CONVERSION OF ELEPHANT GRASS TO

BIOETHANOL AND VALUE ADDED

PRODUCTS

SUBMITTED BY:

UDDIPAN DEKA (16/031)

MRIGANABH SARMA (16/257)

ANURAG KUMAR (16/281)

ISHITA SAIKIA (16/282)

B.E.7​th​ SEMESTER

DEPARTMENT OF CHEMICAL ENGINEERING

ASSAM ENGINEERING COLLEGE,

 JALUKBARI,GUWAHATI-13

CONVERSION OF ELEPHANT GRASS TO BIOETHANOL AND

VALUE ADDED PRODUCTS

SEVENTH SEMESTER B.E. PROJECT

Submitted in partial fulfilment of

the Requirements for the Degree of

BACHELOR OF ENGINEERING

in

CHEMICAL ENGINEERING

of

GAUHATI UNIVERSITY

by

UDDIPAN DEKA (16/031)

MRIGANABH SARMA (16/257)

ANURAG KUMAR (16/281)

ISHITA SAIKIA (16/282)

 DEPARTMENT OF CHEMICAL ENGINEERING

ASSAM ENGINEERING COLLEGE, GUWAHATI-781013

JANUARY 2020

CERTIFICATE

DEPARTMENT OF CHEMICAL ENGINEERING

ASSAM ENGINEERING COLLEGE

GUWAHATI​-​781013

This is to certify that ​Uddipan Deka, Roll No-16/031, Mriganabh Sarma, Roll
No-16/257, Anurag Kumar, Roll No-16/281 and Ishita Saikia, Roll No-16/282 ​of B.E.
7th Semester have jointly carried out the project entitled-

“CONVERSION OF ELEPHANT GRASS TO VALUE ADDED PRODUCTS”

under my supervision and submitted the report in partial fulfilment of the requirement for
the Degree of Bachelor of Engineering in Chemical Engineering of Gauhati University,
which may be accepted​.

DATE: (Signature)

DR. BANDANA CHAKRABORTY

ASSOCIATE PROFESSOR
DEPARTMENT OF CHEMICAL ENGINEERING,
 ASSAM ENGINEERING COLLEGE,
GUWAHATI-781013

ACKNOWLEDGEMENT

A detailed report such as this one would not have been possible without the contribution
of several people who have helped us wholeheartedly. Firstly, we would like to extend
our gratitude to our guide teacher, Dr. Bandana Chakraborty Ma’am, Associate Professor,
Department of Chemical Engineering, Assam Engineering College, for being our pillar of
strength throughout the two semesters. Without her support and pieces of advice, we
would not have been able to progress in our project-work.

Secondly, we would like to thank Dr. Mihir Purkait Sir, Head, Center for the
Environment, IIT Guwahati, for providing us the opportunity to work in his project. We
would also like to thank Dr. Dibyajyoti Haldar, Post-Doctoral Fellow, Simons Dhara and
Prangan Duarah, Junior Research Fellows, IIT Guwahati for helping us with all our lab
work in IIT, Guwahati. The project would never have been possible without the
cooperation and assistance of all the above mentioned people.
 ABSTRACT

Pennisetum purpureum o​ r Elephant grass or Napier grass is a tropical plant ​that has a
great potential to be chosen as a raw material for renewable energy production due to its
fast​-g​ rowing abilities and high carbohydrate content. The aim of this work is to optimize
the ​best pretreatment conditions for maximum yield of bioethanol and thus evaluate the
highest glucose and ethanol yield after subjecting the samples to alkaline pretreatment,
enzymatic hydrolysis and fermentation with dried yeast (​Saccharomyces cerevisiae​). For
pretreatment, the samples were subjected to KOH(1%, 2%, 3% conc.) and were put in an
autoclave for residence times of 30 mins, 60 mins and 90 mins. Enzymatic hydrolysis of
the cellulose in the cake residue after filtration is carried out using carboxy methyl
cellulase. The quantity of fermentable sugars obtained is evaluated using HPLC and the
fermentable sugars subsequently obtained are subjected to fermentation using
Saccharomyces cerevisiae. Thus, a comparative study of the effect of the different
factors- temperature, concentration and time, on the ethanol fermentability of the
cellulosic fraction of elephant grass is done and the best pretreatment conditions are
optimized.
 LIST OF FIGURES

Chapter No. Title Page No.

4 Plot showing the relation
between pretreatment
conditions and residence
time.

LIST OF TABLES

Table Title Page

No. No.
1 Final HPLC Results of Enzymatic Hydrolysis of different
pre-treated biomass for Glucose at 121​0​C and 30minutes

2 Final HPLC Results of Enzymatic Hydrolysis of different

pre-treated biomass for Xylose at 121ºC and 30 mins

3 Final HPLC Results of Enzymatic Hydrolysis of different

pretreated biomass for glucose at 121ºC and 60 mins

4 Final HPLC Results of Enzymatic Hydrolysis of different

pretreated biomass for Xylose at 121ºC and 60 mins

5 Final HPLC Results of Enzymatic Hydrolysis of different

pretreated biomass for glucose at 121ºC and 90 mins

6 Final HPLC Results of Enzymatic Hydrolysis of different

pretreated biomass for Xylose at 121ºC and 90 mins

TABLE OF CONTENTS

Chapter No. Title Page No.

Certificate
Acknowledgement
Abstract
List of figures
List of tables
1 Introduction
1.1 Biofuel types and uses
1.2 Aim and objectives of
present study
 2 Literature Survey
3 Materials and Methodology
4 Results and Discussion
Conclusion
Bibliography

CHAPTER 1

INTRODUCTION

Many in the energy industry view biofuel as vitally important to future energy production
because of its clean and renewable properties. Ironically, most of the major oil companies
are investing millions of dollars in advanced biofuel research. America's largest oil
company, ExxonMobil, says they are funding a broad portfolio of biofuels research
programs including ongoing efforts on algae as well as programs on converting
alternative, non-food-based biomass feed stocks, i.e., cellulosic biomass, to advanced
biofuels.

One of the biggest knocks against fossil fuels is that they give off toxic emissions.
Biofuels do not release as much carbon as fossil fuels do, and because of this, there are
fewer harmful emissions out of biofuels. Biofuels are made out of organic substances,
which are biodegradable. But the concept of using farmland to produce fuel instead of
food comes with its own challenges, and solutions that rely on waste or other feedstocks
haven't yet been able to compete on price and scale with conventional fuels.

1.1 Biofuel types and uses 

There are various ways of making biofuels, but they generally use chemical reactions,
fermentation, and heat to break down the starches, sugars, and other molecules in plants.
The resulting products are then refined to produce a fuel that cars or other vehicles can
use.

Much of the gasoline in the United States contains one of the most common biofuels:
ethanol. Made by fermenting the sugars from plants such as corn or sugarcane, ethanol
contains oxygen that helps a car's engine burn fuel more efficiently, reducing air
pollution. ​However, there are ​some drawbacks​ to these kinds of fuels. Issues include
things like the amount of land space required to grow the crops. This, in particular,
creates problems with higher food prices and deforestation. Additionally, the costs for
converting crops in energy crops, as well as the need to retrofit existing vehicles and
power plants to run on them is not cheap.

Among several alcoholic biofuels that have been investigated, bioethanol has emerged as
a potential fuel. Lignocellulosic biomass, available mainly in the form of agro and forest
residues, is potential cheap substrate for bioethanol production.
These lignocellulosic biomasses are essentially made up of three major constituents, viz.
cellulose, hemicellulose and lignin. Among these, the important components for ethanol
production are cellulose and hemicellulose, as these are made up of the sugar monomers.
The physical, chemical and structural composition of any lignocellulosic biomass is
resistant to the direct action of enzymatic hydrolysis. This is due to high crystallinity of
cellulose and also presence of a complex network of lignin around cellulose, which
reduce exposure of cellulose fraction of biomass to enzyme action and hydrolysis
reaction. This necessitates pretreatment of raw biomass prior to ethanol production via
fermentation. The main objectives of pretreatment are: reducing the crystallinity of
cellulose, increasing the porosity and surface area of biomass, and removal of the lignin
stratum around cellulose entangled with other two components. All of these effects lead
to higher and faster hydrolysis of cellulose, thus maximizing the extent of
saccharification and yield of fermentable sugars. Several pretreatment methods have been
practiced for various lignocellulosic biomasses prior to enzymatic hydrolysis. These
methods are of physical, chemical or physicochemical nature.

1.2 Aim and objectives of present study

In this paper we have dealt with optimization of pretreatment of an abundant

lignocellulosic waste biomass, i.e. the tropical grass ​Pennisetum purpureum o​ r Elephant
grass or Napier grass. We have also studied the subsequent enzymatic hydrolysis of
pretreated biomass. ​The key benefits of elephant grass are financial and environmental.
Elephant grass is a specialised plant that can grow up to four metres in just 100 days,
producing several crops to harvest each year. Another unique feature of elephant grass is
that it can grow on marginal land that is unsuitable for food production and, therefore, is
cheap to lease. Elephant grass also stores a large percentage of the carbon it absorbs from
the atmosphere in its roots.

Existing studies till now are specifically focused in pretreatment and fermentability of
elephant grass varieties, but under such different experimental conditions that make
difficult the comparison of the results. The enzymatic pretreatment (cellulase + esterase)
of elephant grass gave 113 mg sugars/g biomass. The biological delignification
pretreatment of a Colombian species of Penisetum sp. allowed a lignin removal of
10.7–55.9% . Regarding the fermentability, ethanol yields of 45.5 mg/g biomass, using
the strain ​Klebsiella oxytoca ,​ and 97– 107 mg/g biomass, using the strain Saccharomyces
cerevisiae​ D5A, have been reported for two different genotypes of elephant grass.

Our studies have been carried out under comparable conditions that allow the selection of
the best pretreatment conditions for maximum yield of bioethanol. Our studies are
focused on the effects of the factors- concentration and time on the alkaline pretreatment
process, and the ethanol fermentability of the cellulosic fraction of elephant grass.
Furthermore, the conditions for the best pretreatment found were optimized in order to
maximize the ethanol yield.
 CHAPTER 2

LITERATURE SURVEY

Effects of the different pretreatment methods on enzymatic saccharification and

ethanol production using different feedstocks

Eliana, ​et al​. (2013) found that chemical and physicochemical pretreatments like alkaline
delignification, diluted acid hydrolysis, steam explosion, alkaline peroxide and aqueous
ammonia soaking had different effect on the hydrolysis and the fermentability of the
cellulosic fraction of this material. Alkaline pretreatment with NaOH yielded the highest
concentrations of reducing sugars and ethanol from elephant grass, compared to results
obtained with dilute acid, steam explosion, aqueous ammonia soaking and alkaline
peroxide pretreatments. Optimization of the alkaline delignification with NaOH allowed
a lignin removal of 88.4%, total reducing sugars yield of 711.2 mg/g biomass after
enzymatic hydrolysis (82% of theoretical) and ethanol concentration of 26.05 g/L which
corresponds to maximum theoretical yield of 95%.

Tsai, ​et al. (2018) conducted an experiment where the Napier grass after different
pretreatments was tested for simultaneous saccharification and fermentation (SSF) with
dried yeast (​Saccharomyces cerevisiae​) and cellulase (CTec2) to produce ethanol.
Compared with dilute acid and two-stage pretreatments, alkaline pretreatment was found
to be superior based on the ethanol yield from dried biomass of Napier grass.
Alkaline-pretreated Napier grass biomass could be converted to glucose at 0.245 g/g-raw
material in 96 hours through enzyme hydrolysis. Applying SSF to the alkaline pretreated
biomass resulted in an ethanol yield of 0.143 g/g-raw material, resulting in the highest
yield among those that have been published in the literature. Both high lignin and
hemicellulose removal rates by alkaline pretreatment alone could make biomass more
accessible to enzyme hydrolysis and lead to higher ethanol production. The ethanol yield
reached 86.6% of the theoretical yield based on the glucan in pretreated biomass using
sole alkaline pretreatment, which was higher than that from the two-stage pre-treatment
(80.5%).

Phitsuwan, ​et al​. (2016) conducted an experiment where feasible alkaline pretreatment
technologies, including NaOH, Ca(OH)​2​, NH​3​, and alkaline H​2​O​2 were used to delignify
lignocellulose with the aim of improving glucose recovery from Napier grass stem
cellulose via enzymatic saccharification. The influences of the pretreatments on structural
alterations were examined using SEM, FTIR, XRD and TGA, and the relationships
between these changes and the enzymatic digestibility of cellulose were addressed.
Pretreatment of NP stem with NaOH gave the best effect on enzymatic glucose
production, yielding 18.5 g/L of glucose and 94% enzymatic efficiency. This superior
outcome was due to the extensive removal of lignin (84%), which created porosity and
allowed the accessibility of cellulose to cellulases. In addition, the removal of surface
xylan was assisted by xylanase supplement.

Singh, ​et al. (2014) found that among the different methods employed for pretreatment,
the most efficient treatment has been revealed to be autoclaving of biomass at 121ºC and
15 psi pressure for 30 min in acidic (1% v/v, H2SO4) environment. Total reducing sugar
(TRS) yield during this pretreatment, mainly due to hydrolysis of hemicellulosic fraction
of biomass, has been 285.3 mg/g of raw biomass. Further enzymatic hydrolysis resulted
in reducing sugar yield of 187.4 mg/g of pretreated biomass (9.37 g/L). The total
fermentable sugar (TFS) yield from the optimized pretreatment was 397.7 mg/g raw
biomass (39.77 g/100 g raw biomass). The effects of different pretreatment methods on
biomass structure and complexity were investigated by field emission scanning electron
microscopy (FESEM), Fourier transform infrared (FTIR) spectroscopy and X-ray
diffraction (XRD) techniques.

Xu, ​et al. ​(2010) investigated t​he effects of residence time, lime loading, and biomass
washing on the sugar production efficiency in the lime pretreatment method of
switchgrass. ​Under the best pretreatment conditions (50 degrees Celsius, 24 h, 0.10 g
Ca(OH)​2 /g
​ raw biomass, and wash intensity of 100 ml water/g raw biomass), the yields
of glucose, xylose, and total reducing sugars reached 239.6, 127.2, and 433.4 mg/g raw
biomass, which were respectively 3.15, 5.78, and 3.61 times those of untreated biomass.
The study on calcium-lignin bonding showed that calcium ions crosslinked lignin
molecules under alkaline conditions, which substantially decreased lignin solubilization
during pretreatment, but the resulting high lignin contents of the pretreated biomass did
not compromise the improvement of enzymatic digestibility.

Soares, ​et al. ​(2011) evaluated the ​efficiency of ethanol production by fermentation of a
hydrolysate obtained by the enzymatic hydrolysis of purple elephant grass (​Pennisetum
​ chum.) using a blend of cellulases​. ​The hydrolysate obtained after
purpureum S
enzymatic hydrolysis and agitation was fermented using dried ​Saccharomyces cerevisiae
yeast at 30 ​℃ for 10 hours. The liquids obtained after fermentation were analysed using
HPLC to determine the quantity of ethanol produced. After 4 hours of fermentation, the
maximum quantity of ethanol was 1.8 g/L. The stoichiometric yield of ethanol was
approximately 95%.

Menegol, ​et al. ​(2016) evaluated ​the enzymatic hydrolysis of ​Pennisetum purpureum
(elephant grass) at high total solid levels (from 4% to 20% (w/v)) in a concomitant ball
milling treatment in a rotating hydrolysis reactor (RHR). ​It was found that elephant grass
when hydrolyzed in the rotating hydrolysis reactor has about double ethanol production
than that was produced when the biomass was hydrolyzed in a static reactor (SR).
 CHAPTER 3

MATERIALS AND METHODS USED

Biomass Collection​: ​Pennisetum purpureum,​ also known as Elephant grass was collected
from the campus of IIT Guwahati. Whole plant body, except roots, was used as substrate
for pretreatment. Biomass was chopped, washed with water and dried at 60​0​C for 24 h
prior to pretreatment.

Equipments used​: Weighing machine, whatman filter paper, centrifuge tube, pH meter,
HPLC filter, autoclave, centrifuge, ice, enzyme cellulase, HNO​3​, KOH flakes, pipette, hot
air oven, incubator, millipore water, 1M citric acid, 1M NaOH, mortar pestle, litmus
paper etc.

1. ​Base Preparation​: Different concentrations of KOH solutions(1%,2% and 3%) were

prepared.1.5g,3g and 4.5g of KOH pellets were weighed and added to 150mL of
millipore water to obtain 1%,2% and 3% KOH solutions respectively.

● Preparation of 1% KOH(w/v):1.5003 g of KOH flakes is added to 50 mL

millipore water. Another 100 mL water is added to the solution to enhance
mixing.
 ● Preparation of 2% KOH(w/v): 3.0008 g of KOH flakes is added to 50 mL
millipore water. Another 100 mL water is added to the solution to enhance
mixing.
● Preparation of 3% KOH(w/v): 4.5054 g of KOH flakes is added to 50 mL
millipore water. Another 100 mL water is added to the solution to enhance
mixing.
2. ​Biomass weighing​: The biomass obtained from Elephant grass which was chopped,
washed, dried and grinded in advance was weighed using weighing balance.12 g biomass
was weighed and stored in a bottle.

3. ​Base pretreatment​: The alkali pretreatment was carried out to delignify the biomass
and thus separate cellulose, hemicellulose to extract sugar. The alkali pretreatment was
carried out with 1%(w/v),2%(w/v) and 3%(w/v) KOH solutions and analysed for
different times to optimise the production of sugar viz. glucose and xylose.

4​. Preparation of enzyme carboxymethylcellulase​: Carboxymethylcellulase was

prepared by B. amyloliquefaciens SS35 isolated from rhinoceros dung. The enzyme
production comprised of following components: carboxymethylcellulose 18.1 g,peptone
2.1g/L,K​2​HPO​4​ 1.0g/L,NaCl 1.0 g/L and MgSO​4​ 0.1g/L(pH 5.5).

SET 1:

a) Autoclaving for 60 min​:

1) With 1%(w/v) KOH: 120 mL of the 1%(w/v) KOH solution prepared was added
to the weighed biomass and was subjected to steam explosion in a high pressure
reactor at 15 psi and 121​0​C .The product formed in the high pressure reactor was
filtered using whatman filter paper and the solid fraction(cake residue) was
washed until neutral pH. The pH was measured using litmus paper and pH meter.
The washed cake residue was dried and the dry weight was 7.1859 g, then it was
stored. The volume of filtrate lignin obtained was 41 mL.
2) With 2%(w/v) KOH: 120 mL of the 2%(w/v) KOH solution prepared was added
to the weighed biomass and was subjected to steam explosion in a high pressure
 reactor at 15 psi and 121​0​C .The product formed in the high pressure reactor was
filtered using whatman filter paper and the solid fraction(cake residue) was
washed until neutral pH.The pH was measured using litmus paper and pH meter.
The washed cake residue was dried and the dry weight was 5.9123 g, then it was
stored. The volume of filtrate lignin obtained was 42 mL.
3) With 3%(w/v) KOH: 120 mL of the 3%(w/v) KOH solution prepared was added
to the weighed biomass and was subjected to steam explosion in a high pressure
reactor at 15 psi and 121​0​C .The product formed in the high pressure reactor was
filtered using whatman filter paper and the solid fraction(cake residue) was
washed until neutral pH.The pH was measured using litmus paper and pH meter.
The washed cake residue was dried and the dry weight was 5.563 g, then it was
stored. The volume of filtrate lignin obtained was 44 mL.

b) ​Acid preparation+ Hydrolysis​: Acid hydrolysis was carried out to neutralize the
alkaline filtrate lignin.HNO​3 was used for the purpose which when added to the filtrate
lignin formed a precipitate.6M HNO​3 was prepared from 70%(w/v) HNO​3 by adding
37.9746 mL HNO​3​ in 62.0253 mL water to make 100 mL solution.

1) For 1%(w/v) KOH pretreated sample: The filtrate obtained was treated with 6M
HNO​3 .The precipitate obtained was centrifuged at 10000 rpm for 5 min and the
supernatant was drained. The lignin was dried in hot air oven and stored in small
centrifuge tubes. Dry weight of filtrate lignin was 0.5275 g.

2) For 2%(w/v) KOH pretreated sample: The filtrate obtained was treated with 6M
HNO​3 .The precipitate obtained was centrifuged at 10000 rpm for 5 min and the
supernatant was drained. The lignin was dried in hot air oven and stored in small
centrifuge tubes. Dry weight of filtrate lignin was 0.7323 g.
3) For 3%(w/v) KOH pretreated sample: The filtrate obtained was treated with 6M
HNO​3 .The precipitate obtained was centrifuged at 10000 rpm for 5 min and the
 supernatant was drained. The lignin was dried in hot air oven and stored in small
centrifuge tubes. Dry weight of filtrate lignin was 0.9195 g.

c) ​Enzymatic Hydrolysis​:

a) Preparation of 1M NaOH: 0.8 g NaOH was added in 20 mL millipore water to

make 20mL 1M NaOH solution.
b) Preparation of 1M Citric acid:1.9212 g anhydrous citric acid was added in 10 mL
millipore water to obtain 10 mL 1M citric acid solution
The solid fraction obtained from different pretreatment processes was analyzed for sugar
release. The sugar released after physical, chemical or physiochemical treatments was
estimated on a gross basis-only after completion of the process. The individual pentose
and hexose sugar present in the hydrolyzate were confirmed with HPLC analysis. The
HPLC apparatus comprised of a pump, an RI(refractive index) detector, a vacuum
degasser and a HiPlex-H column(Varian,300 mm × 5µm × 4.6 mm).HPLC grade
water(deionized water,Milli Q) was used as the mobile phase at a flow rate of 0.4
mL/min.Samples were diluted appropriately and filtered through a 0.2 µm membrane
filter before HPLC analysis.

● Preparation of buffer:200 mL enzymatic buffer was prepared by mixing 18.61 mL

1M NaOH solution and 10 mL 1M citric acid solution with 171.39 mL millipore
water which gave a pH of 5.The cake residue which was dried and stored was
grinded manually by mortar pastel. The hydrolysis was done on duplicates i.e.,
two counts each of 1.5g for 1%,2% and 3% cellulose. Thus 1.5g cellulose were
taken in 6 conical flasks(100 mL) and 30mL of enzymatic buffer solution was
added to each sample. After that 0.285µL of enzyme carboxymethylcellulase was
added to each sample and then were placed in an incubator.2mL of sample was
collected in every 24 hours in a 2mL centrifuge tube and immediately put on ice
to stop the reaction. The collected sample was centrifuged two times for 5 minutes
at 10000 rpm, each time the precipitate was discarded and supernatant was
 centrifuged. Then in 6 small beakers, 600uL millipore water was taken in each
beaker with a pipette. To this 600uL supernatant was added. These samples were
filtered using HPLC filters and sent for HPLC analysis which continued for 96
hours.
SET 2:

a) Autoclaving for 90 min​:

1) With 1%(w/v) KOH:120 mL of the 1%(w/v) KOH solution prepared was added
to the weighed biomass and was subjected to steam explosion in a high pressure
reactor at 15 psi and 121​0​C .The product formed in the high pressure reactor was
filtered using whatman filter paper and the solid fraction(cake residue) was
washed until neutral pH. The pH was measured using litmus paper and pH meter.
The washed cake residue was dried and the dry weight was 6.89 g,then it was
stored. The volume of filtrate lignin obtained was 43 mL.
2) With 2%(w/v) KOH: 120 mL of the 2%(w/v) KOH solution prepared was added
to the weighed biomass and was subjected to steam explosion in a high pressure
reactor at 15 psi and 121​0​C .The product formed in the high pressure reactor was
filtered using whatman filter paper and the solid fraction(cake residue) was
washed until neutral pH. The pH was measured using litmus paper and pH meter.
The washed cake residue was dried and the dry weight was 5.7833 g, then it was
stored. The volume of filtrate lignin obtained was 43 mL.
3) With 3%(w/v) KOH:120 mL of the 3%(w/v) KOH solution prepared was added
to the weighed biomass and was subjected to steam explosion in a high pressure
reactor at 15 psi and 121​0​C .The product formed in the high pressure reactor was
filtered using whatman filter paper and the solid fraction(cake residue) was
washed until neutral pH.The pH was measured using litmus paper and pH
meter.The washed cake residue was dried and the dry weight was 5.5984 g,then it
was stored.The volume of filtrate lignin obtained was 44 mL.
b).​Acid preparation+ ​Hydrolysis​: Acid hydrolysis was carried out to neutralize the
alkaline filtrate lignin.HNO​3 was used for the purpose which when added to the filtrate
lignin formed a precipitate.6M HNO​3 was prepared from 70%(w/v) HNO​3 by adding
37.9746 mL HNO​3​ in 62.0253 mL water to make 100 mL solution.

1) For 1%(w/v) KOH pretreated sample: The filtrate obtained was treated with 6M
HNO​3 .The precipitate obtained was centrifuged at 10000 rpm for 5 min and the
supernatant was drained. The lignin was dried in hot air oven and stored in small
centrifuge tubes. Dry weight of filtrate lignin was 0.6486 g.

2) For 2%(w/v) KOH pretreated sample: The filtrate obtained was treated with 6M
HNO​3 .The precipitate obtained was centrifuged at 10000 rpm for 5 min and the
supernatant was drained. The lignin was dried in hot air oven and stored in small
centrifuge tubes. Dry weight of filtrate lignin was 0.8724 g.
3) For 3%(w/v) KOH pretreated sample: The filtrate obtained was treated with 6M
HNO​3 .The precipitate obtained was centrifuged at 10000 rpm for 5 min and the
supernatant was drained. The lignin was dried in hot air oven and stored in small
centrifuge tubes. Dry weight of filtrate lignin was 1.0051 g.

c) ​Enzymatic Hydrolysis​:

a) Preparation of 1M NaOH: 0.8076 g NaOH was added in 20 mL millipore water to

make 20mL 1M NaOH solution.
b) Preparation of 1M Citric acid:2.8823 g anhydrous citric acid was added in 15 mL
millipore water to obtain 15 mL 1M citric acid solution
The solid fraction obtained from different pretreatment processes was analyzed for sugar
release. The sugar released after physical, chemical or physiochemical treatments were
estimated on a gross basis-only after completion of the process.
The solid fraction obtained from different pretreatment processes was analyzed for sugar
release. The sugar released after physical, chemical or physiochemical treatments were
estimated on a gross basis-only after completion of the process. The individual pentose
and hexose sugar present in the hydrolyzate were confirmed with HPLC analysis. The
HPLC apparatus comprised of a pump, an RI(refractive index) detector, a vacuum
degasser and a HiPlex-H column(Varian,300 mm × 5µm × 4.6 mm).HPLC grade
water(deionized water, Milli Q) was used as the mobile phase at a flow rate of 0.4
mL/min. Samples were diluted appropriately and filtered through a 0.2 µm membrane
filter before HPLC analysis.

● Preparation of buffer: 200 mL enzymatic buffer was prepared by mixing 18.61

mL 1M NaOH solution and 10 mL 1M citric acid solution with 171.39 mL
millipore water which gave a pH of 5.26.The cake residue which was dried and
stored was grinded manually by mortar pastel. The hydrolysis was done on
duplicates i.e.,two counts each of 1.5g for 1%,2% and 3% cellulose.Thus 1.5g
cellulose were taken in 6 conical flasks(100 mL) and 30mL of enzymatic buffer
solution was added to each sample.After that 0.285µL of enzyme
carboxymethylcellulase was added to each sample and then were placed in an
incubator.2mL of sample was collected in every 24 hours in a 2mL centrifuge
tube and immediately put on ice to stop the reaction.The collected sample was
centrifuged two times for 5 minutes at 10000 rpm,each time the precipitate was
discarded and supernatant was centrifuged.Then in 6 small beakers 600uL
millipore water was taken in each beaker with a pipette.To this 600uL supernatant
was added.These samples were filtered using HPLC filters and sent for HPLC
analysis which continued for 96 hours.
Set 3:

a) Autoclaving for 30 min​:

1) With 1%(w/v) KOH:120 mL of the 1%(w/v) KOH solution prepared was added
to the weighed biomass and was subjected to steam explosion in a high pressure
reactor at 15 psi and 121​0​C .The product formed in the high pressure reactor was
filtered using whatman filter paper and the solid fraction(cake residue) was
washed until neutral pH.The pH was measured using litmus paper and pH
meter.The washed cake residue was dried and the dry weight was 7.2112 g,then it
was stored.The volume of filtrate lignin obtained was 41.5 mL.
2) With 2%(w/v) KOH:120 mL of the 2%(w/v) KOH solution prepared was added
to the weighed biomass and was subjected to steam explosion in a high pressure
reactor at 15 psi and 121​0​C .The product formed in the high pressure reactor was
filtered using whatman filter paper and the solid fraction(cake residue) was
washed until neutral pH.The pH was measured using litmus paper and pH
meter.The washed cake residue was dried and the dry weight was 6.0047 g,then it
was stored.The volume of filtrate lignin obtained was 39 mL.
3) With 3%(w/v) KOH:120 mL of the 3%(w/v) KOH solution prepared was added
to the weighed biomass and was subjected to steam explosion in a high pressure
reactor at 15 psi and 121​0​C .The product formed in the high pressure reactor was
filtered using whatman filter paper and the solid fraction(cake residue) was
washed until neutral pH.The pH was measured using litmus paper and pH
meter.The washed cake residue was dried and the dry weight was 5.5807 g,then it
was stored.The volume of filtrate lignin obtained was 42 mL.

b) Acid preparation+Hydrolysis:​Acid hydrolysis was carried out to neutralize the

alkaline filtrate lignin.HNO​3 was used for the purpose which when added to the
filtrate lignin formed a precipitate.6M HNO​3 was prepared from 70%(w/v) HNO​3
by adding 37.9746 mL HNO​3​ in 62.0253 mL water to make 100 mL solution.
1) For 1%(w/v) KOH pretreated sample:The filtrate obtained was treated with 6M
HNO​3 .The precipitate obtained was centrifuged at 10000 rpm for 5 min and the
 supernatant was drained.The lignin was dried in hot air oven and stored in small
centrifuge tubes.Dry weight of filtrate lignin was 0.5534 g.
2) For 2%(w/v) KOH pretreated sample:The filtrate obtained was treated with 6M
HNO​3 .The precipitate obtained was centrifuged at 10000 rpm for 5 min and the
supernatant was drained.The lignin was dried in hot air oven and stored in small
centrifuge tubes. Dry weight of filtrate lignin was 0.7599 g.
3) For 3%(w/v) KOH pretreated sample:The filtrate obtained was treated with 6M
HNO​3 .The precipitate obtained was centrifuged at 10000 rpm for 5 min and the
supernatant was drained. The lignin was dried in hot air oven and stored in small
centrifuge tubes. Dry weight of filtrate lignin was 0.8627 g.

c) Enzymatic Hydrolysis:
a) Preparation of 1M NaOH:0.8076 g NaOH was added in 20 mL millipore water to
make 20mL 1M NaOH solution.
b) Preparation of 1M Citric acid:2.8823 g anhydrous citric acid was added in 15 mL
millipore water to obtain 15 mL 1M citric acid solution
The solid fraction obtained from different pretreatment processes was analyzed for sugar
release. The sugar released after physical, chemical or physiochemical treatments were
estimated on a gross basis-only after completion of the process.

The solid fraction obtained from different pretreatment processes was analyzed for sugar
release. The sugar released after physical, chemical or physiochemical treatments was
estimated on a gross basis-only after completion of the process. The individual pentose
and hexose sugar present in the hydrolyzate were confirmed with HPLC analysis. The
HPLC apparatus comprised of a pump, an RI(refractive index) detector, a vacuum
degasser and a HiPlex-H column(Varian,300 mm × 5µm × 4.6 mm).HPLC grade
water(deionized water,Milli Q) was used as the mobile phase at a flow rate of 0.4
mL/min. Samples were diluted appropriately and filtered through a 0.2 µm membrane
filter before HPLC analysis.
 ● Preparation of buffer:200 mL enzymatic buffer was prepared by mixing 18.61 mL
1M NaOH solution and 10 mL 1M citric acid solution with 171.39 mL millipore
water which gave a pH of 5.The cake residue which was dried and stored was
grinded manually by mortar pestle. The hydrolysis was done on duplicates i.e.,
two counts each of 1.5g for 1% cellulose. Thus 1.5g cellulose were taken in 6
conical flasks(100 mL) and 30mL of enzymatic buffer solution was added to each
sample. After that 285uL of enzyme cellulase was added to each sample and then
were placed in an incubator.2mL of sample was collected in every 24 hours in a
2mL centrifuge tube and immediately put on ice to stop the reaction. The
collected sample was centrifuged two times for 5 minutes at 10000 rpm, each time
the precipitate was discarded and supernatant was centrifuged. Then in 6 small
beakers 600uL millipore water was taken in each beaker with a pipette. To this
600uL supernatant was added. These samples were filtered using HPLC filters
and sent for HPLC analysis which continued for 96 hours.

Fermentation on a test sample:

a) ​Preparation of YPG medium for Yeast Activation:

Materials required​:

Yeast Extract-0.5 g

Peptone- 1 g

Glucose-2.5 g

H​2​O-50 mL

● The above chemicals were mixed at desired quantity and autoclaved for 20 mins
at 121º C
 ● The autoclaved medium is allowed to cool till the room temperature.

b) ​Activation of Baker's Yeast​:

​Materials required​:

YPG Medium-50 mL
Baker's Yeast-0.1000 g
● The above samples are placed in a shaking incubator at 30º C at 120 rpm.
● 0.1000 g of yeast is added to the prepared YPG medium for activation.
● The conical flask with the prepared sample is then placed in a shaking incubator
at preset temperature and rpm.

c) ​Preparation of the test sugar sample​:

Materials required​:

Buffer solution ( Citric acid and NaOH): 50 mL

Glucose-0.25 g

Xylose-0.25 g

The above samples are mixed together and autoclaved for 20 mins at 121º C.

d) ​Fermentation of the test sample​:

Materials required​:

Sugar sample-45 mL
Yeast extract-5 mL
Peptone
● After autoclaving, peptone and yeast extract are added to the sugar sample.
● The mixture is then placed in a shaking incubator at 35º C and 12 rpm.
 CHAPTER 4

RESULTS AND DISCUSSIONS

Table 1: Final HPLC Results of Enzymatic Hydrolysis of different pretreated biomass for
Glucose at 121​0​C and 30minutes
Time(h) Glucose(g/L) Glucose(g/L) Glucose(g/L)
Enzymatic 1% KOH 2% KOH 3% KOH
Hydrolysis
A(X) C1(Y) B(yEr ​+​ ) C(Y) D(yEr ​+​ ) E(Y) F(yEr ​+​ )
6 5.99 0.39 7.45 0.24 7.97 0.1
12 7.8 0.75 9.4 0.01 10.19 0.15
24 9.66 0.36 12.07 0.15 12.72 0.15
48 11.03 0.17 15.6 0.14 17.56 0.77
72 12.11 0.13 17.6 0.08 19.68 0.41

Table 2: Final HPLC Results of Enzymatic Hydrolysis of different pretreated biomass for
Xylose at 121ºC and 30 mins

Time(h) Xylose(g/L) Xylose (g/L) Xylose (g/L)

Enzymatic 1% KOH 2% KOH 3% KOH
Hydrolysis
A(X) C1(Y) B(yEr ​+​ ) C(Y) D(yEr ​+​ ) E(Y) F(yEr ​+​ )
6 1.81 0.05 2.2 0.01 2 0.01
12 2.07 0.13 2.54 0.06 2.35 0.01
24 2.38 0.09 2.96 0.05 2.54 0.02
48 2.47 0.08 3.33 0.06 3.02 0.18
72 2.85 0.07 3.87 0.13 3.5 0.16
Table 3: Final HPLC Results of Enzymatic Hydrolysis of different pretreated biomass for
Glucose at 121​0​C and 60minutes

Time(h) Glucose(g/L) Glucose(g/L) Glucose(g/L)

Enzymatic 1% KOH 2% KOH 3% KOH
Hydrolysis
A(X) M(Y) N(yEr ​+​ ) O(Y) P(yEr ​+​ ) Q(Y) R(yEr ​+​ )
6 5.93 0.12 7.78 0.22 8.25 0.09
12 7.53 0.06 10.23 0.28 10.96 0.25
24 9.53 0.27 12.73 0.28 13.01 0.04
48 11.15 0.05 15.22 0.41 16.77 0.6
72 12.79 0.12 17.94 0.63 18.96 0.2
Table 4: Final HPLC Results of Enzymatic Hydrolysis of different pretreated biomass for
Xylose at 121​0​C and 60minutes

Time(h) Xylose (g/L) Xylose (g/L) Xylose (g/L)

Enzymatic 1% KOH 2% KOH 3% KOH
Hydrolysis
A(X) G(Y) H(yEr ​+​ ) I(Y) J(yEr ​+​ ) K(Y) L(yEr ​+​ )
6 1.79 0.05 2.36 0.03 2.16 0.01
12 2.04 0.002 2.83 0.08 2.61 0.07
24 2.33 0.07 3.14 0.02 2.84 0.09
48 2.53 0 3.43 0.14 3.14 0.27
72 2.96 0.1 4.06 0.11 3.57 0.06

Table 5: Final HPLC Results of Enzymatic Hydrolysis of different pretreated biomass for
Glucose at 121​0​C and 90minutes

Time(h) Glucose(g/L) Glucose(g/L) Glucose(g/L)

Enzymatic 1% KOH 2% KOH 3% KOH
Hydrolysis
A(X) G(Y) H(yEr ​+​ ) I(Y) J(yEr ​+​ ) K(Y) L(yEr ​+​ )
6 6.33 0.04 7.91 0.08 8.07 0.11
12 8.14 0.09 10.34 0.09 10.64 0.64
24 10.28 0.09 13.1 0.02 13.76 0.39
48 12.49 0.28 16.27 0.17 17.56 1.21
72 13.51 0.73 17.72 0.15 19.21 1.3

Table 6: Final HPLC Results of Enzymatic Hydrolysis of different pretreated biomass for
Xylose at 121​0​C and 90minutes

Time(h) Xylose (g/L) Xylose (g/L) Xylose (g/L)

Enzymatic 1% KOH 2% KOH 3% KOH
Hydrolysis
A(X) M(Y) N(yEr ​+​ ) O(Y) P(yEr ​+​ ) Q(Y) R(yEr ​+​ )
6 1.99 0.02 2.42 0.02 2.17 0.04
12 2.29 0.04 2.97 0.007 2.57 0.17
24 2.59 0.02 3.33 0.007 2.94 0.03
48 2.93 0.09 3.8 0.06 3.43 0.19
72 3.06 0.21 3.95 0.09 3.5 0.15
GRAPH 1: THE PLOT SHOWING THE RELATIONSHIP BETWEEN DIFFERENT
PRETREATMENT CONCENTRATIONS AND DIFFERENT RESIDENCE TIME.

Temperature​- 121​0​C

KOH Concentrations​-1%,2%,3%

Pre-treatment time​- 30min, 60min, 90min

Data analysis of Glucose yield-

Glucose yield increases with increasing concentration of KOH in pre-treatment solution.
After 72 hours of enzymatic hydrolysis maximum, 19.68 g/L Glucose yield has been
achieved from 12.11 g/L pretreated biomass just by increasing KOH concentration from
1% to 3%. Higher concentration of KOH pre-treatment conditions proved out to have
more effective delignification process. However pre-treatment time shows minimal effect
on glucose production. In case of mild alkali (1%KOH) pre-treatment, glucose
production increases with increasing pre-treatment time. But in case of higher
concentration of KOH, Pre-treatment time has no effect on glucose yield. HPLC data of
samples collected after 6h, 12h, 24h, 48h and 72 hours shows exponential growth in
glucose yield although rate of glucose yield slows down after 48 hours of hydrolysis.

Data analysis of Xylose yield-

As cellulose only contains only anhydrous glucose, after hydrolysis it will converted to
glucose. But hemicellulose contains different monomers. As a result, hydrolysis breaks
down hemicellulose to different five carbon sugars like xylose and arabinose. Dominance
of xylose concentration as a constituent of hemicellulose is the reason to focus on xylose
yield instead of arabinose. 2% KOH treated biomass gives highest yield of xylose
followed by 3% and 1%. High concentration alkali pre-treatment removes hemicellulose
along with lignin. Increasing pre-treatment time shows a little increment on xylose
production. Maximum xylose yield has been obtain from 60min 2% KOH pre-treated
biomass(4.06 g/L).
 CONCLUSION

Compared with different concentrations of KOH in pre treatment solution, it was found
that with increasing concentration of KOH (i.e. 1%, 2%, 3%), the yield of glucose also
increases. Hence, pretreatment conditions with higher concentrations of KOH were more
effective for the production of Glucose. While the pretreatment time has very minimal
effect on glucose yield, it increases with increasing pretreatment time in case of lower
concentration of KOH whereas in case of higher concentration of KOH, pre treatment
time has no effect on glucose production. While in case of Xylose, its production is
highest for 2% KOH pretreated biomass followed by 3% and 1% KOH concentration.
With increasing pretreatment time, Xylose production shows a very little increment.
Maximum xylose yield has been obtain from 60min 2% KOH pre-treated biomass(4.06
g/L).
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