Introduction

India is blessed with varied agroclimatic agricultural abundance of agricultural waste and
manpower making it is most suitable for the cultivation of all the types of temperature,
subtropical and tropical crops. Bioethanol production is not only feasible from conventional
substrates like sugarcane molasses but also from lignocellulosic like rice straw, wheat straw,
corn strover and waste from paper industry etc. Apart from the conventional crops residues, new
energy crops (sunflower stalks, alfalfa fibers) have been identified as potential bioethanol
production sources. The conversion of biomass to ethanol includes increased national energy
security, reduction in green house emissions, and use of renewable resources. The lignocellulosic
are abundantly available which may be successfully converted to bioethanol for partial
supplementing gasoline or other petroleum products.

Production of Biofuels from Renewable Biomass

The present-day energy scenario in India and around the globe is precarious, thus driving to the
search of alternative to fossil fuels. Abundant energy, stored primarily in forms of carbohydrates,
can be found in waste biomass from agricultural, municipal and industrial sources. Potential
strategy for deriving useful forms of energy from carbohydrates include production of ethanol.
However, lignocellulosic biomass has long been recognised as potential low cost source of mix
sugars for fermentation to fuel ethanol. In recent years, biomass-derived fuels have received
increasing attention as one solution to our nation’s continued and growing dependence on
imported oil. We set a goal in its Strategic Plan to promote energy security through a diverse
energy supply that is reliable, clean, and affordable. Biomass is the single renewable resource
that has the potential to supplant our use of liquid transportation fuels now and help create a
more stable energy future. Using our indigenous biomass resources, we can potentially fuel our
cars. The project includes conversion of lignocellulosic substrates viz. rice straw, wheat straw,
mushroom stiple, and water hyacinth to bioethanol using several techniques of pretreatment viz.
acid hydrolysis, irradiation, steam explosion, and alkaline hydrolysis, liquid hot-water etc.
followed by sachariffication of pretreated biomass by cellulolytic enzymes produced from the
strains screened and isolated from the locale. The sacharified hydrosylate will be fermented
using the local ethanol producing strains. Distillation of ethanol from fermentation broth by
distillation unit and using the left biomass for further applications for that it can be converted to
lumps and supplied to other organizations where it can be gasified for syngas and water gas
production. Increasing energy consumption creates unbalanced energy management and requires
power sources that are able to sustain for longer periods. More recently, wild plants like Lantana
camara, Prosopis juliflora, and Saccharum spontanum has been found as source of fermentable
sugars which can be converted into bioethanol. Similarly our study will be focused on use of
various new surplus substrates easily available in Haryana.

The overall objectives set for the Project reflect the current strategy of focusing on cellulosic
ethanol as the most immediate path to meet the desired goals and address both the technology
advances required to enable production of cost-competitive cellulosic ethanol and the increase
biofuel production volume needed to meet petroleum fuel displacement goals.

Review of literature

LIGNOCELLULOSIC BIOMASS
Lignocellulosic biomass is the non-starch, fibrous part of plant material and is an attractive
resource fuel production because its abundance and availability [Perlack, R.D., L.L. Wright, A.F.
Turhollow, R.L. Graham, B.J. Sotcks, and D.C. Erbach. 2005. Biomass as feedstock for a
bioenergy and bioproducts industry: the technical feasibility of a billion-ton annual supply.
DOE/GO-102005-2135, April, U.S. Department of Energy and U.S. Department of Agriculture.]
Chemical composition of lignocellulosic feedstocks is a key factor affecting efficiency of biofuel
production during conversion processes [Hamelinck, C.N., G.V. Hooijdonk, and A.P. Faaij.
2005. Ethanol from lignocellulosic biomass: techno-economic performance in short-, middle-,
and long-term. Biomass and Bioenergy 28: 384–410., Hames, B.R., S.R. Thomas, A.D. Sluiter,
C.J. Roth, and D.W. Templeton. 2003]. New tools for compositional analysis of wheat straw,
rice straw, mushroom stipe and water hyacinth feedstocks and process intermediates ethanol
production. The structural and chemical composition of lignocellulosic feedstocks is highly
variable because of genetic and environmental influences and their interactions.

COMPOSITION OF LIGNOCELLULOSIC BIOMASS

Lignocellulose is the term used to describe the three-dimensional polymeric composites formed
by plants as structural material. It consists of variable amounts of cellulose, hemicellulose, and
lignin [Brown, R.C. 2003. Biorenewable resources; Engineering new products from agriculture.
Iowa State Press. Ames]. The composition of lignocellulosic biomass is as follows:
 Cellulose
Cellulose (30–50% of total feedstock dry matter) is a glucose polymer linked by β–1,4
glycosidic bonds. The basic building block of this linear polymer is cellubiose, a glucose-
glucose dimer (dimer: two simpler molecules—monomers—combined to form a
polymer). Hydrolysis of cellulose results in individual glucose monomers. This process is
also known as saccharification.
 Hemicellulose
Hemicellulose (20–40% of total feedstock dry matter) is a short, highly branched
polymer of five-carbon (C5) and six-carbon (C6) sugars. Specifically, hemicellulose
contains xylose and arabinose (C5 sugars) and galactose, glucose, and mannose (C6
sugars). Hemicellulose is more readily hydrolyzed compared to cellulose because of its
 branched, amorphous nature. A major product of hemicellulose hydrolysis is the C5 sugar
xylose.
 Lignin
Lignin (15–25% of total feedstock dry matter), a polyphenolic structural constituent of
plants, is the largest non-carbohydrate fraction of lignocellulose. Unlike cellulose and
hemicellulose, lignin cannot be utilized in fermentation processes.
 Ash
Ash (3–10% of total feedstock dry matter) is the residue remaining after ignition (dry
oxidation at 575 ± 25°C) of herbaceous biomass. It is composed of minerals such as
silicon, aluminum, calcium, magnesium, potassium, and sodium.

PRETREATMENT OF LIGNOCELLULOSIC MATERIALS

Pretreatment aims to get rid of lignin and hemicellulose, reduce crystallinity of cellulose and
increase surface area of materials to improve formations of sugars. To overcome lignocellulose
recalcitrance, pretreatment is required. The objective of pretreatment is to alter the physical
features and chemical composition of the lignocellulose, to make it more digestible.
Specifically, pretreatment improves enzyme access and effectiveness by
a) Removing or altering lignin
b) Removing hemicellulose
c) Decrystallizing cellulose
d) Removing acetyl groups from hemicellulose
e) Reducing the degree of polymerization in cellulose
f) Expanding the structure to increase pore volume and internal surface area

ACID HYDROLYSIS

Dilute Sulfuric Acid Pretreatment

This approach is one of the oldest and most studied pretreatment options, having been known
since 1819 (Galbe and Zacchi 2002). Dilute acid hydrolysis makes use of lower
concentrations of acid (typically 0.5–5% H2SO4 at elevated temperatures and pressures, and
typically achieves hydrolysis of hemicellulose and yields enzymatically accessible cellulose.
Different process options include, batch, plug flow and flow through. The main drawback to
dilute acid hydrolysis is the degradation of C5 sugars to yield furfural and C6 sugars to yield
HMF as well as other toxic byproducts. Other issues include highly corrosive reaction
environment, formation of gypsum (after neutralization with lime), and high use of steam.
Acid-catalyzed hydrolysis of switchgrass and wheat straw biomass was compared with
 alkaline-catalyzed hydrolysis and no-catalyst hydrolysis for release of sugars at various
severities (Nagle et al. 2006). Acid hydrolysis across the severity range was comparable with
both sources of biomass, but wheat straw was more reactive than switchgrass under alkaline
hydrolysis.
Alkaline hydrolysis
Steam explosion
Preconditioning
Sachariffication
Distillation

Broad objectives:

 Isolation and screening of cellulolytic microorganisms

 Optimization of ethanol production
 Ethanol distillation and gasification of lignocellulosic wastes.

Specific objectives:

 Procurement of substrates i.e. plant wastes viz. rice straw, wheat straw, mushroom stiple,
and water hyacinth.
 Development of cost effective methods of pretreatment of lignocellulosic wastes.
 Isolation and screening of cellulolytic microorganisms
 Saccharification of pretreated biomass by cellulolytic microorganism to release
fermentable sugars.
 Ethanol production by specific fermenting microbes from hydrosylate
 Purification of ethanol from fermentation broth and energy generation from left biomass
by gasification in collaboration with public sector undertakings.

11. Work methodology

1. Material collection: The lignocellulosic substrates like straw and wheat straw will be
collected from the agricultural fields of Haryana and stored for processing. Waste
mushroom stipe will be collected from the HAIC Agro R&D center, Murthal. Water
hyacinth, a water weed will be harvested from ponds.

2. Pretreatment: (a) Physical pretreatment by Grinding and milling of lignocellulosic

biomass,

(b) Chemical pretreatment by alkaline (NaOH/KOH) or Acid hydrolysis

(HCl/H2SO4)
 (c) Biological pretreatment: Different microorganism will be screened for
pretreatment

3. Preconditioning: Removal of inhibitory compounds like hemifurfurals, furfurals etc.

and to allow efficient sachharification followed by fermentation.

4. Saccharification: Enzymatic hydrolysis of the pretreated substrates by cellulolytic

microorganisms

5. Fermentation: Cellulosic substrates can be converted to ethanol by either simultaneous

sachharification and fermentation or separate enzymatic hydrolysis and fermentation
process.

6. Ethanol purification: Bioethanol hence generated will be purified using suitable

chromatographic techniques.

7. Gassification of the left biomass: The remaining biomass will be subjected to

gasification for energy generation in collaboration with PSUs.

13. Year wise activities

Ist year

1. Collection of raw material

2. Pretreatment of lignocellulosic substrates
3. Screening of cellulolytic microorganisms

IInd year

1. Saccharification
2. Fermentation
3. Comparison of the various substrates for ethanol production

IIIrd year

1. Ethanol purification
2. Gasification

Rationale for proposed target

With rapid economic growth in the country, India’s energy demands are increasing in every
sphere of life. Thus search should be focused on alternative substrates for generation of clean
fuels like bioethanol. India, a country with massive agricultural potential can become a major
supplier of alternative fuel. Also the rising crude oil prices augment the problem which justifies
the urgent requirement for alternative energy sources. Screening of newer microorganisms, for
the hydrolysis of the specific substrates might help in higher levels of saccharification followed
by higher ethanol production by fermentation. The local indegenious microflora will be
investigated for their cellulolytic and fermentation potential.

Most of the national and international research uses rice straw as a lignocellulosc substrate. The
main drawback of this substrate is high silicon content, which cannot be completely oxidized in
the left over substrates. However, we propose to subject our left over waste to gasification for
energy. Besides, we will also be using other substrates, which might give a higher sugar yield.

Thus in the given project, screening of indigenous cellulolytic and fermentative microorganism
might be a step forward in generation of ecofriendly bioethanol.

10. Justification of proposed project

Bioethanol production from lignocellulosic biomass could avoid competition with food
production and offer considerable potential to promote rural development and increasing the
overall economic situation in emerging and developing regions. Therefore, we would like to
discover the potential for fuel ethanol production from agricultural production from agricultural
residues in this region. In this study, we intend to produce bioethanol from agricultural residues
in state of Haryana. We propose the given project keeping the following observations in mind:

 Lignocellulosic wastes minimizes the potential conflict between land use for food (and
feed) and energy feedstock production
 The raw material is less expensive than conventional agricultural feedstock.
 It can also be produced with lower input of fertilizers, pesticides, and energy.
 Biofuels from biomass are economic and environment friendly (low emission of green
house gases)
 It can also provide employment in rural areas.

The growing need for energy independence and proposed renewable fuels had led
recently to a major expansion of fuel ethanol production. The need to find other cost
effective and efficient grains for ethanol production has increased in significance.
Cereal grain are high in starch and are currently being utilized for ethanol production.
To ensure long term viability of the industry, fermentation strategies that focus on
holistic utilization of the feedback that maximize value addition will increase in
importance.