Dept. Agroindustrial Technology,

Faculty of Agricultural Technology, Udayana University, Bali.

Outline of Presentation

Introduction

Objective of this study

Materials and Methods

Results and Discussion

Conclusion

INTRODUCTION

Bioethanol is a necessary commodity in the present and in the

future and to increase the number of significantly because many the

raw materials that can be used to bioethanol production.

Lignocellulosic biomass will undoubtedly play an important and

increasing role in our future energy.

To achieve enzymatic degradation in production of ethanol by

enzymatic hydrolysis, a pretreatment process is necessary

Classified into physical pretreatment, physico-chemical pretreatment,

chemical pretreatment, and biological pretreatment

Chemical as a solvent: NaOH, KOH, NH3, H2O2, H2SO4, etc.

Enzyme from fungi potential use as catalys for saccharification

before fermentation: A. niger, T. viride, T. resei., etc.

Therefore, in this research will develop a production method that can

improve the efficiency of bioethanol production from

lignocellulose waste especially in pretreatment and saccharification

processes.

Bioethanol Production

Sugary waste

cooking

Starch

Saccarification

(light hydrolysis)

Fermentation

& purification Silage

Flowchart of bioethanol production

Pretratment

Lignocellulosic

Saccarification

(heavy hydrolysis)

BIOETHANOL

OBJECTIVES OF THIS STUDY

P

To find out a source of lignocelluloses

that could be converted to simple

sugars especially glucose as a raw

material in bioethanol production.

To find out the appropriate of chemical

pretreatment of delignification

processes.

Comparison of saccharification process

of a lignocellulosic substrate using

crude enzyme of Aspergillus niger FNU

6018 and Trichoderma viride FNCC

6012.

MATERIALS AND METHODS

P

Slant medium

PDA

Glycerol

stock

1. Aspergillus niger 2. Trichoderma viride

Culture, Media & Chemicals

Equipment

Step of Bioethanol Production from Lignocellulosic Materials

Cellulase

enzyme

Drying & Size

reduction

Chemical

Pretreatment

Saccarification

(hydrolysis)

Fermentation

Distilation

Flowchart of bioethanol production from lignocellulosic materials

Lignocellulosic

materials

Refresh & enzyme

production

BIOETHANOL

Glycerol

stock

P

Pretreatment

• Material size reduction

• Delignification process

• Variation concentration of chemical & selected

materials

Saccharification with fungi

Preparation of A. niger & T. viride

Selection of substrate for cellulase enzyme production

Production of cellulase enzymes

Harvesting and enzyme activity testing

Comparison of saccharification process between

crude enzymes of A. niger & T. viride

Harvesting the saccharification products

Determination of solvent concentrations was performed by soaking of

powdered cellulosic material each of 5 g in a chemical solution selected at a

concentration of 2%, 4%, 6% and 8% (b / v) solution in beaker glass with a

ratio of 1: 15 (w / v) for a certain time (based on preliminary experiments

obtained 8 hours soaking time) at room temperature.

After filtration, washing until neutral and drying at oven temperature 105oC to

constant weight, analyzed the physicochemical properties of the material after

the delignification process.

Variation concentration of chemical solvents and selected materials

Selection of chemicals for the delignification process

Lignocellulosic waste was soaked with NaOH, Ammonia and H2O2 solutions at

a certain concentration (2%). Each type of lignocellulosic waste was weighed 5

g, then put into a beaker glass and each given the above chemical solution at a

ratio of 1:15 (w / v), soaked for 8 h at room temperature. After filtration,

washing until neutral & drying in the oven at 105°C to constant weight.

Pretratment of lignocellulosic materials

Refresh & seed culture reparation

slant medium

Seeds culture

Incubation at

30oC, 4 days,

150 rpm

Refresh in PDA;

incubated at

30oC, 4 days Glycerol

stock Incubation at

30oC, 7 days,

150 rpm

Assay of CMC-ase & FP-ase Activity

Selection of substrate for cellulase enzyme production

Each of the 10 ml of sterile aquades was added to Aspergillus niger and

Trichoderma viride cultures on the agar medium, then shaken to release the

spores into the liquid phase. Then each 1% (w / v) substrate bagasse, corn

straw, rice straw and sawdust put in a 100 ml Erlenmeyer and added a solution

of nutrients and minerals in the ratio 1: 1 to the substrate.

Production, harvesting and enzyme activity testing

Each of the 10 ml of sterile aquades was poured into Aspergillus niger and

Trichoderma viride cultures in a slank medium, then shaken to release the spores

into the liquid phase. Then each spore suspension (10% w/v) was transferred into

a 100 ml Erlenmeyer containing a substrate of 1% (w / v) and a sterile nutrient

solution. The media used is the same as the one used for substrate selection.

The fermentation product in Erlenmeyer are stirred and shaken, then filtered with

filter paper. Filtrate that has been separated from its substrate is a crude enzyme

and ready to be analyzed. The crude enzymes were tested for their activities: test

of endoglucanase / CMC-ase enzyme activity and filter paperase / FP-ase .

Saccharification process

The best pretreated materials

Added aquades until 200 ml

Added citrate buffer (0,05M, pH 4.8, 100 ml)

Weighing 2 g

Incubated at 50oC for 96 h

pH adjusted by citrate solution

Added cellulase enzyme (15 FPU/g)

Filtration

Supernatant (glucose)

Flowchart of saccharification process

Residue

Analysis

Analysis of water content (Sudarmadji, 1984)

Analysis of hot water soluble fraction (LAP), cellulose, hemicellulose,

lignin (Chesson, 1978)

Swelling analysis or water retention value (Lee and Fan, 1982)

Reduction sugar content Nelson-Somogyi method

Testing of endoglucanase activity (Darwis and Sukara, 1990).

Testing activity of filter paperase (Varga et al., 2004)

Measurement of microbial growth by spectrophotometer (Gunam et al.,

2006).

Analysis Procedure

Gambar 1. Lignocellulosic feedstock after physical pretreatment: drying and

size reduction with a milling (BG = Bagasse; CS = Corn Straw; PS = Paddy

Straw; SD = Sawdust)

RESULTS AND DISCUSSION

Physical pretreatment

Selection of Raw Materials and Chemical Prereatment

0

10

20

30

40

50

60

70

Control Soaking withNaOH 2%

Soaking withNH3 2%

Soaking withH2O2 2%

Am

ou

nt

(%)

CS = Corn Strow Moisture content (%)

Water retention value (WRV)

Hot water soluble (%)

Hemicellulose (%)

Cellulose (%)

Lignin (%)

Ash (%)

Chemical solvent:

1. NaOH

2. NH3

3. H2O2

Lignocellulose materials:

1. Bagasse

2. Corn straw

3. Paddy straw

4. Sawdust

Selection of Raw Materials and Chemical Solutions for Prereatment

Figure 2. Effect of chemical treatment on lignoselusa material on its

characteristics: BG = Bagasse; CS = Corn Straw; PS = Paddy Straw; SD =

Sawdust. Each type of lignocellulosic waste was weighed 5 g, then put into a

glass cup and each given the above chemical solution (2%) at a ratio of 1:15 (w

/ v), soaked for 8 hours at room temperature.

0

10

20

30

40

50

60

70

Control Soaking withNaOH 2%

Soaking withNH3 2%

Soaking withH2O2 2%

Am

ou

nt

(%)

PS = Paddy Strow Moisture content (%)

Water retention value(WRV)Hot water soluble (%)

Hemicellulose (%)

Cellulose (%)

Lignin (%)

Ash (%)

0

10

20

30

40

50

60

70

Control Soakingwith NaOH

2%

Soakingwith NH3

2%

Soakingwith H2O2

2%

Am

ou

nt

(%)

SD = Sawdast Moisture content (%)

Water retention value(WRV)

Hot water soluble (%)

Hemicellulose (%)

Cellulose (%)

Lignin (%)

Ash (%)

Chemical and Raw Materials selected treatment

Figure 3. Characteristic substrate of bagasse powder (A) and corn straw powder (B)

after soaking with 2, 4, 6 & 8% NaOH for 8 hours at room temperature.

0

10

20

30

40

50

60

70

80

0 2 4 6 8 10

Am

ou

nt

(%)

NaOH concentration (%)

A

Moisture content (%)

Hot water soluble (%)

Hemicellulose (%)

Cellulose (%)

Lignin (%)

Ash (%)

Selected kinds of substrate on cellulase enzyme production (CMC-ase)

Figure 4. Comparison of CMC-ase activity of Aspergillus niger (A) and Trichoderma

viride (B) on different substrates. The production of crude cellulase enzyme from molds

was tested with 4 different substrates: bagasse (BG), corn straw (CS), paddy straw (PS)

and sawdust (SD). Measurement of CMC-ase activity was performed after 5 and 7 days

of fermentation at 30○C

Endoglucanase Activity (CMC-Ase) of Aspergillus niger & Trichoderma viride

0.000

0.005

0.010

0.015

0.020

0.025

0.030

BG CS PS SD

Un

it (

U)

A. FP-ase activity of Aspergillus niger Day 5

Day 7

Figure 5. Comparison of FP-ase activity of (A) Aspergillus niger and Trichoderma

viride (B). The production of crude cellulase enzyme from molds was tested with 4

different substrates: bagasse (BG), corn straw (CS), paddy straw (PS) and

sawdust (SD). Measurement of FP-ase activity was performed after 5 and 7 days

of fermentation at 30○C.

Filter paperase activity (FP-Ase) of Aspergillus niger & Trichoderma viride

Selected kinds of substrate on cellulase enzyme production (FP-ase)

The comparison of saccharification process of lignocellulosic substrates between A. niger & T. viride

Figure 6. Time course of saccharification of bagasse by crude cellulase

enzyme from A. niger and T. viride at room temperature during 96

h incubation.

CONCLUSION

The NaOH solution is still the most effective chemical reducing lignin

content, increasing water absorption (water retention value) and

cellulose content of the treated chemical lignocelluloses.

Bagasse and corn straw are potential agricultural waste to be used as

raw material for the production of bioethanol based on their

characteristics after being given treatment delignification which the

highest cellulose content of 69.46, the lowest lignin 8.79, and higher

water retention value of 8.42.

The saccharification process obtained by the crude enzyme from

Aspergillus niger is better activity than the enzyme obtained from

Trichoderma viride with its respective activities as follows for the FP-

ase activity of 0.0226 U (bagasse as a substrate) and the CMC-ase

activity of 0.0606 U (corn straw as a substrate).

Clean air