Bioethanol production
from hemi-cellulosic
sugars of rice straw by
Pichia Stipitis
HARIKA VARANASI
12031D9619
Content
s
Introduction
Bioethanol and its production
Rice straw, its components and
pretreatments
Enzyme production
Saccharification of rice straw
Fermentation
Results
Applications
Conclusion
Bioethanol
Bioethanol is an alcohol made from fermentation. It refers to
ethyl alcohol produced from microbial sources as apposed to
synthetically produced ethanol from petrochemical sources.
Bioethanol Production
Bioethanol can mainly be produced in 3 ways:
1. Feedstock rich in sugar
2. First generation process technology:
Starch
Sugar
Ethanol
3. Second generation process technology:
Cellulose and Hemicelluloses
Ethanol
Rice
Straw
Lignocellulose
materials
represent
a
promising option as feedstock for ethanol
production
Rice straw is one of the most abundant
lignocellulose waste materials in the world.
Annually 667.6 million tons of rice straw is
produced in Asia. It can potentially produce
205 billion liters bioethanol per year
Rice straw components
The major components of rice straw are :
Cellulose
Hemicelluloses
Lignin
Silica
Ash
The first two are composed of chains of sugar
molecules. These chains can be hydrolyzed to
produce monomeric sugars, some of which can
be fermented using ordinary bakers yeast.
Structure of major
components
Structure of Cellulose Based on Walker (2010)
Structure of hemicelluloses Based on Walker (2010)
Structure of Lignin
Based on Walker (2010)
Rice Straw Pretreatments
A major problem in the processing of
lignocellulose materials is the natural resistance
of these materials to the conversion process
required to generate fermentable sugars due to
the presence of lignin and the degree of
crystallinity of cellulose.
Pre-treatment is responsible to separate the
components of the lignocellulose biomass,
reducing the crystallinity of the material, making
the cellulose accessible, and removing the lignin
(Sun and Cheng 2002).
Enzyme Production
Saccharification
Enzymatic hydrolysis (i.e., Saccharification) is
one of the major bottlenecks due to the
recalcitrance of plant cell wall.
In the present study Trichoderma ressei and
Aspergillus niger are used
Enzymes involved in the
Saccharification step
1. Cellulose acting enzymes
Different characteristics of the cellulose influence the efficiency of enzymatic hydrolysis
such as the degree of polymerization (DP), crystallinity, particle size , and surface area.
2. Hemicellulose acting enzymes
Hemi cellulose hydrolysis requires the intervention of several types of enzymes with
complementary activities working at different levels of the hemicelluloytic network,
either on the main chain of sugars or to disconnect substitutions.
These enzymes act on rice straw to produce the desired sugars needed for conversion
to bioethanol.
Fermentation
Ethanol fermentation using the hydrolysate obtained after the
saccharification of biomass is the last step in lignocellulosic
bioethanol production process.
The hydrolysate contains large amount of fermentable sugars that
can be directly used by the ethanologenic microorganisms.
The major characteristics of an organism to be used in ethanol
production are the ability to give a high yield of ethanol, to produce
it with a high productivity and to withstand high ethanol
concentration. In addition, the organism should possess the ability
to utilize multiple sugars as well as that to tolerate inhibitors that
are usually present in the hydrolysate obtained after pre-treatment
and enzymatic saccharification.
In the present study pichia stipitis has been used for the
fermentation process.
Glucose standard by DNS
Method
Concentration (g/ml)
OD at 540nm
200
0.22
400
0.39
600
0.55
800
0.69
1000
0.85
Glucose standard by
D.N.S
Method
Protein Standard
Concentration (g/ml)
OD at 595nm
200
0.12
400
0.22
600
0.33
800
0.42
1000
0.52
Protein standard by Bradford's
method
Phenols by FC Reagent
Concentration(g/ml)
O.D at 750nm
200
0.368
400
0.810
600
1.135
800
1.471
1000
1.874
Phenol Standard Curve
Ethanol standard by Potassium
Dichromate method
Ethanol (%)
Absorbance at 590nm
0.12
0.25
0.36
0.48
10
0.59
Ethanol Standard Curve by
Potassium Dichromate method
Assay for Xylanase
Activity
DAY
UNITS(IU/ml)
PROTEIN(g/ml)
14.6
119
2.0
147
4.3
162
6.6
418.2
24.0
462.9
43.3
462.9
22.3
332.9
26.6
310.5
Xylanase Activity
The Xylanase activity was minimum on second day and
reached maximum on sixth day.
Assay for Cellulase
activity
DAY
UNITS(IU/ml)
PROTEIN
(g/ml)
0.2
119.0
0.5
147.0
2.7
162.0
6.1
418.2
4.4
462.9
2.7
462.9
0.5
332.9
3.5
310.5
Cellulase Activity
The highest cellulase activity was observed on fourth day of production.
Assay for Fpase activity
DAY
UNITS(IU/ml)
PROTEIN
( g/ml)
4.6
119.0
11.1
147.0
13.8
162.0
12.7
418.0
13.3
462.9
11.6
462.9
11.1
332.9
10.5
310.5
Fpase Activity
The highest Fpase activity was observed on third day of production.
Holocellulose Content
By using the TAPI methods the cellulose, Pentosans and
lignin contents were determined and their percentages
are as follows:
% of
lignocelluloses
Amount
Lignin
12%
Pentosans
22.5%
Cellulose
39%
Phenols Studies
Rice straw was used as a
lignocellulosic source to provide
rich pentose media. The result
obtained
from
the
hemi
cellulosic hydrolysate (based on
phenol standard graph) is as
following:
The water hydro lysate contains
5.2 grams of phenol per liter
The acid hydro lysate contained
6.3 grams of phenols per liter
Ethanol production
studies
Time(h)
Reducing sugars(mg/ml)
ETHANOL (%)
6.4
16
4.3
0.8
24
2.1
1.6
36
1.5
1.9
48
0.7
2.5
Ethanol production
graph
Applications
Transport fuel to replace gasoline
Fuel for power generation by thermal
combustion
Fuel for fuel cells by thermochemical
reaction
Fuel in cogeneration systems
Feedstock in the chemicals industry
Conclusion
In recent years it has been investigated that, instead of traditional feed
stock (starch crops), cellulosic biomass, including forest and industrial
residues, agriculture waste and municipal waste, could be used as an
ideally inexpensive and sufficient amount of sugar for production of
This preliminary study showed that ethanol production (second
generation of bioethanol) from rice straw is possible by intact fungal
organisms as source of cellulase and hemicellulase enzymes through
fermentation using Trichoderma.R and Aspergillus. This was followed
by hydrolysate fermentation by Pichia Stipitis through separate
hydrolysis and fermentation (SHF) process.
The use of rice straw is cheap, renewable source of energy
(fermentable sugars) to produce ethanol via sustainable technology
promise with great future. The ethanol yield in our study was 2.5 %.