This document discusses biofuels produced from biomass waste sources. It begins with introductions to biomass, biofuels like ethanol and biodiesel, and describes their production processes. The key steps discussed are pretreatment of lignocellulosic biomass using acids, enzymatic hydrolysis to break down cellulose and hemicellulose into sugars, and fermentation of sugars into ethanol. Several biomass sources like sugarcane bagasse are tested. Enzymes and microbes involved in the process are also outlined. Advantages of bioethanol include its environmental feasibility, use as a gasoline supplement, and potential for cost reduction through large scale production.

1. Biofuel from wastes an economic and
environmentally feasible resource
DevendraPratap Singh
Department Of Applied Chemistry
Dr. Ambedkar Institute Of Technology for Handicapped
Kanpur
1

2. CONTENTS
1.Introduction
2.Need of Biofuel
3.Steps for Production of
Biofuel from
Biomass
(a)Pretreatment (Comparative study)
(b)Enzymatic hydrolysis & Fermentation
(c)Kinetic study of production of ethanol
4.Advantage of bioethanol
5.Conclusion & Effective parameters
6.Major concerns/problems in bio ethanol
cellulosic materials
7.Future Prospectus
8.Case Study of total biofuel production
Lignocellulosic
production from
2

3. (1) Introduction
(a) BIOMASS
Biomass is a renewable energy resource derived from the
carbonaceous waste of various human and natural activities.
It is derived from numerous sources, including the byproducts from the timber industry, agricultural crops, raw
material from the forest, major parts of household waste and
wood.
Barley Straw, Rice Straw, Wheat Straw and Rape Straw before Pre-treatment
3

4. (b) Biomass Resources
Untapped Natural
Resource
Agriculture: Rice husk, Rice straw, Wheat
straw, Vegetable residue, etc
Livestock: Animal waste, Butchery waste,
etc.
Agriculture,
Livestock, Forestry
and Fishery group
Other Waste group
Forestry: Forest residue, Thinned wood,
Processing waste, Sawdust, etc.
Fishery: Processing waste, Bowel, Dead
fish, etc.
Industry: Sewage sludge, Organic
processing waste, etc.
Household: Garbage, Human waste, etc.
Plantation
(Production group)
Continental area: Grain, Plant, Vegetable,
Fat and oil, etc.
Water area: Algae, Photosynthetic
bacteria, etc.
4

5. (c) BIOFUEL
A fuel that is produced using biological feedstock's. It is a
renewable energy source derived from biomass, such as plants,
agricultural or forestry waste, animal wastes, or food waste.
Common biofuels include
ethanol and biodiesel.
First generation Biofuels : Corn and sugar to ethanol, Chemical
transesterification of vegetable oils
Second Generation Biofuels : Lignocellulose to ethanol Enzymatic
bioconversion of Vegetable oil
Third generation Biofuels: Energy crops for bio-alcohol, Algal
Ethanol /Biodiesel
5

6. (d)ETHANOL
Produced from hydrolysis of sugar crops, Lignocellulosic
biomasses, and fruit and vegetable waste by suitable enzymes or acid
followed by fermentation of sugars, starch, cellulose and
hemicelluloses using yeast or bacterium. It is used primarily as a
supplement to gasoline.
Fermentation is the process by which cells release energy under
anaerobic conditions .
(C6H10O5)n
Acid Pretreatment
C12H22O11 +
H2O
C6H12O6
zymase(Yeast)
invertase
n C6H12O6
C6H12O6 + C6H12O6
2C2H5OH
+ 2CO2
6

7. (e) BIODISEL
It is Defined as the mono alkyl ester of long chain fatty acids
derived from renewable lipid sources by Transesterification. It is
produced from virgin or used vegetable oils (both edible & non
edible) and animal fats through various chemical process.
Transesterification: Transesterification is the process of reacting a
triglyceride molecule with an excess of alcohol in presence of
catalyst to produce glycerin and fatty aids.
Triglyceride + Methanol
NaOH or KOH
Methyl ester + Glycerol
7

8. 2. Need of Biofuels
It provides a market for excess production of vegetable oils
and animal fats.
It decreases the country's dependence on imported petroleum.
It is renewable and does not contribute to global warming due
to its closed carbon cycle.
8

9. 3. Steps for Production of Biofuel from Lignocellulosic Biomass
(a) Pretreatment
A pretreatment step is necessary for the enzymatic hydrolysis
process. It is able to remove the lignin layer and to decristallize
cellulose so that the hydrolytic enzymes can easily access the
biopolymers.
Need of lignocellulose pretreatment
Tight multi-polymeric complex of cellulose, hemicelluloses
and lignin
Protective action of lignin
Crystalline structure of cellulose
Limited surface area for hydrolysis
The purpose of physical pretreatments is the increase of the
accessible surface area and the size of pores of cellulose and
the decrease of its crystallinity and its polymerization degree.
9

10. Effect of Pretreatment
Pretreatment gives enzyme accessible substrate
10

11. Wheat bran, sugarcane bagasse, Rice bran and rape straw were used for
the treatment process. Before pretreatment these biomasses are reduced
in to smaller particles after milling and crushing (particle size <180 μm).
(b) Dilute Acid pre-treatment
For the comparative result equal amount of biomasses were pretreated
in two ways one is acid trement where 2-3% acid (H2SO4) was used for
the pre-treatment method. In this content acid soaked biomass slurry
was autoclaved at 1210C for 30 minutes. To separate the solid and
liquid fraction centrifuge method was used. The dilution and pH was
maintained at 5 by adding alkali of centrifuged biomass before
fermentation process.
11

12. Different raw materials and their contains after pretreatment
Raw Material
Cellulose
Hexosans (H) %
45
Hemicelluloses
Pentosans (P)%
35
30
50
15
Rice straw
32.1
24
18
Rape straw
33.4
30
17
Rice bran
30.4
22
16
Wheat bran
31.3
23
17.5
Sugarcane bagasse
Wheat straw
Lignin
%
15
12

13. Effect of acid pretreatment on carbohydrate content of sugarcane
baggase
Sample Sample-Acid used for
Cellulose
Available
Pre-treatment (%) Conversion (%) Substrate (%)
S1
0.5
11.8
88.2
S2
1.0
12.8
87.2
S3
1.5
13.6
86.4
S4
2.0
14.2
85.8
S5
2.5
15.2
84.8
S6
3.0
16.0
84
13

14. (c) Enzyme pre-treatment
150 g of each biomass were suspended in 500 mL H2O in ratio of 3:10
(w/v) sugarcane bagasse and added of 0.1 mL of α-amylase enzyme.
The pH of sample was adjusted at pH 5, 5.5, and 6. The sample was
incubated in water bath 100°C for 30 minutes, after that the mixture
was applied for second enzymatic treatment (0.2 ml of glucoamylase).
Finally, hydrolzsate was pressed through cheese cloth. The amount of
reducing sugar in juice was measured.
pH
5
5.5
6
5
5.5
6
Temperature (°C)
30
30
30
40
40
40
Glucose (%)
23.35
22.80
22.00
21.05
19.43
18.95
Effect of enzyme pre-treatment methods on glucose content of sugarcane baggase
From the Table, it is clear that, increasing pH at 400C showed reverse
effect on glucose concentration .
14

15. (d) Fermentation
The pre-treated samples were carried out for fermentation experiments.
The yeast S. cerevisiae was used for fermentation. After 3 fermentation
days the ethanol content was measured by gas chromatography. S.
cerevisiae was also used with Pitchia stipititis for both the fermentation
of pentose and hexose. Equal amount of both the yeast and P. Stipititis
were taken for the efficient hydrolysis and fermentation of both Pentose's
and hexoses sugar present in the hydrolyzed.
Fermentation Medium:- One litre of production medium was prepared
according to the requirement of S. cerevisiae, containing 50.0 gL -1glucose,
1.0 gL-1yeast extract, 5.0 gL-1KH2PO4, 2.0 gL-1(NH4)2SO4 and 0.4 gL1
MgSO4.7H2O. The medium was sterilized and the pH was adjusted to 5.0.
The Preparation of Inoculums:- The micro-organism was cultured in 250
mL Erlenmeyer flasks, containing 100 mL of the (PDA) medium, which has
the same composition as the fermentation medium. The Erlenmeyer flask
was incubated at 280C for 6 hours on a rotary shaker at 200 rpm.
15

16. Production of Ethanol % (v/v) at 300C and pH 5,from enzyme treated biomass
Biomass (150g)
Sugar (%)
Ethanol (%) by
Sugarcane bagasse
Wheat Bran
23.35
20.74
S.cerevisiae
19.25
17.47
S.cerevisiae & P. stipititis
26.75
21.17
Rape Straw
22.75
18.09
25.48
Rice bran
21.03
17.95
24.28
40
Ethanol (%)
35
30
26.75
Glucose (%
)
25.48
25
24.28
21.17
Ethanol (% by S. cerevisie
)
Ethanol (% by P.Stitipititis and
)
S. cerevisie
20
15
10
Sugercane
bagasse
Rape straw
Wheat bran
Typpes of biomass
Rice bran
16

17. SN.
1
2
3
4
Types of biomass
samples
Ethanol %(v/v) by
S.cerevisiae
S.cerevisiae & P. stipititis
Sugarcane baggase
Wheat Bran
Rape Straw
Rice bran
24.25
21.47
23.95
23.05
35.38
31.25
34.37
33.98
Production of Ethanol % (v/v) at 300C and pH 5 of acid treated biomass
40
35.38
35
34.37
33.98
31.25
Glucose (%
)
30
Ethanol (% by S. cerevisie
)
25
Ethanol (% by P.Stitipititis and
)
S. cerevisie
20
15
10
Sugercane bagasse
Rape straw
Wheat bran
Rice bran
T y p p e s o f b io mas s
17

18. (e) Observation of Kinetic study of 100 gm sugar
SN. Raw Materials
Sugarcane
from Bagasse
Rape
Straw
Rice
bran
Wheat
bran
1
Biomass yield YX/S (gg-1)
0.015
0.014
0.040
0.010
2
Ethanol yield YP/S (gg-1)
0.36
0.29
0.33
0.26
3
Final biomass, (gl-1)
1.49
1.32
1.28
0.92
4
Final ethanol (gl-1)
34.6
26.5
31.5
22.9
5
Substrate utilized, (%)
95.90
90.80
93.52
85.42
6
Fermentation efficiency (% of
theoretical)
99.01
96.5
89.2
84.6
7
Fermentation time, (h)
24
24
24
24
* Theoretical yield based on total sugars is 0.511 gg-1
18

19. Fermentation of 100 gl­-1 sugar by yeast (temp 30°C, pH5) (•) total reducing sugars, () ethanol, ()
biomass.
19

20. Formula used
Specific growth rate (µ) h-1 =
Specific Ethanol productivity (qp) gg-1h-1 =
Specific substrate uptake rate (qs) gg-1h-1 =
Cell Yield, YX/S (gg-1) =
Ethanol Yield, YP/S (gg-1) =
Fermentation Efficiency (%) =
20

21. Enzymes that are able to hydrolyze the cellulose (C6 Sugar) and
hemicelluloses (C5 sugars) These are:Yeast (Saccharomyces cerevisiae ): is able to utilize only hexose.
Z. mobilis: has the ability to decompose both hexose and pentose
Trichoderma resei: produces cellulase enzymes needed to convert
cellulose and hemicellulose in to sugars .
Clostridium thermocellum (C. thermocellum): this bacterium will
convert cellulose directly to ethanol, but has some other byproducts that
can reduce efficiency during fermentation.
Pitchia stipititis : also able to decompose both hexose and pentose
Aspergillus Niger: For saccharification of algal biomass. Aspergillus
Niger is cellulolytic and amylolytic in nature as it produces cellulases and
amylases.
 Fungi- Saccharomyces cerevisiae (Strain 1), Kluyveromyces
marxianus (Strain 2), Candida tropicalis (Strain 3), , a strain of Pitchia
(Strain 4) and Candida krusei (Strain 5).
 Saccharomyces cerevisiae simultaneously combination with Pitchia
stipititis and Pitchia tenofiller for the complete fermentation of sugars.
21

22. (f) Complete process of production of ethanol
BIOMASS
SUGAR & NUTRIENTS
Handling
Enzyme
Production
Grinding
Pretreatment
CELLULOSE
LIGNIN
Inoculation
Enzymatic
Hydrolysis
Fermentation
XYLOSE
Distillation
Acid Pretreatment
ETHANOL
22

23. (4) ADVANTAGES OF BIOETHANOL
Environmental feasibility: Befouled are biodegradable and far less toxic
that fossil fuels, Benefit over fossil fuels is the greenhouse gas emissions
reduced.
As motor fuel: The principle fuel used as a petrol substitute is bioethanol in
terms of E85, E10. The most common blend is 10% ethanol and 90%
petrol .Blending bioethanol with petrol will ensure greater fuel security,
avoiding heavy reliance on oil producing nations.
Calorific value: Although the Gross calorific value of ethanol is
(29,700kj/kg) lower than petrol (48,000kj/kg) and diesel (44,800 kj/kg),
yet is less toxic than bothers. Ethanol is a high octane fuel and has
replaced lead as an octane enhancer in petrol , also it has low tendency to
create knocking in spark engines.
Minimum expenses: The cost of Ethanol production from lignocelluloses is
approx Rs.55/- per liter at this time but in a large scale and using modern,
techniques it will be minimized.
23

24. Fuel properties of anhydrous ethanol and comparison with petrol and
diesel fuel
Property
Composition, weight %
C
H
O
Density, kg/m3
Lower heating value, MJ/kg
Octane number
Cetane number / n-Heptane
Rapid vapour pressure (kPa)
Stoichiometric air/fuel ratio, weight
Boiling temperature, °C
Flash point, closed cup, °C
Ethanol
52.2
13.1
34.7
794
26.7
100
8
15.6
9:1
78
13
Petrol
Diesel
85-88
12-15
0
750
42.9
85-90
5-15
55-103
14:1
80-225
-42
84-87
13-16
0
825
43
30-40
1.4
16:1
188-343
74
Sources: JEC, 2005; Joseph, 2007
24

25. Effects of Blend on Octane Rating
120
115
Octane Number
110
105
100
95
90
85
80
75
0
20
40
60
80
100
Volume % Ethanol
Research Octane No.
Motor Octaane No.
As the ethanol blending (% of ethanol) increases in the gasoline,
Octane rating becomes slightly higher. From the figure the Motor
Octane number was found maximum for 100% blend, 112. While
Research Octane number was found 118 on 100% blending (Bailey
25
and John Russell ).

26. 5- Conclusion & Effective Parameters
(a) Effect of sugar concentration: Ethanol Production slightly decreases with
increase in sugar concentration . It can be concluded that it is possible to
successively use sugarcane bagasse, wheat bran and rape straw for
bioethanol. Enzyme treatment at 30ºC and pH 5 is an effective treatment
method for converting biomass to glucose.
(b) Effect of temperature: Ethanol production is significantly reduced by
increasing the temperature .
(c) Effect of Ph: In case of different pH ethanol fermentation is more
favourable at pH 5-6.
(d) Effect of nutrient supplementation on ethanol production: In order to
improve the bio ethanol production nitrogen source in fermentation
medium such as NaNO3 , KNO3 enhances the enzymatic growth as well as
fermentation.
(e) Effect of Inoculums size: Enzymatic growth and Ethanol production is
significantly increases by greater inoculums size.
26

27. 6-Major concerns/problems in bio ethanol production from
cellulosic materials
 The ability to ferment pentose (five-carbon sugars), especially
xylose and arabinose, into ethanol is important for the efficiency and
economics of the process.
 Recently, special microorganisms have been genetically engineered
which can ferment these sugars into ethanol with relatively high
efficiency.
 Formation of inhibitors is also the effective parameter which
decreases the ethanol productivity. Inhibitors decreases the
enzymatic growth.
 Some of the methods which are required to enhance the production
of ethanol from biomass are: Evaporation, Extraction with organic
solvents and Adsorption on activated charcoal, molecular sieves,
Neutralization, Alkaline Detoxification .
27

28. Future Prospects
1.
2.
3.
4.
5.
The production of ethanol from lignocellulosic materials can be made
cost-effective and done in large scale provided the following
conditions are satisfied:
Raw materials can be produced insufficiently large amounts and
costs of production and collection are acceptable.
Pretreatment of lignocellulosic materials is cost-effective.
High yields of ethanol from hexoses and pentose's are attainable.
Environmental pollution due to the process is minimized.
If bio fuels continue their rapid growth around the globe, the impact
on the agricultural sector can be significant. Increased jobs and
economic development for rural areas in both industrialised and
developing countries is one possibility, if governments put the
appropriate policies in place and enforce them. The more involved
farmers are in the production, processing, and use of bio fuels, the
more likely they are to benefit from them.
28

29. A Case Study
Total Biomass (Agricultural Residue) and projected biofuel (Ethanol) production
upon total Area (In thousand hect.) of main crops in Uttar Pradesh (2012-13).
S.N
Agricultural
Residue
Biomass
(dry
ton/acre/y
ear)
1-3
EtOH
Yield
(liter/dry
ton)
333
EtOH Yield
(liter/acre/year)
Wheat Straw
Agricultural
area
(In Thousand
Hect.)
9485
1.
2.
Rice Straw
4372
3-4
335
1173
3.
Barley Straw
58
3-5
345
1380
4.
Sugar cane
Bagasse
Maize Straw
1970
5-6
360
1980
274
3-4
345
1208
Rape (Sarson)
Straw
622
3-5
355
1180
5.
6.
666
Biomass data Source: U.P. all agricultural sankhyikiya spatrika
29

30. Benefits of Biofuels:
Using alternative energy is better for the envoirment.
Using homegrown energy is better for health.
Using Biofuels is better for the (Rural) economy.
It makes us less dependent on foreign imports.
 Private Sector Investments
 Rural Employment
 Reduction in Petroleum Imports
 Energy Security
 Earnings from Carbon Credits
dr.pratap2012@gmail.com
30

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