to blend with gasoline, the remaining 5%

water must be removed by other methods. Modern ethanol plants use a molecular

sieve system to produce absolute (100%, or 200 proof) ethanol. The anhydrous

ethanol is then blended with approximately 5% denaturant (such as gasoline) to render

it undrinkable and thus not subject to beverage alcohol. (Bothast and Schlicher, 2005

34

Fig. 3.7 Distillation of ethanol obtained from fermentation of substrates

3.7 Feed products from stillage processing-

The solid and liquid fraction remaining after distillation is referred to

as whole stillage. Whole stillage includes the fiber, oil, and protein components of the

grain, as well as the non-fermented starch. This co product of ethanol manufacture is a

valuable feed ingredient for livestock, poultry, and fish.

First, the thin stillage was separated from the insoluble solid fraction

using centrifuge. Between 15% and 30% of the liquid fraction (thin stillage) was

recycled as backset. The remainder was concentrated further by evaporation and

mixed with the residual solids from the fermentation. After evaporation, the thick,

viscous syrup was mixed back with the solids to create a feed product known as wet

distillers. Distillers containing 65% moisture can be used directly as a feed product. In

fact, it is often favored by dairy and beef feeders because cattle seem to prefer the

moist texture. However, moist feedstock has a shelf-life of only 1–2 weeks. Unless

the feedlot is within about 80 km of the ethanol plant, handling and storage can be a

35

challenge, especially in hot summer months when shelf-life is very limited. To

increase shelf-life, wet distiller was dried to 10–12% moisture, to produce dry

distiller. (Bothast and Schlicher, 2005)

Fig. 3.8 Stillage from corn

3.8 Quantitative estimation:

Substrate solution was distilled in alcohol distillation unit for

quantitative estimation of bioethanol. For the quantity estimation of bioethanol, the

pure ethanol series were oxidized by Jones reagent [K2Cr2O7+H2SO4]. Optical

density (OD) was measured through spectrophotometer at 600 nm (Tiwari et al, 2011)

36

4. RESULTS AND DISCUSSION

The national priorities for bioethanol production in ranked order are

new co-products, plant emissions, fermentation, feedstock, fiber recovery, dry

distillers, separation, pretreatment, saccharification, germ recovery, distillation, starch

hydrolysis, and carbon dioxide. For the sake this review, we highlighted three areas:

high fermentable hybrids, conversion of biomass to ethanol, and recovery of new and

high-value ethanol co-products with the best near-term opportunities to produce

ethanol more cost-efficiently.

The Saccharomyces cerevisiae culture showed single celled microorganism

found on the YPD medium when observed under microscope. It grew rapidly and

matured in three days. Flat, smooth, moist, and cream in color colonies were selected

for the fermentation.

37

Fig. 4.1 Microscopic view of Saccharomyces cerevisiae

The overall energy balance of sugarcane molasses conversion to ethanol

demonstrates that 11% bioethanol is produced when sugar concentration in molasses

was reached to 20% (increase in sugar concentration=decrease in conversion of

ethanol). When sugar level ranged from 4.0-6.0 (w/v) ethanol production was 55ml

per 500ml of molasses. Based on the practical fermentation efficiency, ethanol

production from rice straws by alkaline treatment was estimated as 5.1%. It produced

25.5ml bioethanol from 500ml alkali treated rice straws while from corn substrate

solution 166.65ml bioethanol was estimated.

It was observed that after supplementation of Saccharomyces cerevisiae

in all three substrate solutions (corn, sugarcane molasses and rice straws), the

cellulosic plant material i.e. rice straws had produced the least bioethanol production.

The data obtained from glucose fermentation obtained from liquefaction of corn

starch by S. cerevisiae show that about 33.33% (v/v) in the fermented solution was

achieved after 24 hrs. The fermented mash contains about 3.6 g yeast which can be

used as animal feed.

Table 4.1: Amounts of bioethanol at 37°C

Sr. no. Substrate Estimation of bioethanol (ml)

1 Corn 166.65

2 Sugarcane molasses 55

3 Rice straws 25.5

38

Corn Sugarcane molasses Rice straws0

20

40

60

80

100

120

140

160

180

Amount of Bioethanol Prduced (ml)

Substrare

Fig.no. 4.2 Amount of bioethanol produced in ml

39

Fig. 4.3 Ethanol obtained

Table 4.2: Percentage of bioethanol at 37°C

Sr.no. Substrate Ethanol Percent

1. Corn 33.33 %

2. Sugarcane Molasses 11 %

3. Rice Straws 5.1 %

33.33%

11%

5.1%

Bioethanol %

CornSugarcane molassesRice Straws

Fig.no. 4.4 Bioethanol %

Fig. 4.5 Measurement of distilled ethanol by volume fraction

40

5. SUMMARY AND CONCLUSION

5.1 Summary

Present investigation entitled "Bioethanol production from different

biomass by using strain of Saccharomyces cerevisiae " was carried out in vitro

conditions during Nov 2012- April 2013 in MITCON Biotechnology &

Pharmaceutical Center laboratory, Pune

Experiment was laid out in Completely Randomized Block Design with

three treatments (corn, sugarcane molasses and rice straws used as substrates for

bioethanol production). No any replication is done with the experiment.

Bioethanol is a form of renewable energy that can be produced from

agricultural feedstocks. The basic steps used for production of bioethanol from

different substrates are saccharification of starch or cellulose, yeast fermentation of

sugars, distillation and dehydration. Prior to fermentation, crops require

saccharification or hydrolysis of carbohydrates such as cellulose and starch into

sugars so cellulase was used to treat rice straws and α-amylase for corn. A

Saccharomyces cerevisiae culture was produced by using baker’s yeast. It was

inoculated into the liquefied substrates. Ethanol was produced by fermentation of the

sugar as microbial fermentation only work with sugars. Currently, only the sugar

(sugar cane) and starch (corn) portions can be economically converted. There is much

activity in cellulosic ethanol (rice straws), where the cellulose part of a plant is broken

down to sugars and subsequently converted to ethanol. For the ethanol to be usable as

a fuel, most of the water is removed by distillation. This mixture is called hydrous

ethanol and can be used as a fuel alone, but unlike anhydrous ethanol, hydrous ethanol

is not miscible in all ratios with gasoline, so the water fraction is typically removed in

41

further treatment of dehydration in order to burn in combination with gasoline in

gasoline engines. Dehydration of ethanol was done by adding benzene to the mixture.

Present investigation showed, corn in combination with

Saccharomyces cerevisiae fermentation recorded highest result in yield of bioethanol

(166.65ml) followed by sugarcane molasses (55ml) and rice straws (25.5ml).

5.2 Conclusion

The findings of the present study can show that bioethanol can be a

promising fuel and can overcome the energy crisis in the future. The cereals, straws

and molasses, which are wasted in croplands, can be used to produce bioethanol.

Producing ethanol from cellulose is a difficult technical problem to solve but requires

less energy for conversion than starch fermentation. Today, the world is facing the

problem of health, energy and environment, all of which can be solved by bioethanol

because bioethanol is eco-friendly, less polluting and can be a useful alternative

source of energy. And, as per the bioethanol estimation in this study, corn ethanol can

be a promising fuel for bulk production while agro-wastes (sugarcane molasses and

rice straws) also can be converted into fuel rather than its wastage.

5.3 Future line of work

1. Ethanol is most commonly used to power automobiles, though it may be used

to power other vehicles, such as farm tractors, boats and airplanes. Ethanol

consumption in an engine is approximately 51% higher than for gasoline since

the energy per unit volume of ethanol is 34% lower than for gasoline.

42

2. Carbon dioxide a greenhouse gas, is emitted during fermentation and

combustion. When compared to gasoline, depending on the production

method, ethanol releases less greenhouse gases.

3. The Clean Air Act requires the addition of oxygenates to reduce carbon

monoxide emissions.

4. Cellulosic ethanol production is a new approach which may alleviate land use

and related concerns. In the future, ethanol produced from cellulose has the

potential to cut life cycle greenhouse gas emissions by up to 86 percent

relative to gasoline.

5. For bioenergy to become a widespread climate solution, technological

breakthroughs are necessary.

6. As ethanol yields improve or different feedstocks are introduced, ethanol

production may become more economically feasible. Also, as long as oil

prices remain high, the economical use of other feedstocks, such as cellulose,

become viable. By-products such as straw or wood chips can be converted to

ethanol. Fast growing species like switch grass can be grown on land not

suitable for other cash crops and yield high levels of ethanol per unit area.

43

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