Optimizing the Conversion of Pretreated Sila Sorghum Stalks to Simple Sugars ...
Project report Kshitij Agarwal (1) (1) (1)
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INDUSTRIAL TRAINING REPORT
Determination of Structural Carbohydrates and Lignin in Biomass
By
KshitijAgarwal
Undergraduate, Batch of 2018
Bachelor in Technology, IDD
Department of Biochemical Engineering
Indian Institute of Technology (BHU) Varanasi
(DATE: 15/05/2015- 07/07/2015)
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Acknowledgement
I have taken efforts in this project. However, it would not have been possible without the
kind support and help of many individuals. I would like to extend my sincere thanks to all of
them.
I am highly indebted to Dr Harshad Ravindra Velankar for his guidance and constant
supervision as well as for providing necessary information regarding the project & also for
their support in completing the project.
I would like to express my gratitude towards Dr Anu Jose Mattam for her kind co-operation
and encouragement which helped me in completion of this project.
I would like to express my special gratitude and thanks to Dr Arindam Kuila for giving me
such attention and time.
My thanks and appreciations also go to Mr Ananth Kishore for his support and guidance.
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Abstract
Lignocellulose refers to plant dry matter (biomass), so called lignocellulosic biomass. It is
the most abundantly available raw material on the Earth for the production of bio-fuels,
mainly bio-ethanol. It is composed of carbohydrate polymers (cellulose, hemicellulose), and
an aromatic polymer (lignin). These carbohydrate polymers contain different sugar
monomers (six and five carbon sugars) and they are tightly bound to lignin. Lignocellulosic
biomass can be broadly classified into virgin biomass, waste biomass and energy crops.
Virgin biomass includes all naturally occurring terrestrial plants such as trees, bushes and
grass. Waste biomass is produced as a low value by-product of various industrial sectors
such as agricultural (corn stover, sugarcane bagasse, straw etc.), forestry (saw
mill and paper mill discards). Energy crops are crops with high yield of lignocellulosic
biomass produced to serve as a raw material for production of second
generation biofuel examples include switch grass (Panicum virgatum) and Elephant grass.
Biomass
Carbohydrate
Polymers(Cellulos+Hemicellu
lose)and aromatic
Polymer(lignin)
Virgin
Biomass
Naturally
Occuring
Terrestrialplants
Waste Biomass
Low value
byproducts of
various industrial
sectors
Energy Crops
High yield of
lignocellulosic
biomass
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Introduction
Biomass, or plant derived material, is of interest as a fuel source for several reasons.
Foremost, when managed wisely, it has the potential to become a sustainable source of
hydrocarbon fuels. It is a leading near-term solution to fill the gap between growing global
energy demand and dwindling petroleum availability. The conversion of biomass to
renewable fuels has the potential to be carbon neutral, where carbon dioxide produced
during fuel production and consumption is utilized by the next generation of plants during
growth cycles. Finally, many geographic areas contain some type of plant material that can
be utilized as a fuel source, eliminating the need for long-distance fuel transport.
Many types of biomass are inherently heterogeneous, especially lignocellulosic biomass, or
non-edible plant material. Biomass derives from living, growing plants that change during
their life cycle. Since plants are a living organism, the polymer matrix of the material is very
complex and difficult, or impossible to control. The variable nature of biomass feed stocks
represent a risk in processing environments, as processes can be difficult to optimize
without steady state input.
Cellulosic biomass feed stocks can be processed in several ways to make fuels. In the
biochemical conversion process, the cellulosic biomass is converted to monomeric
carbohydrates, which are then fermented to ethanol, butanol, or other liquid fuels.
Alternative conversion techniques include thermochemical conversion to either pyrolysis oil
or synthesis gas, or catalytic conversion of the monomeric carbohydrates in aqueous
solution. The techniques for biomass feedstock compositional analysis are largely
independent of the conversion process, although the analyses of process intermediates are
obviously dependent on the conversion process.
Biomass
Combustion
CO2+Energy
CO2 consumed by
next generation
plants.
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Biomass Composition
Plant derived biomass consists of many different constituents, but the principal constituents
are structural carbohydrates, lignin, protein, ash, and non-structural materials.
The structural carbohydrates are typically divided into two groups, cellulose and
hemicellulose. Cellulose is a polymer with a rigid structure of repeating glucose units, and is
highly stable and resistant to chemical attack. It has a high degree of hydrogen bonding,
which contributes to the rigidity of the structure. Hemicellulose is a polymer consisting of
shorter, highly branched chains of sugars. Hemicellulose can contain five-carbon sugars,
such as xylose and arabinose, as well as six-carbon sugars, such as glucose, galactose,
and mannose. The backbone may be mannose or xylose, with a variety of side chain
sugars. The branched character of hemicellulose causes it to be more amorphous and
easier to break down compared to cellulose.
Aside from carbohydrates, the major structural materials present in lignocellulosic biomass
include lignin, ash, and protein. Lignin is a polymeric structure that is highly aromatic and
branched. It has a high molecular weight and a complex structure. Lignin assists in holding
the cells together, provides the plant with rigidity, and gives it some resistance to insect and
biological degradation. Ash is any inorganic matter, typically silica. Protein is a compact
structure made up of chains of amino acids.
Materials that are not a part of the cellular structure and can be removed with solvents are
termed extractives for the purpose of biomass compositional analysis. Extractives can
include waxes, saps, and fats.
Lignocellulosic
Biomass
Structural
Carbohydrates
Cellulose
Hemicellulose
Lignin
Proteins
Ash
Non-
Structural
materials
Fats
Waxes
Saps
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Lignin Estimation
Chemicals Required:
a) Potassium Permanganate solution(0.1N)
b) Sulphuric Acid solution(4N)
c) Potassium Iodide Solution(1N)
d) Sodium thiosulphate Solution(0.1N)
e) Starch Indicator solution(0.2%)
Procedure:
Lignin estimation was done by standard method (Hussain et al., 2002) in which kappa
number was determined based on 50% consumption of the permanganate. About 0.05 g of
dry sample was taken and dispersed in 3 mL distilled water and ground to fine paste by
using mortar and pestle. The disintegrated sample was transferred to 100 mL conical flask
and distilled water was added to make the total volume to 60 mL. Then, 7.5 mL of
potassium permanganate solution and 7.5 mL of sulfuric acid solution were mixed together
and added immediately to disintegrate the sample. Thus, the total volume was made to 75
mL. The reaction was allowed to proceed at 25 °C for exactly 10 min. Then, 1.5 mL of
potassium iodide solution was added and the free iodine was titrated with standard sodium
thiosulphate solution using starch indicator. A blank titration was carried out using the same
volume of water and reagent. The kappa number was then calculated from the following
equation:
Kappa Number (k) = P×f / W
Where,
P = mL of 0.1N potassium permanganate consumed by the experimental sample
W = Weight of dry sample in g
f = Factor for correction to 50% permanganate consumption
Lignin content was determined by using following equation
Lignin content (%) = (Kappa number × 0.155) × 100
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Flow chart:
Take 0.5 g of drysubstrate and
dispersedin30 ml distilled
waterand groundto fine paste
byusingmortar andpestle.
Transferredit to 1000ml conical
flaskanddistilledwater was
added to make the total volume
to 600ml.
Seventyfive ml of potassium
permanganate solution and75
ml of sulfuric acidsolutionwere
mixed together andadded
immediatelyto disintegrate the
sample.
Take 1ml of that solutionand
dilutedto 100 ml and 1ml of
dilutedsolution wasadded to
10 ml of anthrone reagent and
well mixed
Tubes were heated inboiling
water bath for 10 minutes
Absorbance was measuredat
630 nm bytaking a blank with
anthrone reagent andwater
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Cellulose Estimation
Chemicals Required:
a) Acetic/Nitric Reagent (150 ml of 80% Acetic acid with 15 ml of conc.Nitric acid is
mixed)
b) Sulphuric Acid (67%)
c) Anthrone Reagent(200mg Anthrone+100ml H2SO4)
d) Standard Cellulose Solution (100mg of Cellulose+10ml 67% H2SO4. 1ml of this is
added to 100ml H2O)
Procedure:
A dry sample of 0.5-1.0 g was taken in a dry test tube and 3 mL of acetic/nitric reagent was
added and the sample was well mixed using a vortex mixture. The solution was placed in a
water bath at 100 °C for 30 min. Then the mixture was cooled and centrifuged for 15-20 min
at 10,000 rpm and the supernatant was discarded. The residue was washed with water and
10 mL of 67% H2SO4 was added and kept aside for 1 h. From the solution, 1 mL was taken
and diluted to 100 mL and 1 mL of diluted solution was then added to 10 mL of anthrone
reagent and mixed well. Tubes were heated in boiling water bath for 10 min and the
absorbance was measured at 630 nm by taking a blank with anthrone reagent and water.
Amount of cellulose was calculated from standard graph of cellulose which was prepared
by taking 0.4 to 2 mL standard cellulose solution (Viles and Silverman, 1949).
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Flow Chart:
A weighed dry sample(0.5-
1.0 g) was taken in a dry test
tube and 3 ml of
acetic/nitric reagentwas
added
Keep it in a water bath at
100 oC for 30 min
Cooled to room
temperature and centrifuge
at 5000 rpm for 10 minutes
The residuewas washed
with water and 10ml of 67%
H2SO4 was added and kept
for 1
Take 1ml of that solution
and diluted to 100 ml and
1ml of diluted solution was
added to 10 ml of anthrone
reagent and well mixed
Tubes were heated in boiling
water bath for 10 minutes
Absorbancewas measured
at 630 nm by takinga blank
with anthrone reagent and
water
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Hemicellulose Estimation
Chemicals Required:
a) Sulphuric Acid- 1%
b) Anthrone Reagent (200mg Anthrone+100ml H2SO4)
Procedure:
For hemicellulose estimation, dry lignocellulosic biomass was treated with 1% sulphuric
acid, at 100 °C for 4 h.Then, the treated lignocellulosic biomass was dried overnight.
Hemicellulose content was determined from the difference in total soluble sugar content
between control and treated lignocellulosic biomass (Marlett and Lee, 2006).
Flow chart:
Take driedlignocellulosic
substrate
Treatedwith1% sulphuric
acid,at 100 oC for 4 h
Treatedlignocellulosic
biomasswasdried
overnight
Measure the total soluble
sugar byanthrone
methodof control and
treatedsample
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Results and Discussions
Before Pre-treatment:
Cellulose Estimation:
Concentration OD at
630nm
10 0.05
25 0.15
50 0.24
75 0.34
100 0.44
OD of Sample- 0.162
Using:
y=0.0045x
x=36 ug/ml
% Cellulose in Wheat Bran= 36%
A standard curve was plotted using the OD of different compositions at 630 nm. The slope
of this curve was used to find out the Concentration of the sample whose OD is known at
630 nm. The OD divided by the slope of the curve gives the concentration of the sample.
Lignin Estimation:
Sample Titre
Value
1 21.1
2 22
3 22
Blank- 23.05 ml
Using:
y = 0.0045x
R² = 0.9738
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
0 20 40 60 80 100 120
OD@630nm
Concentration(ug/ml)
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Lignin Content (%)=(k*0.155)*100
k= ((Blank)-(Titre Value))=1.35ml
Lignin Content= 20.925%
Using the titration method triplets of sample were titrated against Sodium Thiosulphate
sulphate solution. The purple colour of KMnO4 disappears and the solution becomes
transparent in colour. Thus, substituting the value of point of neutralisation in the formula
we get th concentration of Lignin in biomass.
Hemicellulose Estimation:
Concentration OD at
630nm
10 0.05
25 0.15
50 0.24
75 0.34
100 0.44
OD of Sample- 0.099
y=0.0045x
x=22ug/ml
% Hemicellulose in Wheat Bran= 22%
Using the Anthrone reagent method used in Cellulose estmation the standard curve of OD
vs Concentration is plotted. Using the equation of the curve the concentration of
hemicelluloses is found and by means of back calculation the content of Hemicellulose is
estimated in Biomass.
y = 0.0045x
R² = 0.9738
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
0 20 40 60 80 100 120
OD@630nm
Concentration(ug/ml)
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After Pre-treatment
Cellulose Estimation:
Concentration OD at
630nm
10 0.05
25 0.15
50 0.24
75 0.34
100 0.44
OD of Sample- 0.18
Using:
y=0.0045x
x=40ug/ml
% Cellulose in Wheat Bran= 40%
In case of weak acid pre treatment, the biomass is pre treated at temperature of 150oC. For
hydrolysis of cellulose the temperature required is 180oC. Thus, it is observed that the
cellulose concentration does not change drastically even after pre-treatment.
Lignin Estimation:
Sample Titre
Value
1 9.7
2 9.8
3 9.7
Blank- 10.41
Using:
Lignin Content (%)=(k*0.155)*100
k= ((Blank)-(Titre Value)) = 0.68 ml
y = 0.0045x
R² = 0.9738
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
0 20 40 60 80 100 120
OD@630nm
Concentration(ug/ml)
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Lignin Content= 10.54%
Lignin Hydrolysis occurs at very low temperature, thus on pre-treatment at 150oC hydrogen
bonds between molecules break and 50% hydrolysis occurs. Thus, the concentration of
Lignin reduces to half.
Hemicellulose Estimation:
Concentration OD at
630nm
10 0.05
25 0.15
50 0.24
75 0.34
100 0.44
OD of Sample- 0.05
y=0.0045x
x=22ug/ml
% Hemicellulose in Wheat Bran= 11.12%
Hydrolysis of Hemi cellulose occurs at temperature between 150oC-1600C releasing sugars
and soluble oligomers from the cell wall matrix into the hydrolysate. Thus hemi-cellulose
content reduces by half.
Conclusion
% Lignin Cellulose Hemicellulose
Before Pre-
treatment
20.925 36 22
After Pre-
treatment
10.54 40 11.12
y = 0.0045x
R² = 0.9738
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
0 20 40 60 80 100 120
OD@630nm
Concentration(ug/ml)
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The net content of Structural carbohydrates, namely cellulose and hemi-cellulose, and
lignin in the biomass before and after pre-treatment was found to vary. As it was
theoretically reported that there is a decrease in concentrations of Lignin and Hemi-
cellulose and a slight increase in the concentration of Cellulose, the practical
experimentation also yielded the same results. The decrease in Lignin and hemi-cellulose
was found to be half whereas only 5% increase in Cellulose occurred. This change in
concentrations of lignocelluloses was observed due to varying degrees of Hydrolysis. The
method used for pre-treatment was weak-acid hydrolysis.
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