Bioethanol production from hemi-cellulosic sugars of rice straw by Pichia Stipitis HARIKA VARANASI

12031D9619

Contents

•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 ProductionBioethanol 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 componentsThe 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 baker’s yeast.

Structure of Cellulose Based on Walker (2010)

Structure of hemicelluloses Based on Walker (2010)Structure of Lignin Based on Walker (2010)

Structure of major components

Rice Straw Pre-treatments

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 (µg/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 (µg/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(µg/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

2 0.12

4 0.25

6 0.36

8 0.48

10 0.59

Ethanol Standard Curve by Potassium Dichromate method

Assay for Xylanase Activity

DAY UNITS(IU/ml) PROTEIN(µg/ml)

1 14.6 119

2 2.0 147

3 4.3 162

4 6.6 418.2

5 24.0 462.9

6 43.3 462.9

7 22.3 332.9

8 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

(μg/ml)

1 0.2 119.0

2 0.5 147.0

3 2.7 162.0

4 6.1 418.2

5 4.4 462.9

6 2.7 462.9

7 0.5 332.9

8 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

( μg/ml)

1 4.6 119.0

2 11.1 147.0

3 13.8 162.0

4 12.7 418.0

5 13.3 462.9

6 11.6 462.9

7 11.1 332.9

8 10.5 310.5

Fpase Activity The highest Fpase activity was observed on third day of production.

Holocellulose Content

% of lignocelluloses Amount

Lignin 12%

Pentosans 22.5%

Cellulose 39%

By using the TAPI methods the cellulose, Pentosans and lignin contents were determined and their percentages are as follows:

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 (%)

0 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 %.