PROJECT REPORTONCHARACTERIZATION OF BIOMASS & ETHANOL PRODUCTION FROM BIOMASS BY MICROBIAL HYDROLYSIS

UNDER THE GUIDANCE OF UNDER THE SUPERVISION OF Er. Mohit Kamthania Dr. Maya Kumari Lecturer, IBMER Scientist DMangalayatan University, Aligarh Defence Institute of Bio-Energy Research, Haldwani, Uttarakhand

SUBMITTED BYHansa SaraswatM. Sc. BiotechnologyEnrolment No-20120612

For the partial fulfillment of the requirementFor the award ofMaster of Science(Biotechnology)

INSTITUTE OF BIOMEDICAL EDUCATION & RESEARCH,MANGALAYATAN UNIVERSITY,BESWAN, ALIGARHUTTAR PARDESH

Approval Sheet

This dissertation entitled Characterization of Biomass & Ethanol Production from Biomass by Microbial Hydrolysis by Hansa Saraswat is approved for the degree of Master of Science in Biotechnology.

Examiners

___________________________

___________________________

___________________________

Supervisor (s)(Dr. Maya Kumari)Scientist D, DIBERHaldwani, Uttarakhand

(Er. Mohit Kamthania)

Faculty, Department of BiotechnologyInstitute of Biomedical Education and Research Mangalaytan University, Aligarh, Uttar Pradesh

Chairman

___________________________

Date: Place: Beswa, Aligarh, Uttar Pradesh, India Declaration

I declare that this written submission represents my ideas in my own words and where other ideas or words have been included; I have adequately cited and referenced the original sources. I also declare that I have adhered to all principles of academic honesty and integrity and have not misrepresented or fabricated or falsified any idea/data/fact/source in my submission. I understand that any violation of the above will be cause for disciplinary action by the Institute and can also evoke penal action from the sources which have thus not been properly cited or from whom proper permission has not been taken when needed.

Hansa Saraswat Roll No.-20120612

Date:

SUPERVISORS CERTIFICATE

This is to certify that the dissertation titled Characterization of Biomass & Ethanol Production from Biomass by Microbial Hydrolysis by Hansa Saraswat in partial fulfillment of the requirements for the award of the Degree of Master of Science in Biotechnology is an original work carried out by her under my supervision and guidance. It is certified that the work has not been submitted anywhere else for the award of any other diploma or degree of this or any other University.

Supervisor(s)

(Dr. Maya Kumari)Scientist D, DIBER Haldwani, Uttarakhand

(Er. Mohit Kamthania) Faculty, Department of Biotechnology Institute of Biomedical Education and Research Mangalaytan University, Aligarh, Uttar Pradesh ACKNOWLEDGEMENTThe preparation of this document would not have been possible without the support, hard work and endless efforts of a number of individuals. I owe my gratitude to all those people who gave me the possibility to complete this thesis.I express my gratefulness and great sense of respect to Dr. M. Nasim, Director, DIBER, Haldwani for giving me permission to carry out my thesis work. I sincerely thank Dr. Ankur Agarawal, Scientist D, DIBER, Haldwani for his support during my work. I express my deep sense of gratitude to my elegant guide Dr. Maya Kumari, Scientist D, DIBER, Haldwani. Her deep interest in planning the experiment, inspiring guidance, constant encouragement, debonair discussion and constructive criticism helped me to complete this thesis work.It is my proud privilege to thank Brig. (Dr.) Surjit Singh Pabla, Vice- Chancellor, Mangalayatan University, Aligarh for his relentless efforts and constant inspiration throughout the study. It gives me immense pleasure in expressing my sincere gratitude to Prof. Lokesh Upadhaya, Head of Institute of Bio-medical Education and Research, Mangalayatan University, Aligarh for their encouragement, inspiring guidance and motivation during my work.It is my pleasure to pen down my sincere thanks to Er. Mohit Kamthania, Faculty of Institute of Bio-Medical Education and Research, Mangalayatan University, Aligarh for his thoughtful guidance and co-operation during the study. I would also like to thank Dr. Kamal Kishore Chaudhary, Co-ordinate Head of Institute of Biomedical Education and Research who inspired me at every step during my study.I would like to express my heartfelt gratitude to Maa and Papa who brought me to this position. I find myself enveloped in warmth of love of my sister and brother for their immaculate affection and motivation.My special thanks to my friends, colleagues and juniors, for providing assistance when needed and made difficult times easier. At last but not the least I record my sincere thanks to all the people who directly or indirectly helped me in my endeavour. Hansa SaraswatINDEX

LIST OF FIGURESLIST OF TABLESABSTRACT

1. INTRODUCTION1.1 Introduction1.2 Motivation1.3 Objectives of study1.4 Outline of Thesis

2. REVIEW OF LITERATURE2.1 Lignocelluloses Biomass2.2 Plant Cell Wall Composition2.3 Pre-treatment of Lignocelluloses Biomass.2.4 Microbial Hydrolysis2.5 Fermentation for Ethanol production

3. METHODOLOGY AND CALCULATIONS 3.1 Materials 3.2 Methodology 3.3 Calculations 4. RESULT, DISCUSSION AND CONCLUSION 4.1 Result and discussion 4.2 Conclusion

REFERENCESList of Figures

Figure No.Title

1.1Structure of Plant Cell Wall

1.2Aspergillus niger on NA Plate

1.3Trichoderma reesei on NA Plate

1.4Saccharomyces cerevisiae on NA Plate

1.5Standard Curve for Reducing Sugar Estimation through DNSA Method

1.6Standard Curve for Total Carbohydrate Estimation through Anthrone Method

1.7Standard Curve for Ethanol Estimation through Potassium Dichromate Assay

1.8Characterization of Biomass for its Different Components of Cell Wall

1.9(a) Cellulose and Lignin (b) Composition in Different Biomass

1.10Reducing Sugar Estimation for Jatropha before Microbial Incubation for Hydrolysis

1.11Reducing Sugar Estimation for Jatropha Seed Coat after Microbial Incubation

1.12Reducing Sugar Estimation for Jatropha Fruit Husk with Microbes Incubation

1.13Total carbohydrate estimation for Jatropha without Microbial Incubation

1.14Total Carbohydrate Estimation for Jatropha Seed Coat after Microbial Incubation

1.15Total Carbohydrate Estimation for Jatropha Fruit Husk after Microbial Incubation

1.16Ethanol estimation for Jatropha biomass from Non- hydrolyzed Fractions

1.17Ethanol Estimation for Jatropha Seed Coat after Microbial Hydrolysis and after Fermentation

1.18Ethanol estimation for Jatropha Fruit Husk with Microbes Incubation and after Fermentation

List of Tables

Table No.Title

1.1Comparison of First and Second Generation Bio-fuel

1.2Standard Curve for Reducing Sugar Estimation through DNSA Method

1.3Standard Curve for Total Carbohydrate Estimation through Anthrone Method

1.4Standard Curve for Ethanol Estimation through Potassium Dichromate Assay

1.5Characterization of Different Biomass

1.6Cell Wall Composition of Different Biomass

1.7Cellulose and Lignin Composition of Different Biomass Characterized in this Study

1.8Hydrolysis of Characterized Biomass and Reducing Sugar Estimation for through DNSA Method

1.9Reducing Sugar Estimation for Jatropha Seed Coat and Jatropha Fruit Husk before Microbial Hydrolysis

1.10Reducing Sugar Estimation for Jatropha Seed Coat after Microbial Hydrolysis

1.11Reducing Sugar Estimation for Jatropha Fruit Husk after Microbial Hydrolysis

1.12Total Carbohydrate Estimation for Characterized Biomass through Anthrone Method

1.13Total Carbohydrate Estimation for Jatropha Seed Coat and Jatropha Fruit Husk before Microbial Hydrolysis

1.14Total Carbohydrate Estimation for Jatropha Seed Coat after Microbial Hydrolysis

1.15Total Carbohydrate Estimation for Jatropha Fruit Husk after Microbial Hydrolysis

1.16Ethanol Estimation for Characterized Biomass through Potassium Dichromate Assay

1.17Ethanol Estimation for Jatropha Seed Coat and Jatropha Fruit Husk for Non- hydrolyzed Fractions

1.18Ethanol Estimation for Jatropha Seed Coat after Microbial Hydrolysis and after Fermentation

1.19Ethanol estimation for Jatropha Fruit Husk with Microbial Hydrolysis and after Fermentation

ABSTRACTFirst generation bio-fuels produced primarily from food crops such as grains, sugar beet and oil seeds are under scanner, due to the possibility of creating undue competition for land and water used for food and fiber production. The cumulative impacts of these concerns have increased the interest in developing bio-fuels produced from non-food biomass. Second-generation bio-fuels production from lignocelluloses biomass which includes agricultural residues (Corn Stover, crop straws and bagasse), herbaceous crops (alfalfa, switch grass), short rotation woody crops, forestry residues, waste paper and other wastes (municipal and industrial)cereal straw, bagasse, forest residues, purpose-grown energy crops such as vegetative grasses and short rotation forests, could avoid many of the concerns facing first-generation bio-fuels and potentially offer greater cost reduction potential in the longer term and do not interfere with food security. Moreover, bio-ethanol is very important in terms of energy security, environmental concern, employment opportunities, agricultural development, foreign exchange saving, socioeconomic issues etc. Lignocelluloses biomass consists of three main structural units: cellulose, hemicelluloses and lignin. Cellulose is crystalline glucose polymer and hemicellulose is amorphous polymers of xylose, arabinose, and lignin a large poly aromatic compounds. The characterization of different biomass viz. Pine needle, Camelina, Jatropha stem, Jatropha petiole, Jatropha leaves, Jatropha seed coat, Jatropha fruit husk, Jatropha seed cake, Wheat straw, Paddy, Sugarcane and Sugarcane trash was carried out in the current study with the aim to explore its potential for use as feedstock for bio-ethanol production. Camelina gave good result in the composition of cellulose and Jatropha seed coat gave in lignin composition. The maximum amount of cellulose was seen in Camelina (70.2 %) and maximum amount of lignin in Jatropha Seed Coat (42.9 %). Trichoderma reesei gave good result in the production of bio-ethanol from Jatropha fruit husk and its seed coat. The maximum amount of ethanol produced (168.2 l/ml) was on 5th day of incubation with Trichoderma reesei. The result obtained here gives an indication of the usefulness of Jatropha crop for its potential use for ethanol production through microbial hydrolysis and fermentation. However, it needs to be validated through further experimentation and analyses.

Key Words: Lignocelluloses Biomass, Bio-ethanol, Hydrolysis, Reducing Sugar, Total Carbohydrate, Fermentation.

CHAPTER 1

1.1 INTRODUCTIONIncreased concern for the security of oil supply and the negative impact of fossil fuels on the environment, particularly greenhouse gas emissions has put pressure on society to find renewable alternatives [Jacques; 1999]. Bio-energy from renewable resources is already today a viable alternative to fossil fuels; however, to meet the increasing need for bio-energy several raw materials have to be considered. Resources for biological conversion of energy to forms useful to humanity include majorly the plant biomass. Among forms of plant biomass, lignocelluloses biomass is particularly well suited for energy applications because of its large scale availability, low cost and environmentally benign production. Efficient ethanol production processes and cheap substrates are needed. Current ethanol production processes using crops such as sugar cane and corn are well established; however utilization of a cheaper substrate such as lignocelluloses could make bio-ethanol more competitive with fossil fuel, the processing and utilization of this substrate is complex and there is a requirement for efficient micro-organisms to ferment a variety of sugars [Gunaseelan; 1997].The amount of petrochemical like diesel and petrol is limited and the whole world is under the shade of energy crisis and looking for the alternative fuel source. Many countries have used bio-fuel as alternative to the petrochemicals like diesel and petrol. Ethanol is a kind of bio-fuel which can be blended with the gasoline in fixed proportion and can be used as alternative to fuel like diesel and petrol. Maximum of 20 % ethanol can be blended with the gasoline and can be used as alternative fuel source with same carbonator engine [Midilli; 2006]. Bioethanol is renewable source of energy which is generally produced by the fermentation of carbohydrate. Biomass such as cellulose, animal fat, etc is used for the production of ethanol through fermentation. Main source for such kind of biomass is sugarcane, corn, wheat bran, cassava, sweet potato etc. These are used for the ethanol production but they are mainly used for the food source and if these sources will be used for the ethanol production the whole world is going to face food crisis as the world population is increasing rapidly. To prevent the world from fuel crisis and food crisis, research has been focused on the production of bio-fuel from the waste biomass and the plant source which is not for the food purpose like waste generated in sugar mill, chemical pulp generated in paper industry and weed plant.

Bio-fuels produced from biomass such as plants or organic waste could help to reduce both the worlds dependence on oil and CO2 production. These bio-fuels have the potential to cut CO2 emission because the plants they are made from use CO2 as they grow [Osamu and Carl; 1987]. Bio-fuels and bio-products produced from plant biomass would mitigate global warming. This may due to the CO2 released in burning equals the CO2 tied up by the plant during photosynthesis and thus does not increase the net CO2 in the atmosphere. Additionally, bio-fuel production along with bio-products can provide new income and employment opportunities in rural areas. 21st Century is looking for a shift to alternate industrial feedstock and green processes to produce these chemicals from renewable biomass resources [Stevens and Verhe; 2000].First generation bio-fuels can offer some CO2 benefits and can help to improve domestic energy security. But concerns exist about the sourcing of feedstock, including the impact it may have on biodiversity and land use and competition with food crops. There are concerns about environmental impacts and carbon balances, which sets limits in the increasing production of bio-fuels of first generation. The main disadvantage of first generation bio-fuels is the food-versus-fuel debate; one of the reasons for rising food prices is due to the increase in the production of these fuels [Laursen; 1993].Second-generation bio-fuels produced from plant biomass refers largely to lignocelluloses materials, as this makes up the majority of the cheap and abundant nonfood materials available from plants. But, at present, the production of such fuels is not cost effective because there are a number of technical barriers that need to be overcome before their potential can be realized [Eisberg; 1887]. Plant biomass represents one of the most abundant and underutilized biological resources on the planet, and is seen as a promising source of material for fuels and raw materials. At its most basic, plant biomass can simply be burned in order to produce heat and electricity. However, there is great potential in the use of plant biomass to produce liquid bio-fuels. However, bio-fuel production from agricultural by-products could only satisfy a proportion of the increasing demand for liquid fuels. This has generated great interest in making use of dedicated biomass crops as feedstock for bio-fuel production [Gomez et.al; 1978]. The examples of 2nd generation bio-fuels are cellulosic ethanol and FischerTropsch fuels. The production of 2nd generation bio-fuels is non-commercial at this time, although pilot and demonstration facilities are being developed. Therefore it is anticipated that, these 2nd generation bio-fuels could significantly reduce CO2 production, do not compete with food crops and some types can offer better engine performance. When commercialized, the cost of second generation bio-fuels has the potential to be more comparable with standard petrol, diesel, and would be most cost effective route to renewable, low carbon energy for road transport [ www.shell.com.].Therefore due to many advantages and disadvantages of the 1st generation bio-fuels and obvious advantages of 2nd generation bio-fuels as shown in Table. 1, the approaches to integral utilization of biomass for sustainable development are more reasonable, where all parts of the plant such as leaves, bark, fruits, and seeds can be utilized to useful products.

1st Generation Fuel2nd Generation Bio-fuel

Technology- Economical Feedstock- Vegetable oils, Corn sugar, etc.Feedstock- Non-food, Cheap and Abundant Plant Waste Biomass (Agricultural and Forest Residue, Grass, Aquatic Biomass and Water Hyacinth, etc.

Products- FAME or Bio-diesel, Corn ethanol, sugar alcoholProducts- Hydro treating oil, Bio-oil, FT oil, Lignocelluloses Ethanol, Butanol, Mixed Alcohols

Benefits- Environmental friendly, Economic and Social SecurityAdvantages-1. Not competing with food2. Advance technology still under development to reduce the cost of conversion3. Environmentally friendly

Problems-1. Limited feedstock (Food Vs Fuel)2. Blended partly with conventional fuel

Table1.1- Comparison of First and Second Generation Bio-fuel

1.2 Motivation- The amount of petrochemical like diesel and petrol is limited and the whole world is under the shade of energy crisis and looking for the alternative fuel source. Many countries have used bio-fuel as alternative to the petrochemicals like diesel and petrol. Ethanol is a kind of bio-fuel which can be blended with the gasoline in fixed proportion and can be used as alternative to fuel like diesel and petrol. Maximum of 20 % ethanol can be blended with the gasoline and can be used as alternative fuel source with same carbonator engine. Bioethanol is renewable source of energy which is generally produced by the fermentation of carbohydrate. Biomass such as cellulose, animal fat, etc is used for the production of ethanol through fermentation. Main source for such kind of biomass is sugarcane, corn, wheat bran, cassava, sweet potato etc. These are used for the ethanol production but they are mainly used for the food source and if these sources will be used for the ethanol production the whole world is going to face food crisis as the world population is increasing rapidly. To prevent the world from fuel crisis and food crisis, research has been focused on the production of bio-fuel from the waste biomass and the plant source which is not for the food purpose like waste generated in sugar mill, chemical pulp generated in paper industry and weed plant.

1.3 Objectives of Study-1. Biomass characterization for bio-ethanol production2. Hydrolysis of characterized biomass using microbes3. Fermentation for ethanol production of characterized biomass

1.4 Outline of Thesis-The outlines of the thesis work are structured as mentioned below:

Chapter 1:This chapter relates to the Introduction/ foundation of the thesis work, motivation and objectives of the study.

Chapter 2:This chapter relates to the details about the project work carried out and contains the literature survey pertaining to the project work.

Chapter 3:This chapter relates to the detail about materials and methods used for biomass characterization, hydrolysis, fermentation and calculations.

Chapter 4:This chapter relates to the details of results obtained and discussion of the results in light of previous findings.

CHAPTER 2

2. REVIEW OF LITERATURE2.1 Lignocelluloses biomass-First generation bio-fuels produced primarily from food crops such as grains, sugar beet and oil seeds are under scanner, due to the possibility of creating undue competition for land and water used for food and fiber production. The cumulative impacts of these concerns have increased the interest in developing bio-fuels produced from non-food biomass. Second-generation bio-fuels production from lignocelluloses biomass which includes agricultural residues (Corn Stover, crop straws and bagasse), herbaceous crops (alfalfa, switch grass), short rotation woody crops, forestry residues, waste paper and other wastes (municipal and industrial)cereal straw, bagasse, forest residues, purpose-grown energy crops such as vegetative grasses and short rotation forests, could avoid many of the concerns facing first-generation bio-fuels and potentially offer greater cost reduction potential in the longer term and do not interfere with food security [Salman ; 2011]. Moreover, bio-ethanol is very important in terms of energy security, environmental concern, employment opportunities, agricultural development, foreign exchange saving, socioeconomic issues etc. Lignocelluloses biomass consists of three main structural units: cellulose, hemicelluloses and lignin. Cellulose is crystalline glucose polymer and hemicellulose is amorphous polymers of xylose, arabinose, and lignin a large poly aromatic compounds.The term characterization describes the measurement of the chemistry, structure, and interactions of the biomass components [Pauly and Keegstra; 2008]. The characterization of different biomass viz. Pine needle, Camelina, Jatropha stem, Jatropha petiole, Jatropha leaves, Jatropha seed coat, Jatropha fruit husk, Jatropha seed cake, Wheat straw, Paddy, Sugarcane and Sugarcane trash has been conducted by different researchers aiming to explore its potential for use as feedstock for bio-ethanol production.Some details of different biomass:-Pinesareconifertreesin thegenusPinusin thefamily Pinaceae. They are the only genus in thesub-familyPinoideae [Barnes and Wagner; 2004].Needles, the adult leaves, which are green (photosynthetic), bundled in clusters (fascicles) of 16, commonly 25, needles together, each fascicle produced from a smallbudon a dwarf shoot in the axil of a scale leaf. Camelina sativais aflowering plantin the familyBrassicaceaeand is usually known in English asCamelina,gold-of-pleasure, orfalseflax, also occasionally wild flax, linseed dodder, German sesame, and Siberian oilseed. It is native to Europe and to Central Asian areas. This plant is cultivated asoil seedcrop [Jones et.al; 2005]. The crop is now being researched due to its exceptionally high levels (up to 45%) ofomega-3 fatty acids, which is uncommon in vegetable sources. Seeds contain 38 to 43% oil and 27 to 32% protein. Wheat(Triticumspp.)is acereal grain, originally from theLevantregion of theNear East. This grain is grown on more land area than any other commercial food. Wheat is widely cultivated as acash cropbecause it produces a good yield per unit area, grows well in a temperate climateeven with a moderately shortgrowing season [Francis et.al; 2009]. Sugarcaneis species of tallperennialtrue grassesof the genusSaccharum, tribeAndropogoneae. Sugarcane belongs to the grass family (Poaceae), an economically important seed plant family that includes maize, wheat, rice, andsorghum and manyforagecrops. The main product of sugarcane issucrose, which accumulates in the stalk internodes. Sucrose, extracted and purified in specialized mill factories, is used as raw material in human food industries or is fermented to produceethanol [Galloway; 1914]. Ethanol is produced on a large scale by the Brazilian sugarcane industry. Jatropha curcasis a species of spurgefamily,Euphorbiaceae. J. curcasis apoisonous, semi-evergreenshrubor smalltree. It is resistant to a high degree ofaridity, allowing it to be grown indeserts. Theseedscontain 27-40%oil (average: 34.4%) [Achten et.al; 2008] that can be processed to produce a high-qualitybio-dieselfuel, usable in a standard diesel engine. The seeds are also a source of the highly poisonous toxalbumincurcin or jatrophin.

2.2 PLANT CELL WALL COMPOSITION-2.2.1 Cell Wall- The cell wall is a tough, flexible but sometimes fairly rigid layer that surrounds some types of cells [Alexopoulos et.al; 1996]. It is located outside the cell membrane and provides these cells with structural support and protection, in addition to acting as a filtering mechanism. A major function of the cell wall is to act as a pressure vessel, preventing over-expansion when water enters the cell [Howland and John; 2000]. Cell walls are found in plants, bacteria, fungi, algae, and some Achaea. Animals and protozoa do not have cell walls [Koivikko et.al; 2005]. The material in the cell wall varies between species, and can also differ depending on cell type and developmental stage.2.2.2 Cell Wall Components- The main ingredient in cell wall is polysaccharides (or complex carbohydrates or complex sugars) which are built from monosaccharide (or simple sugars).Eleven different monosaccharide are common in these polysaccharides including glucose and galactose. Carbohydrates are good building blocks because they can produce a nearly infinite variety of structures [Raven; 1983]. There are a variety of other components in the wall including protein, and lignin.Some of the wall components include as given below and the structure of cell wall is shown in Fig. 1.1.1. Cellulose Cellulose is made up of as many as 25,000 individual glucose molecules. Cellobiose (glucose-glucose disaccharide) is the basic building block. Cellulose readily forms hydrogen bonds with itself (intra-molecular H-bonds) and with other cellulose chains (inter-molecular H-bonds). A cellulose chain will form hydrogen bonds with about 36 other chains to yield micro-fibrils [Hudler and George; 1998]. Micro-fibrils are 5-12 nm wide and give the wall strength - they have a tensile strength. Some regions of the micro-fibrils are highly crystalline while others are more "amorphous".2. Cross linking glycans (=Hemicelluloses) Diverse group of carbohydrates is called hemicelluloses. It is characterized when being soluble in strong alkali. They are linear (straight), flat, with a -1, 4 backbone and relatively short side chains. Two common types include xyloglucans and glucuronarabinoxylans. Other less common ones include glucomannans, galactoglucomannans, and galactomannans. The main feature of this group is that they dont aggregate with themselves- in other words, they dont form micro-fibrils. However, they form hydrogen bonds with cellulose and hence the reason they are called "cross-linking glycans" [Van Heijenoort; 2001]. There may be a fructose sugar at the end of the side chains which may help keep the molecules planar by interacting with other regions of the chain. 3.Lignin- Polymer of phenolics, especially phenyl propanoids. Lignin is primarily a strengthening agent in the wall. It also resists fungal/pathogen attack [Joseleau et.al; 2007]. Fig. 1.1- Structure of Plant Cell Wall 2.3 PRE-TREATMENT OF LIGNOCELLULOSES BIOMASS-Pretreatment is an important tool for practical cellulose conversion processes. It is necessary in order to alter the structure of cellulosic biomass, to make cellulose more accessible for conversion of carbohydrate polymers into fermentable sugars. The goal of pretreatment is to break the lignin seal, solubilize hemicelluloses, and disrupt the crystalline structure of cellulose [Taherzadeh et.al; 2004]. Various pretreatment options are available now to fractionate, solubilize, hydrolyze, and separate cellulose, hemicelluloses, and lignin components. The biomass is treated to reduce its size and open its structure. Pretreatment usually hydrolyzes hemicelluloses to the sugars (xylose, L-arabinose, and others) that are water soluble [Saha and B.C.; 2004].Pretreatment must meet the following requirements [Sun; 2002]:1. Improve the formation of sugars or the ability to subsequently form sugars by hydrolysis,2. avoid the degradation or loss of carbohydrate,3. Avoid the formation of byproducts that are inhibitory to the subsequent hydrolysis and fermentation processes, and4. Be cost-effective.Pre-treatment are of following types:-1. Physical Pre-treatment2. Chemical Pre-treatment3. Biological Pre-treatment

Physical Pre-treatment-Physical pretreatments do not use any chemicals. Size reduction by mechanical methods such as grinding or milling is one of them, through which the surface area of biomass is increased, and the degree of polymerization (DP) and crystallinity of cellulose is decreased to some extent, but the power requirement for reducing the feedstock from millimeter size to fine particles of micrometers is extremely high, which is unacceptable from the engineering point of view. Radiation such as microwaves that can penetrate and heat the feedstock instantly has also been studied [Binod et.al; 2011]. However, it is problematic to process the feedstock in large quantities, not to mention the power requirement to generate the radiation. Therefore, more attention regarding physical pretreatment has been focused on the hydrothermal processes of steam explosion (SE) and liquid hot water (LHW) treatment. SE involves heating the feedstock at elevated temperature and pressure for a short duration, followed by depressurizing the system to disrupt the structure of LCCs. Due to lower capital investment, less impact on the environment, and simple process design and operation, the SE process has been tested at pilot scales worldwide. The mechanism underlying the pretreatment is assumed to be the partial degradation of LCCs catalyzed by acetic acid released from acetylated hemicelluloses and other organic acids such as formic and levulinic acids, making the process auto hydrolytic in nature [Ramos; 2003].

Chemical Pre-treatment-High temperatures applied during the hydrothermal pretreatments under SE and LHW conditions dehydrate sugars and produce inhibitors such as furfural from xylose and hydroxyl-methyfurfural from glucose. To address this problem, acids can be supplemented to facilitate the deconstruction of LCCs under less severe conditions, either lower temperature or shorter reaction time. Among various acids, sulfuric acid is most commonly used. Although the temperatures in concentrated acid pretreatment are much lower, acid recovery presents a big challenge for the economic viability of the process. Therefore, dilute acid with concentrations less than 2% is preferred, which can be conveniently neutralized by lime or ammonium during the conditioning process [Jennings and Schell; 2011]. Dilute acid pretreatments have been intensively studied over the years with various feedstock and reactors at different scales [Lloyd and Wyman; 2005].

Biological Pre-treatment-Compared with physical and chemical pretreatments in which expensive equipment, chemicals and intensive energy consumption are needed, biological pretreatment by solid fermentation employs microorganisms that degrade lignocelluloses biomass at mild conditions without special requirements for equipment [Keller et.al; 2003]. Both bacteria and fungi have been explored, but rot fungi associated with wood decay are the predominant species in lignocelluloses degradation for the purpose of bio-fuel production, particularly white-rot fungi due to their abundant ligninolytic enzymes, including lignin peroxidase, manganese peroxidase, laccases and other enzymes, and better selectivity in lignin degradation [Dashtban et.al; 2010)]. Although biological pretreatment is energy-saving and environmentally friendly, its disadvantages are apparent. Firstly, the extremely low degradation rate requires times as long as weeks for a significant change in the structure of the lignocelluloses biomass, making the process mismatched with the subsequent hydrolysis of cellulose and fermentation of sugars. Secondly, significant biomass is lost during the process, not only the lignin which is mineralized into low molecular- weight fragments that might be further catabolized into the useless final product CO2 [Steffen et.al; 2000], but also sugars released from hemicelluloses and even cellulose by the hydrolytic enzymes (simultaneous decay with lignin degradation) as a carbon source to support the growth of the microorganisms [Hammel; 1997]. Finally, the control of microbial growth and metabolism under open and solid fermentation conditions with mixture species is unreliable, which inevitably affects the subsequent processes such as cellulose hydrolysis and ethanol fermentation.

2.4 MICROBIAL HYDROLYSIS-2.4.1 Hydrolysis-Hydrolysis(From Greekhydro-meaning "water", andlyses, meaning "separation") usually means the cleavage of chemical bonds by the addition ofwater. Where acarbohydrateis broken into its component sugar molecules by hydrolysis (e.g.sucrosebeing broken down into glucoseandfructose), this is termedsaccharification. Generally, hydrolysis or saccharification is a step in the degradation of a substance.Hydrolysis is a chemical reaction in which water is being used to break the bonds of certain substances. In biotechnology and living organisms, these substances are often polymers. In a hydrolysis reaction involving an ester link, such as that found between two amino acids in a protein, the products that result include one that receives the hydroxyl (OH) group from the water molecule, and another that becomes a carboxylic acid with the addition of the remaining proton (H+).Hydrolysis reactions in living organisms are performed with the help of catalysis by a class of enzymes known as hydrolases. The biochemical reactions that break down polymers such as proteins (peptide bonds between amino acids), nucleotides, complex sugars and starch, and fats are catalyzed by this class of enzymes. Within this class, lipases, amylases and proteinases hydrolyze fats, sugars and proteins, respectively.Cellulose-degrading bacteria and fungi play a special role in paper production and other everyday biotechnology applications, because they have enzymes (cellulases and esterases) that can break cellulose into polysaccharides (polymers of sugar molecules) or glucose.2.4.2 Enzymatic Hydrolysis-The enzymatic hydrolysis, aims at the hydrolysis of the hemicellulose and the delignification. In the case of the hydrolysis of the hemicelluloses, in spite of the specificity of xylanases, where the action is carried out through the synergy of the - xylosidase, endo 1,4--xylanases, acetylxylanaesterase, -glucoronidase and Larabinofuranosidase enzymes, hurdles exist in the form of high cost of enzyme and adequate scale up to industrial level. There are two major types of lignolytic enzymes widely used: phenol oxidase (laccase) and peroxidases (lignin peroxidase, LiP and manganese peroxidase, MnP) [Krause et.al; 2003]. The other enzymes also used, but whose mode of action are not clearly known, are glyoxal oxidase, glucose oxidase, oxido-reductase and methanol oxidase [Eriksson; 2000]. The main area of application was the paper and pulp industry, where such enzymes are used to convert to chlorine and chorine derivative substances. This creates another problem due to the irreversible tendency of total chlorine free bleaching (TCF systems) and elemental chlorine free (ECF systems) [Viikari et.al; 1994].2.4.2.1 Cellulases Enzyme System-Natural cellulosic substrates (primarily plant cell materials) are composed of heterogeneous intertwined polysaccharide chains with varying degrees of crystallinity, hemicelluloses and pectin, embedded in lignin. Microorganisms produce multiple enzymes to degrade plant cell materials, known as enzyme systems [Acebal et.al; 1986]. For microorganisms to hydrolyze and metabolize insoluble cellulose, extracellular cellulases must be produced that are either free or cell associated. The biochemical analysis of cellulases systems from aerobic and anaerobic bacteria and fungi has been comprehensively reviewed during the past two decades. Components of cellulases systems were first classified based on their mode of catalytic action and have more recently been classified based on structural properties. Three major types of enzymatic activities are found [Aho et.al; 1990]:

1. Endoglucanases or 1,4-D-glucan-4-glucanohydrolases,2. Exoglucanases, including 1,4-D-glucan glucanohydrolases (also known as cellodextrinases) and 1,4-Dglucan cellobiohydrolases (cellobiohydrolases), and 3. Glucosidases or glucoside glucohydrolases

1. Endoglucanases or 1,4-D-glucan-4-glucanohydrolases- Endoglucanases cut at random at internal amorphous sites in the cellulose polysaccharide chain, generating oligosaccharides of various lengths and consequently new chain ends [Barras et.al; 1994].

2. Exoglucanases, or cellodextrinases or cellobiohydrolases-Exoglucanases act in a possessive manner on the reducing or non-reducing ends of cellulose polysaccharide chains, liberating either glucose (glucanohydrolases) or cellobiose (cellobiohydrolases) as major products [Boussaid et.al; 1999]. Exoglucanases can also act on micro-crystalline cellulose, presumably peeling cellulose chains from the micro-crystalline structure.

3. Glucosidases or glucoside glucohydrolases-Glucosidases hydrolyze soluble cellodextrinases and cellobiose to glucose [Adney et.al; 1994].

Cellulases are distinguished from other glycoside hydrolases by their ability to hydrolyze 1, 4-glucosidic bonds between glucosyl residues. The enzymatic breakage of the 1,4-glucosidic bonds in cellulose proceeds through an acid hydrolysis mechanism, using a proton donor and nucleophile or base. The hydrolysis products can either result in the inversion or retention (double replacement mechanism) of the anomeric configuration of carbon-1 at the reducing end. The insoluble, recalcitrant nature of cellulose represents a challenge for cellulases systems. A general feature of most cellulases is a modular structure often including both catalytic and carbohydrate-binding modules (CBMs) [Bagnara et.al; 1985]. The CBM effects binding to the cellulose surface, presumably to facilitate cellulose hydrolysis by bringing the catalytic domain in close proximity to the substrate, insoluble cellulose. The presence of CBMs is particularly important for the initiation and processivity of exoglucanases. Revisiting the original model of cellulose degradation proposed by Reese.et.al. a possible additional non-catalytic role for CBMs in cellulose hydrolysis was proposed: the sloughing off of cellulose fragments from cellulosic surfaces of, e.g., cotton fibers, thereby enhancing cellulose hydrolysis. Cellulases systems exhibit higher collective activity than the sum of the activities of individual enzymes, a phenomenon known as synergism. Four forms of synergism have been reported [Lemaire et.al; 1996]:

I. Endo-exo synergy between Endoglucanases and exoglucanases, II. Exo-exo synergy between exoglucanases processing from the reducing and non-reducing ends of cellulose chains, III. Synergy between exoglucanases and glucosidases that remove cellobiose (and cellodextrinases) as end products of the first two enzymes, and Intramolecular synergy between catalytic domains and CBMs.2.4.3 Microbial Hydrolysis-Lignin, due to its recalcitrant structure, decreases the catalytic power and may cause inactivation of the cellulases. Palonen; 2004 showed that the localization and the structure of lignin affect more the enzymatic hydrolysis than the absolute lignin quantity in the lignocellulosics complex. The study revealed, furthermore, that modifications of the lignin surface by oxidants treatments with lactase led to a rise of the cellulose hydrolysis. There are various microorganisms present in nature which are able to attack and degrade lignin, thus exposing the cellulose to easy action by cellulases. These microorganisms are found in forest leaf litter/composts and include the wood rotting fungi, actinomycetes and bacteria. They have specialized enzyme systems to attack, depolymerize and degrade the lignocellulosics matrix to release the lignin and other polymers. Phanerochaete chrysosporium has been the main microorganism studied for lignin degradation by white rot fungi [Kirk and Farell; 1987]. Saritha et.al; 2012 have studied the biological treatments with white rot fungi and Streptomyces sp. For delignifying pulp, increasing digestibility of lignocellulosics as animal feed and for bioremediation of paper mill effluents. Such lignocellulolytic microbes are extremely useful supplements in production of bio-ethanol as they help in removal of lignin from lignocellulosics substrate and also aid in cellulase production. They further studied the treatment of hardwood and softwood residues with Streptomyces griseus isolated from leaf litter and showed that it increased the mild alkaline dissolution of lignins and produced high levels of the cellulase complex when grown on wood substrates. The observed loss of lignin was 10.5% and 23.5% in case of soft wood and hard wood, respectively. The fungal breakdown of lignin is anaerobic and uses a family of extracellular enzymes termed as lignases [Howard et.al; 2005]. Some bacterial laccases have also been isolated from Azospirillum lipoferum, Bacillus subtilis etc. [Kunamneni et.al; 2007]. Recent researches have elucidated that microorganisms like Lentinus edodes (Songulashvilli et al, 2005), Pleurotes spp. [Ragunathan and Swaminathan; 2004], Penicillium camemberti cultured at 2535C for 322 days resulted in 4575% and 6580% holocellulose and lignin degradation, respectively. Thus, biological pretreatment process for lignocellulosic substrate using lignolytic organisms such as actinomycetes and white rot fungi can be developed for facilitating efficient enzymatic digestibility of cellulose. Microbial hydrolysis not only offers advantages such as low capital cost and low energy, but also added advantages of little dependence of chemicals, and mild environmental conditions. However, the main flaw in the process is the low hydrolysis rate obtained in most biological processes. Thus, an economic biological hydrolysis process of lignocelluloses having improved hydrolysis leading eventually to improved fuel yields has to be found out by researching more micro-organisms for their ability to delignify the plant material quickly and efficiently [Saritha et.al; 2012].2.4.3.1 Aspergillus niger-Aspergillus nigeris a haploid filamentous fungus and is a very essential microorganism in the field of biology. In addition to producing extracellular enzymes and citric acid,A. nigeris used for waste management and bio-transformations [Adams et.al; 1997]. The fungi are most commonly found in mesophilic environments such as decaying vegetation or soil and plants.Aspergillus nigerwas isolated from the plantWelwitschia mirabilisin Namibia and Angola, a plant estimated to be about 3000 years old [Takahashi et.al; 1991].A. nigeris easily isolated from common thing such as dust, paint, and soil. Aspergillus nigeris usually found in common mesophilic environments such as soil, plants, and enclosed air environments.A. nigeris not only a xerophilic fungi (mold that doesnt require free water for growth, can grow in humid environments), but is also a thermotolerant organism (capable of growing at high temperatures). Because of this property, the filamentous fungi exhibits a high tolerance to freezing temperatures.The production of ochratoxin A fromA. niger, is liable to cause immunotoxcitiy in animals [Santhiya et.al; 2005]. The effects on animals include a decrease in antibody responses, a size reduction in immune organs, and an alteration in the production of cytokine which are proteins and peptides specifically used in signaling. Food that has been contaminated byA. niger has toxic metabolite which had a major affect on the poultry industry. The culture plate is being shown in Fig. 1.2.

Fig. 1.2- Aspergillus niger on NA Plate2.4.3.2 Trichoderma reesei- Trichoderma reeseiis amesophilicandfilamentousfungus. It is ananamorphof the fungusHypocrea jecorina.T.reesei has the capacity to secrete large amounts ofcellulolyticenzymes(cellulasesandhemicellulases).Microbialcellulaseshave industrial application in the conversion ofcellulose, a major component of plantbiomass, intoglucose. Recent advances in thebiochemistryof cellulasesenzymology, the mechanism of cellulosehydrolysis(cellulolysis),strainimprovement, molecular cloning and process engineering are bringingT. reeseicellulases closer to being a commercially viable route to cellulose hydrolysis. The genome of this organism was released in 2008. . The culture plate is being shown in Fig. 1.3.

Fig. 1.3- Trichoderma reesei on NA Plate

2.5 FERMENTATION FOR ETHANOL PRODUCTION-2.5.1 Ethanol fermentation-The chemical equation below shows the alcoholic fermentation of glucose, whose chemical formula is C6H12O6.One glucose molecule is converted into two ethanol molecules and two carbon dioxide molecules:C6H12O6 2 C2H5OH + 2 CO2C2H5OH is the chemical formula for ethanol.Before fermentation takes place, one glucose molecule is broken down into two pyruvate molecules. This is known as glycolysis.

2.5.2 Saccharomyces cerevisiae-Saccharomyces cerevisiae is a eukaryotic microbe. More specifically, it is globular-shaped, yellow-green yeast belonging to the Fungi kingdom, which includes multicellular organisms such as mushrooms and molds. Natural strains of the yeast have been found on the surfaces of plants, the gastrointestinal tracts and body surfaces of insects and warm-blooded animals, soils from all regions of the world and even in aquatic environments. Most often it is found in areas where fermentation can occur, such as the on the surface of fruit, storage cellars and on the equipment used during the fermentation process.Saccharomyces means "sugar fungus", which provides a clue to the function of this yeast. Saccharomyces have the ability to ferment sugars, meaning they can convert sugar into carbon dioxide and alcohol [Stewart and Graham; 1987]. S. cerevisiae is famously known for its role in food production. It is the critical component in the fermentation process that converts sugar into alcohol; an ingredient shared in beer, wine and distilled beverages [Freeman and Scott; 2005]. It is also used in the baking process as a leavening agent; yeast releasing gas into their environment results in the spongy-like texture of breads and cakes. Archaeologists have found evidence of a fermented beverage in a pot in China as early as 7000BC, and molecular evidence of yeast being used in fermentation was found in a wine jar dating back to 3150BC. Isolation of the species did not occur until 1938, when Emil Mrak isolated it from rotten figs found in Merced, California. S. cerevisiae is also considered to be a "model organism" by scientists [Osley et.al; 2007]. Its big advantage is that it is both a unicellular and eukaryotic organism. Another advantage is its fast growth rate. The culture plate is being shown in Fig. 1.4.

Fig. 1.4- Saccharomyces cerevisiae on NA Plate2.5.2.1 Fermentation of Hexoses by Saccharomyces cerevisiae-Wild-type S. cerevisiae ferments glucose, the dominant sugar in all plant hydrolysates, at high rates even under anaerobic conditions. S. cerevisiae contains an elaborate system for hexose transport [Baldoma and Aguilar; 1988]. S. cerevisiae all transport glucose via facilitated diffusion; glucose uptake only requires a concentration gradient across the plasma membrane. After uptake, glucose dissimilation proceeds via the Embden-Meyerhof glycolytic pathway [Barnett et.al; 1990]. This pathway oxidizes glucose to two pyruvate, resulting in the net formation of two ATP per glucose. In anaerobic, fermentative cultures of S. cerevisiae, the NADH formed by glyceraldehyde-3-phosphate dehydrogenase is re-oxidized via alcoholic fermentation. This essential redox balancing involves the combined activity of pyruvate decarboxylase and alcohol dehydrogenase. But obviously glucose is not the only carbohydrate present in the hydrolysates. In order to ferment such non-glucose carbohydrates with S. cerevisiae, three key criteria have to be met: 1. Presence of a functional transporter in the plasma membrane, 2. Presence of enzyme(s) that couple metabolism of the carbohydrate to the main glycolytic pathway and 3. Maintenance of a closed redox balance. Mannose and fructose are two isomers of glucose that occur in all plant-derived biomass hydrolysates and that can be fermented by all wild-type S. cerevisiae strains. The general observation that yeast capable of fermenting glucose can also ferment fructose and mannose is known as the Kluyver rule. After phosphorylation by hexokinase, mannose-6-phosphate is isomerizes to fructose-6-phosphate by phosphomannose isomerase, encoded by the PMI40 gene. Hexokinase is also responsible for phosphorylation of fructose to fructose-6-phosphate, which is subsequently metabolized through glycolysis. As mannose and glucose compete for the same hexose transporters, kinetics of mixed-substrate utilization is determined by their relative and absolute concentrations in hydrolysates. The galactose permease Gal2p subsequently converted into glucose-6-phosphate via the Leloir pathway [Blank et.al; 2005]. This pathway, which links galactose catabolism to the main glycolytic pathway, consists of three reactions. After phosphorylation of galactose by galactokinase (Gal1p), galactose-1-phosphate uridylyltransferase (Gal7p) converts UDP-glucose and galactose-1-phosphate to UDP-galactose and glucose-1-phosphate. UDP galactose is reconverted into UDP-glucose by the uridine-diphosphoglucose 4-epimerase (Gal10p). Finally, glucose-1-phosphate is converted to glucose-6-phosphate by phosphoglucomutase, major and minor isoforms of which are encoded by PGM2 (also called GAL5) and PGM1, respectively. In wild-type S. cerevisiae strains, growth rates on galactose are generally lower than those on glucose.2.5.3 Zymomonas mobilis-Zymomonas mobilisis a bacterium belonging to the genusZymomonas. It is notable for itsbioethanol-producing capabilities, which surpassyeastin some aspects. It was originally isolated from alcoholic beverages. Z. mobilisdegrades sugars topyruvateusing theEntner-Doudoroff pathway. The pyruvate is thenfermentedto produceethanolandcarbon dioxideas the only products (analogous to yeast).The advantages ofZ. mobilisover S. cerevisiaewith respect to producingbio-ethanol: Higher sugar uptake and ethanol yield (up to 2.5 times higher), Lower biomass production, Higher ethanol tolerance up to 16% (v/v), Does not require controlled addition of oxygen during the fermentation, Amenability to genetic manipulations.However, in spite of these attractive advantages, several factors prevent the commercial usage ofZ. mobilisin cellulosic ethanolproduction. The foremost hurdle is that its substrate range is limited toglucose,fructoseand sucrose [Zhang et.al; 1995].

CHAPTER 3

3. MATERIALS AND METHODOLOGY-3.1 Materials-1. Biomass- Different biomass has been used for the characterization. Listing below different biomass used for characterization:-1. Pine Needle2. Paddy 3. Sugarcane4. Jatropha Stem5. Jatropha Leaves6. Jatropha Petiole7. Camelina8. Sugarcane trash9. Wheat straw10. Jatropha seed cake11. Jatropha fruit husk12. Jatropha seed coat

2. Microbes- Hydrolysis was carried on different biomass with the incubation of different microbes. Each biomass is treated with separate microbe for hydrolysis. Microbes used were Aspergillus niger and Trichoderma reesei. The incubation was done on Jatropha Seed Coat and Jatropha Fruit Husk. The fermenting microbe used was Saccharomyces cerevisiae.

3. Chemicals- Chemicals of Hi-Media and Sigma-Aldrich were used for making different solutions.

4. Glassware- Glassware of Borosil was used for conducting experiment.

3.2 Methodology-1. Characterization of Biomass- The biomass is characterized into holo-cellulose, cellulose, hemi-cellulose and lignin.

a. Holo Cellulose Determination-To 2 g of the extractive free sample, 180 ml distilled water; 8.6 g sodium chloride, 6.0ml ethanoic acid and 6.6 g sodium chloride were added. The mixture was then digested in a 250 ml conical flask under reflux at 70C for 3 hours. It was then allowed to cool, filtered and the residue washed with five 20 ml portions of 100 ml distilled water, the residue was then dried at 105C for 24 hours to attain constant weight [Dubois et.al; 1956].

2 g of the extractive free sample

Add 180 ml distilled water 8.6 g sodium chloride 6.0ml ethanoic acid 6.6 g sodium chloride

Mixture digested in a 250 ml conical flask under reflux at 70C for 3 hours

Cool, filtered and the residue washed with five 20 ml portions of 100 ml distilled water

Residue dried at 105C for 24 hours to attain constant weight

b. Cellulose Determination-To 2 g of the extractive free sample was taken and put into a 250 ml beaker, 100 ml of 17.5% NaOH solution was added and stirred at 25C for 30 minutes. The content of the beaker was then filtered, washed with 25 ml of 9.5% NaOH solution and 20 ml portions of 100 ml distilled water. The residue was again washed with distilled water and 40 ml of 10% acetic acid and further with 1 L distilled water. The residue was then dried at 105C for 24 hours to constant weight [Association of Official Analytical Chemists; 1990].

2 g of the extractive free sample

Add 100 ml of 17.5% NaOH solution and stirred at 25C for 30 minutes

Content filtered & washed 25 ml of 9.5% NaOH solution 20 ml portions of 100 ml distilled water

Residue washed Distilled water 40 ml of 10% acetic acid1 L distilled water

Residue dried at 105C for 24 hours to constant weight

c. Hemi-cellulose Determination-On the basis of solubility in 17.5% NaOH solution, holo-cellulose is sub divided into:- 1. Insoluble cellulose 2. Soluble hemi-cellulose

Hemi-cellulose -Difference between the weight of holo-cellulose and weight of cellulose present [Raghuramulu et.al; 1983].

d. Lignin Determination-To 1 g of the extractives free sawdust sample, 14 ml of cold 72% sulphuric acid was added and stirred. The mixture was left to stand for 2 hours. After the 2 hours, the mixture was then washed in a 1 L conical flask and diluted to 3% sulfuric acid. The mixture was then boiled for 4 hours under reflux. The insoluble material was al- lowed to settle and filtered. The residue was washed and dried in an oven at 105C after 2 hours this then cooled and weighed as the lignin content [Templeton and Ehrman; 1995].

1 g of the extractives free sawdust sample

Add 14 ml of cold 72% sulphuric acid and stirred

Left mixture to stand for 2 hours

After the 2 hours Mixture was then washed in a 1 L conical flask Diluted to 3% sulfuric acidBoil mixture for 4 hours under reflux

Insoluble material settled and filtered

Residue was washed and dried in an oven at 105C

After 2 hours cooled and weighed as the lignin content2. Hydrolysis- To 1 gm powdered biomass 100 ml Mandels Media was added. 1ml sample was collected in two appendrof each to estimate total carbohydrate and reducing sugar. Three replicates of each sample were made and each microbe was incubated in each sample.1 ml sample was taken in each day incubation. 1 gm sample + 100 ml Mandels MediaMandels Media-Urea-0.3 gm(NH4)2SO4-1.4 gmKH2PO4-2.0 gmCaCl2.2H2O-0.4 gmMgSO4.7H2O-0.15 gmBactological Peptone-1.0 gmYeast Extract-0.25 gmFeSO4.7H2O-0.015 gmMnSO4.H2O-0.16 gmZnSO4.7H2O-0.14 gmCo.Cl2-0.2 gmDistill Water-1000 mlpH- 5.5 to 6.0a. Total Carbohydrate Estimation- To 1 ml sample 4 ml Anthrone Reagent was added. Mix and boil in water bath 10 mins. Cool for 10 mins. Keep at room temperature for 20 mins. Optical density was calculated at 620 nm.1 ml sample + 4 ml Anthrone Reagent

Mix and Boil for 10 mins

Cool for 10 mins

Kept at Room Temperature for 20 mins

OD at 620 nm[Anthrone Reagent Preparation- 0.2 gm Anthrone in 100 ml 92% conc. H2SO4][92% conc. H2SO4 Preparation- 93.6 ml conc.H2SO4 + 6.4 ml Distill water]b. Reducing Sugar Estimation-To 2 ml sample 1 ml distill water was added along with 3 ml DNSA (Di-nitro salicylic acid). Place in water bath for 15 mins. Add 1ml Rochelle salt. OD was recorded at 510 nm.2 ml sample + 1 ml distill water

Add 3 ml DNSA

Put in water bath for 15 mins

Add 1 ml Rochelle salt OD at 510 nm[DNSA Preparation - 1 gm DNSA dissolved in 100 ml of 1% NaOH + Add 0.05 gm Sodium Sulphite + 0.2 gm Crystalline Phenol][Rochelle Salt Preparation- 40 gm Sodium Potassium Tartarte + 100 ml Distill water

3. Fermentation-Ethanol Estimation-The replicates were incubated with Saccharomyces cerevisiae. The observations were made for next 6 days.Potassium Dichromate Assay-To 1ml sample 3ml chromic acid was added. Place in water bath at 90C for 10 mins. Add 1ml Rochelle salt. Optical density of sample in each day observation was calculated at 600 nm.1 ml sample + 3 ml Chromic Acid

Put in water bath at 90C for 10 mins

Add 1 ml Rochelle salt

OD at 600 nm[Chromic Acid Preparation- 1 gm Potassium Dichromate + 100 ml 5M H2SO4][5 M H2SO4- 27 ml H2SO4 in 100 ml distill water]

4. Standard Curve Preparation-The stock is prepared by making solution of 50 mg glucose in 50 ml distill water.Stock Solution- 50 mg/ 50 ml solution

a. Reducing Sugar Estimation- The reducing sugar estimation is done by DNSA method. The observations are made by determining the optical density. The control OD obtained is 0.004. The observation made for the estimation of reducing sugar for standard curve preparation is shown in Table 1.2. Fig. 1.5 shows graphical analysis of reducing sugar estimated for standard curve preparation. Control OD- 0.004 S.No.Sample(l)Distill Water (ml)DNSA (ml)Rochelle Salt (ml)OD at 510 nm

1.50950310.156

2.100900310.310

3.150850310.442

4.200800310.589

5.250750310.738

6.300700310.885

Table 1.2- Standard Curve for Reducing Sugar Estimation through DNSA Method

Fig. 1.5- Standard Curve for Reducing Sugar Estimation through DNSA MethodSlope- 0.002900571Factor- 344.7596533b. Total Carbohydrate Estimation-The total carbohydrate estimation is done by Anthrone method. The observations are made by determining the optical density. The control OD obtained is 0.003. The 20 l of the sample is being taken from original sample and further dilution is being done. The original sample is made by different concentration of glucose dissolved in distill water making the solution of 1 ml. The observation made for the estimation of reducing sugar for standard curve preparation is shown in Table 1.3. Fig. 1.6 shows graphical analysis of reducing sugar estimated for standard curve preparation. Control OD- 0.003S.No.Sample from Original sample (l)Distill Water (ml)OD at 620 nm

1.209800.132

2.209800.233

3.209800.342

4.209800.457

5.209800.575

6.209800.695

Table 1.3- Standard Curve for Total Carbohydrate Estimation through Anthrone Method

Fig. 1.6- Standard Curve for Total Carbohydrate Estimation through Anthrone MethodSlope- 0.002260571Factor- 442.3660263

c. Ethanol Estimation-S.No.Sample (l)Chromic Acid (ml)Rochelle Salt (ml)OD at 600 nm

1.50310.107

2.100310.218

3.150310.315

4.200310.405

5.250310.524

6.300310.625

The ethanol estimation is done by Potassium dichromate assay. The observations are made by determining the optical density. The control OD obtained is 0.001. The observation made for the estimation of reducing sugar for standard curve preparation is shown in Table 1.3. Fig. 1.6 shows graphical analysis of reducing sugar estimated for standard curve preparation. Control OD- 0.001.Table 1.4- Standard Curve for Ethanol Estimation through Potassium Dichromate Assay

Fig. 1.7- Standard Curve for Ethanol Estimation through Potassium Dichromate Assay

3.3 Analysis-1. Characterization of Biomass-The characterization of biomass is done. The sample collected in dry form; so the amount of sample is calculated in grams. The value in gram is converted in percentile to get the exact composition of different biomass. The conversion of gram into percentile is being done for the values of holo-cellulose, hemi-cellulose, cellulose and insoluble lignin. The conversion is done by using the formula as given below:-Conversion of composition of biomass in gram into percentile = Amount of the dry biomass* 100/ (Total biomass used * Total Volume)The value of soluble lignin is obtained in gram/litre. The soluble lignin composition is obtained by using formula:-Acid Soluble Lignin (g/l) = A205*Dilution Factor/ Absorpitivity Co-efficient * Cell Path Length{Where Absorpitivity co-efficient = 110 g/L-cm and Cell Path Length = 1 cm}The value in g/l in converted into percentile by the multiplication with 100 of the obtained composition.

2. Hydrolysis-The value of reducing sugar and total carbohydrate is determined in the process of hydrolysis. The concentration of unknown sample is calculated by using formula as below:-

Concentration of unknown sample = OD * Standard Factor{Where Standard Factor = 1/ Slope}

3. Fermentation-The value of ethanol is determined in the process of fermentation. The concentration of unknown sample is calculated by using formula as below:-Concentration of unknown sample = OD * Standard Factor{Where Standard Factor = 1/ Slope}

CHAPTER 4

4.RESULT AND DISCUSSION-1. Characterization of Biomass-The characterization of different biomass was done to find the different composition of plant cell wall viz. holo-cellulose, celulose, hemi-cellulose and lignin for its use in the production of bioethanol through microbial hydrolysis. Lignin content is important to know as this component is non degradable and also limits the access of enzymes for cellulose and hemicellulose degradation. The different biomass characterized was pine needle, paddy, sugarcane, jatropha stem, jatropha petiole, jatropha leaves, camelina, sugarcane trash, wheat straw, jatropha seed coat and jatropha seed cake. The contents of holo-cellulose was observed maximum in jatropha fruit husk, cellulose was maximum in camelina, hemi-cellulose was maximum in paddy and lignin observed to be greater in jatropha seed coat than other biomass characterized [Table 1.5].S.No.SampleHolo-cellulose (%)Cellulose (%)Hemi-cellulose (%)Lignin (%)

1.Pine Needle59.250.58.625.7

2.Paddy74.859.825.613.6

3.Sugarcane75.861.7148.9

4.Jatropha Stem50.5473.49.2

5.Jatropha Leaves60.758.12.619.8

6.Jatropha Petiole54.4495.311

7.Camelina77.570.26.414.9

8.Sugarcane Trash71.867.34.524.2

9.Wheat Straw7966.912.114.9

10.Jatropha Seed Coat74.467.37.1542.9

11.Jatropha Fruit Husk84.566.817.716.1

12.Jatropha Seed Cake70.356.413.919.2

Table 1.5- Characterization of Different BiomassAnalysis was done of composition of plant cell wall in different biomass. The biomass was grouped in three categories:-1. Grasses: Wheat straw, Paddy leaves, Sugarcane leaves and Sugarcane trash. The maximum holo-cellulose content was observed in wheat straw (71.8), cellulose in sugarcane trash (67.3), hemi-cellulose was maximum in paddy (25.6) and lignin was found to be higher in sugarcane trash (24.2) than other biomass characterized [Fig 1.8.1].

Cell Wall ComponentsWheat StrawPaddySugarcaneSugarcane Trash

Holo-cellulose7974.875.871.8

Cellulose66.959.861.767.3

Hemi-cellulose12.125.6144.5

Lignin14.913.68.924.2

Table1.6.1: Cell Wall Composition of Different Grasses

Fig 1.8.1- Characterization of Different Biomass (Grasses) for its Different Components of Cell Wall

2. Shrub-Tree: Jatropha. Different plants parts of Jatropha viz. Jatropha leaves, Jatropha petiole, Jatropha stem, Jatropha seed coat, Jatropha fruit husk and Jatropha seed cake were characterized for its cell wall components. Holo-cellulose, cellulose and lignin content was observed to be maximum in Jatropha seed coat (74.4, 67.3 and 7.15 respectively) than other biomass characterized [Fig 1.8.2]. The study shows that Jatropha seed coat has maximum lignin component in the plant cell wall composition than other Jatropha plant parts.

Cell wall componentsJatropha LeavesJatropha PetioleJatropha StemJatropha Seed CoatJatropha Fruit HuskJatropha Seed Cake

Holo-cellulose60.754.450.574.484.570.3

Cellulose58.1494767.366.856.4

Hemi-cellulose2.65.33.47.1517.713.9

Lignin19.8119.242.916.119.2

Table 1.6.2: Cell Wall Composition of Different Jatropha Plant Parts Fig 1.8.2- Characterization of Different Plant Parts of Jatropha

3. Tree-Shrub: Tree: Pine needle, Shrub: Camelina. The maximum content of holo-cellulose was observed in Jatropha fruit husk (84.5), cellulose in Camelina (70.2), hemi-cellulose in Jatropha fruit husk (17.7) and lignin in pine needle (25.7) than other biomass characterized [Fig 1.8.3]. The results show that Jatropha fruit husk cell wall has maximum of cellulose component.

Cell wall componentsPine NeedleCamelina

Holo-cellulose59.277.5

Cellulose50.570.2

Hemi-cellulose8.66.4

Lignin25.714.9

Table 1.6.3: Cell wall composition of Pine Needle and Camelina Fig 1.8.3- Characterization of Pine Needle and CamelinaIt is important to know cellulose and lignin components for microbial hydrolysis and production of ethanol. The result shows that the content of cellulose was highest in Jatropha Petiole and lignin has highest in Pine Needle. The Jatropha Petiole has greater value of cellulose than all other biomass and it has an increment of 1.43 fold than wheat straw [Table 1.7].TreatmentCellulose %Lignin %

PN50.5723.92

C59.8716.85

PA61.779.08

SC47.0711.87

SC TR58.1718.67

WS496.47

JP70.2719.63

JL67.322.97

JS66.9315.43

JSD & JSC61.8822.08

J F66.811.37

SE2.495151.51

LSD7.267254.41

Table 1.7- Cellulose and Lignin Composition of Different Biomass Characterized.

Cellulose and Lignin composition of different biomass is indicated in Table 1.7 and Fig 1.9 a & b. Least Significant Difference (LSD) test indicates significant differences at 5% level [PN- Pine Needle, C- Camelina, PA- Paddy, SC- Sugarcane, SC TR- Sugarcane Trash, WS- Wheat Straw, JP- Jatropha Petiole, JL- Jatropha Leaves, JS- Jatropha Stem, JSD & JSC- Jatropha Seed Coat & Jatropha Seed Cake , JF- Jatropha Fruit Husk]. The result shows that grasses are low in lignin content compared to shrubs and trees. since lignin imparts recalcitrance for hydrolysis, hence, it can be concluded that grasses are more amicable to microbial degradation compared to shrubs and tree species.(a) (b) Fig. 1.9- (a) Cellulose and Lignin (b) Composition in Different Biomass2. Hydrolysis-a. Reducing Sugar Estimation-Hydrolysis is the process which leads to break down of sucrose to glucose. In this project Jatropha seed coat and Jatropha fruit husk were subjected to microbial hydrolysis using microbes Aspergillus Niger and Trichoderma Reesei, studied by the lab earlier for its cellulose and laccase activities. The reducing sugar content was estimated before incubation and after incubation of microbes for 10 days at 2 days interval. The result shows that reducing sugar content in Jatropha fruit husk was maximum (237.1mg/ml) before incubation compared to Jatropha seed coat. The reducing sugar estimated was maximum in Jatropha seed coat (267.8mg/ml) with the incubation of A. niger after 2 days, and in Jatropha fruit husk (245.4mg/ml) with the incubation of T. reesei after 4 days. After 6 days reducing sugar content showed higher value in Jatropha fruit husk (241.6mg/ml) and with the incubation of T. reesei for 8 days showed the maximum value in Jatropha seed coat (225.4mg/ml) with the incubation of T. reesei and the 10 day result showed the maximum reducing sugar content in Jatropha seed coat (218.2mg/ml) with the incubation of T. reesei [Table 1.8].

S.N.SampleMicrobe Reducing Sugar Concentration (mg/ml) (mean values of 3 replicates)

Days to incubation0 day2 days4 days6 days8 days10 days

1Jatropha seed coatAspergillus niger

196.9264.23227.27219.8213.03191.53

Trichoderma reesei199.67258.53236.7228.87221.5215.2

2Jatropha Fruit HuskAspergillus niger229.13249.33243.23227.03206.8180.17

Trichoderma reesei230.23249.77234.07227.6208.9178.17

Table 1.8- Hydrolysis of Biomass and Reducing Sugar Estimation through DNSA MethodThe reducing sugar estimation for Jatropha seed coat and Jatropha fruit husk was carried out before incubation. The reducing sugar content in Jatropha fruit husk was greater than Jatropha seed coat by 1.15 fold [Table 1.9 and Fig. 1.10].TreatmentReducing Sugar (mg/ml)

JSC198.283

JFH229.683

SE2.716

LSD8.557

Table 1.9- Reducing Sugar Estimation for Jatropha Seed Coat and Jatropha Fruit Husk before microbial hydrolysis is estimated. Least Significant Difference (LSD) test indicated significant differences at 5% level [JSC- Jatropha Seed Coat and JFH- Jatropha Fruit Husk]. Fig. 1.10- Reducing Sugar Estimation for Jatropha before Microbial Incubation for Hydrolysis

The reducing sugar estimation for Jatropha seed coat was done before and after incubation with A. niger and T. reesei. The reducing sugar estimated for Jatropha seed coat with incubation of A. niger was greater (1.02 fold) than Jatropha seed coat incubated with T. reesei after 2 days. Reducing sugar content in Jatropha seed coat incubated with T. reesei was greater than Jatropha seed coat incubated with A. niger by 1.04 fold after 4 days. Reducing sugar content in Jatropha seed coat incubated with T. reesei was greater than Jatropha seed coat incubated with A. niger by 1.04 fold after 6 days. Reducing sugar content in Jatropha seed coat incubated with T. reesei was greater than Jatropha seed coat incubated with A. niger by 1.03 fold after 8 days. Reducing sugar content in Jatropha seed coat incubated with T. reesei was greater than Jatropha seed coat incubated with A. niger by 1.12 fold after 10 days [Table 1.10 and Fig. 1.11].TreatmentReducing sugar content after incubation(mg/ml)

Days to incubation2 46810

Js An264.233227.267219.8213.033191.533

Js Tr258.533236.7228.867221.5215.2

SE2.2131.9163.6193.7513.051

LSD8.6737.5114.18814.70311.957

Table 1.10- Reducing Sugar Estimation for Jatropha Seed Coat after Microbial Hydrolysis. Least Significant Difference (LSD) test indicates significant differences at 5% level. [Js An- Jatropha Seed Coat with A. niger incubation & Js Tr- Jatropha Seed Coat T. reesei incubation].

Fig. 1.11- Reducing Sugar Estimation for Jatropha Seed Coat after Microbial Incubation

The reducing sugar estimation for Jatropha Fruit Husk was done after incubation. The incubation was done with A. niger and T. reesei. The least significant difference is not observed. The reducing sugar estimated for Jatropha Fruit Husk with incubation of T. reesei is greater than Jatropha Fruit Husk with incubation of A. niger by 1.0 fold after 2 days, Jatropha Fruit Husk with incubation of T. reesei is greater than Jatropha Fruit Husk with incubation of A. niger by 1.03 fold after 4 days, Jatropha Fruit Husk with incubation of T. reesei is greater than Jatropha Fruit Husk with incubation of A. niger by 1.0 fold after 6 days, Jatropha Fruit Husk with incubation of T. reesei is greater than Jatropha Seed Coat with incubation of A. niger by 1.01 fold after 8 days and Jatropha Fruit Husk with incubation of T. reesei is greater than Jatropha Fruit Husk with incubation of A. niger by 1.01 fold after 10 days [Table 1.11 and Fig. 1.12].TreatmentReducing sugar content after incubation(mg/ml)

Days to incubation246810

Jf An249.333243.233227.033206.8180.167

Jf Tr249.767234.067227.6208.9178.167

SE1.5671.5315.2431.760210.936

LSD6.1445.99820.5516.89942.866

Table 1.11- Reducing Sugar Estimation for Jatropha Fruit Husk after Microbial Hydrolysis is estimated. Least Significant Difference (LSD) test indicates significant differences at 5% level. [Jf An- Jatropha Fruit Husk with A. niger incubation & Jf Tr- Jatropha Fruit Husk T. reesei incubation].

Fig. 1.12- Reducing Sugar Estimation for Jatropha Fruit Husk with Microbes IncubationReducing sugar content increased after microbial incubation. After 2 days of microbial hydrolysis, the reducing sugar content declined with increasing days which may be beacause of the consumption of sugar produced by the microbes for their growth. b. Total Carbohydrate Estimation-The hydrolysis leads to break down of sucrose to glucose. In this project hydrolysis is performed by using microbes viz. Aspergillus Niger and Trichoderma Reesei, i.e., microbial hydrolysis. Jatropha seed coat and Jatropha Fruit Husk were undergone microbial hydrolysis in this project. The observations were made in different days before and after incubation of microbes. The result shows that Jatropha Fruit Husk has maximum of total carbohydrate contents (412.6mg/ml) before incubation. The total carbohydrate estimated was maximum in Jatropha Fruit Husk (600.9mg/ml) with the incubation of T. reesei after 2 days, contents was greater in Jatropha Fruit Husk (556.7mg/ml) with the incubation of T. reesei after 4 days, after 6 days contents shows higher value in Jatropha Seed Coat (496.5mg/ml) with the incubation of A. niger, 8 day observation shows the maximum value in Jatropha Seed Coat (441.2mg/ml) with the incubation of T. reesei and the 10 day result shows the maximum contents value in Jatropha Fruit Husk (381.0mg/ml) with the incubation of A. niger [Table 1.12].

S.NSampleMicrobeTotal Carbohydrate Concentration (mg/ml) (mean values)

Days to incubation0 day2 days4 days6 days8 days10 days

1Jatropha seed coatAspergillus niger282.17318.43466.83476.23383.87317.9

Trichoderma reesei244.73329.37449.9462.6414.67340.6

2Jatropha Fruit HuskAspergillus niger389.23490.53533.53420.8344.37330.23

Trichoderma reesei363.33541.73510.8424.4426.73221.13

Table 1.12- Total Carbohydrate Estimation for Characterized Biomass through Anthrone Method.LSD (Least Significant Difference) test and SE (Standard Error) was estimated. The reducing sugar estimation for Jatropha Seed Coat and Jatropha Fruit Husk was done before incubation. The reducing sugar estimated for Jatropha Fruit Husk is greater than Jatropha Seed Coat by 1.4 fold [Table 1.13 and 1.13].TreatmentTotal carbohydrate (mg/ml)

JSC263.45

JFH376.283

SE14.1113

LSD44.4652

Table 1.13- Total Carbohydrate Estimation for Jatropha Seed Coat and Jatropha Fruit Husk before Microbial Hydrolysis. [JSC- Jatropha Seed Coat and JFH- Jatropha Fruit Husk].

Fig. 1.13- Total carbohydrate estimation for Jatropha without Microbial IncubationThe total carbohydrate estimation for Jatropha seed coat was done after incubation with A. niger and T. reesei. The total carbohydrate estimated for Jatropha seed coat with incubation of T. reesei was greater than Jatropha seed coat incubated with A. niger by 1.03 fold after 2 days. Total carbohydrate in Jatropha seed coat incubated with A. niger was greater than Jatropha seed coat incubated with T. reesei by 1.03 fold after 4 days. Total carbohydrate in Jatropha seed coat incubated with A. niger was greater than Jatropha seed coat incubated with T. reesei by 1.02 fold after 6 days. Total carbohydrate in Jatropha seed coat incubated with T. reesei was greater than Jatropha seed coat incubated with A. niger by 1.08 fold after 8 days. Total carbohydratein Jatropha seed coat incubated with T. reesei was greater than Jatropha seed coat incubated with A. niger by 1.07 fold after 10 days [Table 1.14 and Fig. 1.14].

TreatmentTotal Carbohydrate estimated after incubation (mg/ml)

Days to incubation2 days4 days6 days8 days10 days

Js An318.433466.833476.233383.867317.9

Js Tr329.367449.9462.6414.667340.6

SE14.5388.6058.61813.28914.991

LSD56.98533.72933.78152.09158.763

Table 1.14- Total Carbohydrate Estimation for Jatropha Seed Coat after Microbial Hydrolysis.[Js An- Jatropha Seed Coat with A. niger incubation & Js Tr- Jatropha Seed Coat T. reesei incubation].

Fig. 1.14- Total Carbohydrate Estimation for Jatropha Seed Coat after Microbial IncubationThe total carbohydrate estimation for Jatropha fruit husk was done after incubation with A. niger and T. reesei. The total carbohydrate estimated for Jatropha fruit husk incubated with T. reesei was greater than Jatropha fruit husk with incubation of A. niger by 1.1 fold after 2 days, Jatropha fruit husk with incubation of A. niger is greater than Jatropha fruit husk with incubation of T. reesei by 1.04 fold after 4 days, Jatropha fruit husk with incubation of T. reesei is greater than Jatropha fruit husk with incubation of A. niger by 1.0 fold after 6 days, Jatropha fruit husk with incubation of T. reesei is greater than Jatropha seed coat with incubation of A. niger by 1.23 fold after 8 days and Jatropha fruit husk with incubation of A. niger is greater than Jatropha fruit husk with incubation of T. reesei by 1.49 fold after 10 days [Table 1.15 and Fig. 1.15].

TreatmentTotal Carbohydrate Estimated after incubation (mg/ml)

Days to incubation2 days4 days6 days8 days10 days

Jf An490.533533.533420.8344.367330.233

Jf Tr541.733510.8424.4426.733221.133

SE29.67816.38812.7249.501620.247

LSD116.33264.23949.87337.24479.365

Table 1.15- Total Carbohydrate Estimation for Jatropha Fruit Husk after Microbial Hydrolysis. [Jf An- Jatropha Fruit Husk with A. niger incubation & Jf Tr- Jatropha Fruit Husk T. reesei incubation].

Fig. 1.15- Total Carbohydrate Estimation for Jatropha Fruit Husk after Microbial Incubation3. Fermentation-Microbial fermentation of released sugars after hydrolysis of biomass leads to formation of bio-ethanol. In this project hydrolysis of Jatropha seed coat and Jatropha fruit husk was performed by using microbes viz. Aspergillus Niger and Trichoderma Reesei. After hydrolysis the hydrolysates was treated with yeast viz. Saccharomyces cerevisiae for fermentation. The fermented fractions were estimated for ethanol produced by potassium dichromate assay starting from day one till six days. The result shows that the ethanol produced was maximum in Jatropha fruit husk (61.2l/ml) with the incubation of A. niger after 1 day, contents was greater in Jatropha seed coat (89.9l/ml) with the incubation of T. reesei after 2 days, after 3 days contents shows higher value in Jatropha fruit husk (157.5l/ml) with the incubation of T. reesei, 4th day observation shows the maximum value in Jatropha fruit husk (172.1l/ml) with the incubation of T. reesei, 5th day result shows the maximum value in Jatropha fruit husk (168.2l/ml) with the incubation of T. reesei and the 6th day result shows the highest value of ethanol in Jatropha fruit husk (166.3l/ml) with the incubation of T. reesei [Table 1.16].

S.N.SampleMicrobe(S. cerevisiae for fermentation)Ethanol Concentration (l/ml) (mean values)

Days to incubation1 day2 days3 days4 days5 days6 days

1Jatropha Seed CoatAspergillus niger56.06785.06798.36798.36797.86795.6

Trichoderma reesei54.56782.133102.433100104.2103.7

2Jatropha Fruit HuskAspergillus niger55.56781.333124.133130.433127.53395.333

Trichoderma reesei56.03376.967130.133123.3132.867134.833

Table 1.16-Ethanol Estimation for Characterized Biomass through Potassium Dichromate AssayThe ethanol estimation is being done for Jatropha. LSD (Least Significant Difference) test and SE (Standard Error) was estimated. The ethanol estimated for Jatropha fruit husk was greater than Jatropha seed coat by 1.18 fold [Table 1.17 and Fig. 1.16].

TreatmentEthanol from Non- hydrolyzed Fractions (l/ml)

J S C21.2667

J F H25.25

SE4.15702

LSD13.0989

Table 1.17- Ethanol estimation for Jatropha seed coat and Jatropha fruit husk for non- hydrolyzed fractions. [JSC- Jatropha Seed Coat and JFH- Jatropha Fruit Husk].

Fig. 1.16- Ethanol estimation for Jatropha biomass from Non- hydrolyzed FractionsThe ethanol estimation for Jatropha Seed Coat was done after incubation. The incubation was done with A. niger and T. reesei. The ethanol estimated for Jatropha Seed Coat with incubation of T. reesei was greater than Jatropha Seed Coat after S. cerevisiae incubation with incubation of A. niger by 1.02 fold after 1 day, Jatropha Seed Coat with incubation of A. niger was greater than Jatropha Seed Coat with incubation of T. reesei by 1.03 fold after 2 days, Jatropha Seed Coat with incubation of T. reesei is greater than Jatropha Seed Coat with incubation of A. niger by 1.04 fold after 3 days, Jatropha Seed Coat with incubation of T. reesei was greater than Jatropha Seed Coat with incubation of A. niger by 1.01 fold after 4 days, Jatropha Seed Coat with incubation of T. reesei was greater than Jatropha Seed Coat with incubation of A. niger by 1.06 fold after 5 days and the result of 6 day shows that Jatropha Seed Coat with incubation of T. reesei has greater value of ethanol than Jatropha Seed Coat with incubation of A. niger by 1.08 fold [Table 1.18 and Fig. 1.17].TreatmentEthanol produced after fermentation (l/ml)

Days to incubation1 days2 days3 days4 days5 days6 days

Js An56.06785.06798.36798.36797.86795.6

Js Tr54.56782.133104.2103.7102.433100.1

SE2.0512.92823.1093.3823.4743.831

LSD8.03911.47812.18913.25713.61615.018

Table 1.18- Ethanol Estimation for Jatropha Seed Coat after Microbial Hydrolysis and after Fermentation. [Js An- Jatropha Seed Coat with A. niger incubation & Js Tr- Jatropha Seed Coat T. reesei incubation].

Fig. 1.17- Ethanol Estimation for Jatropha Seed Coat after Microbial Hydrolysis and after Fermentation.The ethanol estimation for Jatropha Fruit Husk was done after hydrolysis with A. niger and T. reesei and fermentation by incubation with T. reesei. Ethanol produced was greater (1.0 fold) in seed coat than fruit husk after S. cerevisiae incubation after incubation of A. niger after 1 day, Jatropha fruit husk with incubation of A. niger was greater (1.05 fold) than Jatropha fruit husk incubated with T. reesei after 2 days, Jatropha fruit husk incubated with T. reesei was greater (1.04 fold) than Jatropha fruit husk incubated with A. niger after 3 days, Jatropha fruit husk incubated with A. niger was greater (1.05 fold) than Jatropha fruit husk incubated with T. reesei after 4 days, Jatropha fruit husk incubated with T. reesei was greater (1.04 fold) than Jatropha fruit husk incubated with A. niger after 5 days and the result of 6th day shows that Jatropha fruit husk incubated with T. reesei gave greater (1.41 fold) value of ethanol than Jatropha fruit husk incubated with A. niger by [Table 1.19 and Fig. 1.18].

TreatmentEthanol produced after fermentation (l/ml)

Days to incubation1 days2 days3 days4 days5 days6 days

Jf An55.566781.3333124.133130.433127.53395.3333

Jf Tr56.033376.9667132.867134.833130.133123.3

SE3.412883.4062811.361118.863419.045419.0264

LSD13.377813.351944.53373.940474.653774.5793

Table 1.19- Ethanol estimation for Jatropha fruit husk with Microbial Hydrolysis and after Fermentation. [Jf An- Jatropha Fruit Husk with A. niger incubation & Jf Tr- Jatropha Fruit Husk T. reesei incubation].

Fig. 1.18- Ethanol estimation for Jatropha Fruit Husk with Microbes Incubation and after Fermentation

The ethanol production delined after 4 days of incubation with yeast. This may be due to ethanol toxicity which leads to the killing of yeast and hence decline in rate of fermentation.

4.2 SUMMARY AND CONCLUSION- Lignocellulosic biomass is the most abundant feed stock available for biofuel production. Biofuel generation from 1st generation feedstocks had limitations inviting food vs fuel debate which is being overcome by 2nd generation feedstocks which include lignocellulosic biomass. The present study was conducted with the aim to characterize different biomass from different categories of plants like, shrubs, semi-tress and trees to find their cell wall composition for their suitability for microbial hydrolysis and fermentation for ethanol production. The characterization of different biomass viz. pine needle, Camelina, Jatropha Stem, Jatropha Petiole, Jatropha Leaves, Jatropha Seed Coat, Jatropha Fruit Husk, Jatropha Seed Cake, Wheat Straw, Paddy, Sugarcane and Sugarcane Trash was carried out in the current study with the aim to explore its potential for use as feedstock for bio-ethanol production. Camelina gave good result in the composition of cellulose and Jatropha Seed Coat gave in lignin composition. The maximum amount of cellulose was seen in Camelina (70.2 %) and maximum amount of lignin in Jatropha Seed Coat (42.9 %). Lignin content was high in pine needles which is the reason for its slow degradation even in nature. Grasses are comparatively low in lignin content than trees and even shrubs which makes it more amicable for microbial hydrolysis, since lignin acts as a barrier for enzymes to act on other cell wall components.Two different microbes Aspergillus niger and Trichoderma reesei evaluated by this lab earlier for its cellulase and lacase activity were used for hydrolysis of two different biomass namely Jatropha Seed Coat and Jatropha Fruit Husk since these biomass are available in abundant with the lab. Trichoderma reesei gave good result in the production of bio-ethanol from Jatropha fruit husk and its seed coat. The maximum amount of ethanol produced (168.2 l/ml) was on 4th day of incubation with Trichoderma reesei. The ethanol production delined after 4 days of incubation with yeast. This may be due to ethanol toxicity which leads to the killing of yeast and hence decline in rate of fermentation. The result obtained here gives an indication of the usefulness of Jatropha crop for its po