project report kshitij agarwal (1) (1) (1)

HPCL Green R&D Centre

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)


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.


Contents Acknowledgement...................................................................... II

Abstract ...................................................................................... 4

Introduction ................................................................................ 5

Biomass Composition ................................................................ 6

Lignin Estimation........................................................................ 7

Chemicals Required ............................................................... 7

Procedure ............................................................................... 7

Flow chart ............................................................................... 8

Cellulose Estimation .................................................................. 9

Chemicals Required ............................................................... 9

Procedure ............................................................................... 9

Flow Chart ............................................................................ 10

Hemicellulose Estimation......................................................... 11

Chemicals Required ............................................................. 11

Procedure ............................................................................. 11

Flow chart ............................................................................. 11

Results and Discussions.......................................................... 12

Before Pre-treatment............................................................ 12

Cellulose Estimation: ........................................................ 12

Lignin Estimation: ............................................................. 12

Hemicellulose Estimation: ................................................ 13

After Pre-treatment............................................................... 14

Cellulose Estimation: ........................................................ 14

Lignin Estimation: ............................................................. 14

Hemicellulose Estimation: ................................................ 15

Conclusion ............................................................................... 15

References ............................................................................... 16


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

Natura lly Occuring

Terrestrial plants

Waste BiomassLow value

byproducts of various industrial

sectors

Energy Crops

High yield of lignocellulosic

biomass


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+EnergyCO2 consumed by next generation

plants.


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

Structura l Carbohydrates

CelluloseHemicellulose

Lignin

Proteins

Ash

Non-Structural materials

Fats

Waxes

Saps


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


Flow chart:

Take 0.5 g of dry substrate and dispersed in 30 ml distilled

water and ground to fine paste by us ing mortar and pestle.

Transferred i t to 1000ml conical flask and distilled water was

added to make the total volume to 600ml.

Seventy five ml of potassium permanganate solution and 75

ml of sulfuric acid solution were mixed together and added

immediately to disintegrate the sample.

Take 1ml of that solution and di luted to 100 ml and 1ml of

di luted solution was added to 10 ml of anthrone reagent and

wel l mixed

Tubes were heated in boiling water bath for 10 minutes

Absorbance was measured at 630 nm by taking a blank with anthrone reagent and water


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).


Flow Chart:

A weighed dry sample (0.5-1.0 g) was taken in a dry test

tube and 3 ml of acetic/nitric reagent was

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 residue was 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

Absorbance was measured at 630 nm by taking a blank with anthrone reagent and

water


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 dried lignocellulosic substrate

Treated with 1% sulphuric acid, at 100 oC for 4 h

Treated lignocellulosic biomass was dried

overnight

Measure the total soluble sugar by anthrone

method of control and treated sample


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 = 0.0045xR² = 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

@ 6

30

nm

Concentration(ug/ml)


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:


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 = 0.0045xR² = 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

@ 6

30

nm



After Pre-treatment

Cellulose Estimation:


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 = 0.0045xR² = 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

@ 6

30

nm



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:


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 = 0.0045xR² = 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

@ 6

30

nm



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.

References

KeikhosroKarimi, Yusuf Chisti, Future of bioethanol, Biofuel research journal,

2015

AnsaToivola, David Yarrow, Eduard Van Den Bosch, Johannes P. Van Dijken,

W. Alexander Scheffersi, Alcoholic Fermentation of D-Xylose by Yeasts, Applied

And Environmental Microbiology, 1984

Karin Ohgren, Oskar Bengtsson, Marie F. Gorwa-Grauslund, Mats Galbe, Barbel

Hahn-H agerdal , Guido Zacchi, Simultaneous saccharification and co-

fermentation of glucose and xylose in steam-pre-treated corn stover at high fibre

content with Saccharomyces cerevisiae TMB3400, Journal of Biotechnology

DNV, Biofuels 2020

Mingyu Wang, Zhonghai Li, Xu Fang, Lushan Wang and YinboQu, Cellulolytic

enzyme production and enzymatic hydrolysis for second generation bioethanol

production, AdvBiochemEngin/Biotechnol (2012).

project report kshitij agarwal (1) (1) (1)

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