TEKNOLOGI BIOETANOL BERBASIS LIMBAH LIGNOSELULOSA … · TEKNOLOGI BIOETANOL BERBASIS LIMBAH LIGNOSELULOSA Ina Winarni Pusat Penelitian dan Pengembangan Keteknikan Kehutanan dan Pengolahan

28/11/2014

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TEKNOLOGI BIOETANOL BERBASIS LIMBAH LIGNOSELULOSA

Ina Winarni

Pusat Penelitian dan Pengembangan Keteknikan Kehutanan dan Pengolahan Hasil Hutan Kementrian Lingkungan Hidup dan Kehutanan

Bogor

• Chapter 1. Pendahuluan

• Chapter 2. Amphipathic lignin derivatives dari lindi hitam

sebagai limbah proses pulping

• Chapter 3. Hidrolisis enzimatis pulp sagu dengan penambahan

SSL-derivatives sebagai surfaktan

• Chapter 4. Penutup

Contents

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Contents







High level pollution

Developing biomass as an alternative feedstock to biofuels : • Abundant, produced about 10-50 billion tons/year • Renewable

Chapter 1. General introduction

Petroleum, coal, natural gas, etc

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starch

1st generation

• Bioethanol

• Simple process to produce bioethanol • But, compete with production of daily food.

raising the price of the food feedstock

2nd generation

Agricultural and forestry residues (waste)

• Never compete with food production. • Produced from all kinds of plant.

Effective utilization of unused lignocellulose

lignocellulose

Fibrous sago waste from sago mills

Promising lignocellulosic for bioethanol production

Sago palm

• Sago palm

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1. Acid hydrolysis 2. Enzymatic hydrolysis

• Bioethanol of lignocellulose

•Fermentation

•Distillation

Bioethanol

Advantages : •Fast process •Cheap reagent

Disadvantages :

•Corrosive •High cost of maintenance

Advantage : •Mild condition • Environmental

friendly

Disadvantage : •High cost of enzyme

•Pretreatment

Glucose

Lignocellulose

Advantage : •Mild condition • Environmental

friendly

Disadvantages : •High cost of enzyme

•Pretreatment

1. Acid hydrolysis

Advantages : •Fast process •Cheap reagent

Disadvantages :

•Corrosive •High cost of maintenance

• Enzymatic hydrolysis or saccharification

Delignification Without delignification,

enzymatic saccharification is very low (< 20%)

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Pretreatment

Alkaline pulping

Physical (crushing or milling)

Biological (white-rot fungi)

Chemical (alkaline,acid)

Combined method (steam explosion +

alkaline)

Enzymatic

saccharification

• Delignification for substrates

Raw materials lignocellulosic

Delignified pulp

・ CBH I (Cel7A) Mw : 59-68 kDa pI : 3.5-4.2

・ CBH II (Cel6A) Mw : 50-58 kDa pI : 5.1-6.3

・ EG I (Cel7B) Mw : 50-55 kDa pI : 4.0-6.0

・ EG II (Cel5A) Mw : 48 kDa pI : 5.5

・ BGL (Cel3A)

Mw : 75 kDa pI : 8.7

Cellobiohydrolase (CBH) …hydrolyze cellulose to cellobiose

Endoglucanse (EG) …hydrolyze randomly internal glycosidic bonds

β – Glucosidase …hydrolyze cellobiose to glucose

• What is the enzyme used for saccharification ? BGL

Non reducing end Reducing end

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11 cellulose

lignin lignin

Cellu-lase

Cellu-lase

Cellu-lase

Cellu-lase

Cellu-lase

Cellu-lase

Cellu-lase

Cellu-lase

Cellu-lase

Cellulase associates with cellulose by the specific interaction. Cellulase associates with lignin by hydrophobic interaction.

Normal saccharification (no additives)

• Why cellulase activity decreased during saccharification ?

One of obstacles in enzymatic saccharification is irreversible adsorption of cellulase to cellulose

• Immobilized cellulase

Immobilized

enzyme

Polymer

support

E

P

P

(solid)

E

E

S

S

S

Water-soluble immobilized

enzyme

E S S S S

P P P

(solid)

E: Enzyme, S: Substrate, P: Product

E E

polymer support

(soluble) Amphiphilic lignin derivative

Cellulose

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• Positive effect of surfactant as a support of water immobilized enzyme

1

• Faster hydrolysis rate and enabling lower enzyme dosage. (Cantanon and Wilke, 1981;

Ooshima et al, 1986; Helle at al, 1998; Zheng et al, 2008)

2 • SSF (Simultaneous saccharification

and Fermentation). (Alkasrawi et al, 2003)

3 • Re-use of cellulase is possible.

(Otter et al, 1989; Tu et al, 2007)

Developed amphipathic lignin derivatives as alternative surfactant from lignin

14 cellulose lignin lignin

Cellu-lase

Cellulase

Cellu-lase

Cellulase

Cellu-lase

■ It is expected that lignin derivatives promotes the release of cellulase from substrate.

With lignin derivatives Possible to repeat a cellulase hydrolysis

•Hypothesis on the mechanism of cellulase saccharification using lignin derivatives

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0

40

60

80

100

20

5 4 3 2 1

Reaction time (times)

Polyhydric alcohol

(PHA) pulp

Hydrolysis conditions: Substrate, 3 g; Cellulase, 240 mg; PEGDE-AL, 0 wt.% (●) and 0.2 wt.% (●) based on 300 mL of buffer solution

Y. Uraki, N. Ishikawa, M. Nishida, Y. Sano (2001) J. Wood Sci. 47, 301. E

ffic

ien

cy o

f sa

cch

ari

fica

tio

n (%

)

PEGDE-AL is an amphipathic lignin derivative prepared from acetic acid lignin(AL) and polyethylene glycol derivative.

80%

14%

• Repeated cellulase saccharification

(No additive)

(With PEGDE-AL)

PEGDE Polyethylene glycol diglycidylether

13

• Objectives of the research

To synthesize amphipathic lignin derivatives from sago lignin

1)

To evaluate the effect on the sago lignin derivatives to the saccharification of sago pulp

2)

To examine the possibility of re-use of cellulase that recovered from hydrolysate of sago pulp saccharification

3)

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Contents







Sago (Metroxylon sp.)

Starch from sago plants used as various types of dishes or as main daily food in Maluku and Papua provinces.

•Introduction

One of Indonesian native plants Food and energy

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(Irian Jaya) 1,206,469 ha

(Pariaman) 95,790 ha

(Maluku) 50,000 ha

(Sulawesi) plantation 30,000 ha

(Riau) plantation 20,000 ha

(Mentawai) 56,000 ha

(Borneo) 2,795 ha

Sago potency : 1.25 million ha

•Introduction

Pretreatment (4% HCl; 80°C; 1 h)

Fibrous sago waste

Black liquor

Sago soda pulp (substrate) (Yield = 30-40%)

Sago soda Lignin (SSL)

Enzymatic hydrolysis (GC220 ,10 FPU/g substrate; 48 h)

glucose

Starch-free sago waste

Epoxylated PEGs

Amphipathic lignin derivatives

(Yield = 51.2%)

Ultrafiltration

Soda pulping (wood to liquor ratio = 1 : 5); 165° C (heating time: 2h;

holding time: 1.5 h)

(adjust to pH 4; filtration freeze-drying)

NaOH 15% KL : 10.2%

NaOH 20% KL : 1.8%

1.

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1. Preparation of sago soda pulp

pulp

Fibrous sago waste (9.7% starch); 4% HCl (aq) to remove and recovered the starch

Black liquor

Filtration

Washing

Centrifugation

Dry

Unbleached Sago pulp

Starch free sago waste

Alkaline pulping

Pulp


Fibrous sago waste

Black liquor




glucose


Epoxylated PEGs


(Yield = 51.2%)

Ultrafiltration




2.

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2. Preparation of sago soda lignin (SSL)

Fig. Separation scheme of sago soda lignin

Black liquor

Concentration Precipitation until pH 2

Evaporator

Centrifuge and washing

Freezing

Lignin(SSL)

Freeze- dry

1) PEGDE Polyethylene glycol diglycidylether

2) EPEG ethoxy-(2-hydroxy)-propoxy-polyethylene glycol glycidylether

3) DAEO dodecyloxy-polyethylene glycol glycidylether

SSL +

Polyethylene glycol derivatives

Homma et al, 2010; J. Wood Chem. Technol. 30 : 164-174

13

13

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2. Preparation of (PEGDE-; EPEG-; DAEO-) SSL

SSL and 1 M NaOH

PEGDE, EPEG, DAEO added to the solution

Stirring at 70oC for 2 hr

1) pH 4 with AcOH

2) Ultrafiltration

3) Lyophilization

Stirring 24 h

• Characterization of lignin derivatives-SSL

Amphipathic SSL derivatives (PEGDE-; EPEG-; DAEO-SSL) Dissolve in water or amphipathic

PEGDE-SSL (13, 1 )

EPEG-SSL (13, 1.5)

DAEO-SSL (3) (n,m)

n : the number of EO unit

m : charge ratio of PEG (g) to

1 g of SSL

Checking the solubility in water

Du Nouy

surface tensiometer

Surface tension

Water (at 20℃)：72.8 mN/m

aqueous solution of commercial surfactant：30~40 mN/m

Intersection point of 2 approximate line

・・・・・critical micelle concentration (CMC)

The lower CMC, the better surfactant

Log concentration

Surface

tension

3. Measurement of surface activity

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20

30

40

50

60

70

80

1.E-06 1.E-04 1.E-02 1.E+00

Su

rfa

ce t

en

sio

n （

mN

/m）

Concentration （g/mL)

PEGDE-SSL(13,1)

10-6 10-4 10-2 120

30

40

50

60

70

80

1.E-06 1.E-04 1.E-02 1.E+00

Su

rfa

ce t

en

sio

n （

mN

/m）


EPEG-SL(13,1.5)

10-6 10-4 10-2 120

30

40

50

60

70

80

1.E-06 1.E-04 1.E-02 1.E+00

Su

rface

ten

sion

（m

N/m）


DAEO-SSL(3)

10-6 10-410-2 1

• Surface Activity

CMC: n.d Surface tension: n.d

CMC: 1.3 x 10-2 g/mL Surface tension: 40 mN/m


PEGDE-SSL < EPEG-SSL <<DAEO-SSL

Lower critical micelle concentration (CMC) and lower surface tension indicate higher surface activity.


PEGDE-AL < EPEG-AL < DAEO-AL


20

30

40

50

60

70

80

Concentration (g/mL)

Surf

ace

tensi

on (

mN

/m)

10-6

10-4

10-2

10


20

30

40

50

60

70

80


Surf

ace

tensi

on (

mN

/m)

10-6

10-4

10-2

10


20

30

40

50

60

70

80


Surf

ace

tensi

on (

mN

/m)

10-6 10

-410

-21

0

DAEO-AL(6)


EPEG-AL(13,4) PEGDE-AL(13,1)

Contents • Chapter 1. Pendahuluan






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Fibrous sago waste

Black liquor




glucose


Epoxylated PEGs


(Yield = 51.2%)

Ultrafiltration




NaOH 20% KL : 1.8%

NaOH 15% KL : 10.2%

3.

• Saccharification of unbleached sago pulp

★measurement of

Saccharification efficiency

Cellulase (Genencor GC 220)： 10 FPU/g of pulp

Buffer solution: 50 mL (pH 4.8 citrate buffer )

amphipathic lignin derivative (PEGDE-SSL, EPEG-SSL, DAEO-SSL)： 0.05 g

Substrate (unbleached sago pulp): 0.5 g (1.8% and 10.2% lignin content)

ultrafiltration filtration

filtrate Glass

Filter Sugar solution

←pressure

Remove the monosaccharide and other low molecular compound

remained cellulase solution

Incubating for 48 h at 50°C

Sugar content by HPLC

Saccharification efficiency (%) = WS – WR

WS WS : weight of substrate WR : weight of residue

× 100

Washed and reuse until 4 times, check the residual activity using FPU method

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■DAEO-SSL showed the highest SE among the others.

■ All lignin derivatives showed higher efficiency than without additive .

Run (times)

Sacc

har

ific

atio

n e

ffic

ien

cy (

%)

No additive

12%

With PEGDE-SSL

81%

No additive

12%

With DAEO-SSL

86%

Run (times) Run (times)

No additive

12%

With EPEG-SSL

82%

Lignin content 1.8%

• Saccharification efficiency of sago pulp

Lignin content 10.2%

Run (times)

Sacc

har

ific

atio

n e

ffic

ien

cy (

%)

No additive

3%

With PEGDE-SSL

72%

Run (times)

3%

No additive

With DAEO-SSL

69%

Run (times)

3%

No additive

With EPEG-SSL

71%

■ All lignin derivatives showed higher SE than without lignin derivatives

■PEGDE-SSL showed the highest efficiency among others

• Saccharification efficiency of sago pulp

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Res

idu

al a

ctiv

ity

(%)

1.8 % Lignin content 10.2 % Lignin content

■ DAEO-SSL showed the highest residual cellulase activity after 4 times saccharification for unbleached sago pulp (1.8% lignin content).

■ EPEG-SSL showed the highest residual cellulase activity after 4 times saccharification of unbleached sago pulp (10.2% lignin content).

• Residual cellulase activity after 4 times saccharification

• SE of unbleached sago pulp was improved by the addition

of amphipathic lignin derivatives, particularly DAEO-SSL. • The recovered cellulase in the presence of the lignin

derivatives was able to be used again and maintained until 4 times saccharification.

• DAEO-SSL showed a superior performance to others

derivatives; can maintain cellulase activity particularly for pulp with lignin content of 1.8%.

It is possible to re-use cellulase for enzymatic

saccharification of unbleached sago pulp.

Chapter 4. Penutup

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Acknowledgement

1. Prof. Dr. Uraki Yasumitsu

2. Dr. Keichii Koda

3. All my lab mates during my doctoral course

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・Different types of commercial cellulases (no additives)

substrate Meicelase (unit/ FPU)

GC 220 (unit/ FPU)

CMC (EG)

67.0 162.8

p-nitrophenyl lactoside

(CBH)

1.4

3.5

p-nitrophenyl glucoside

(β-glucosidase)

8.2 2.7

Effi

cien

cy o

f sa

cch

arif

icat

ion

(%

)

51.8

71.0

57.9

71.8

0

20

40

60

80

100

120

Meicelase GC220 10 FPU 20 FPU 10 FPU 20 FPU

Saccharification efficiency without lignin derivatives of Meicelase and GC220 and cellulase content

CMC ρNPL ρNPG

Genencor GC220 showed a little superior performance to Meicelase

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• AL-derivatives maintained the residual cellulase

activity at a higher level than PEG4000 and without additive after one-time saccharification.

• Amphipathic AL derivatives showed directly

associated with CBH II, whereas PEG4000 did not. • DAEO-SSL showed a superior performance to others

derivatives for improvement SE and maintained cellulase activity until 4 times saccharification.

• Therefore, I confirmed that amphipathic lignin derivatives are very good materials as a cellulase aid-agent, because they can maintain and recover the cellulase activity.

This research will contribute to provide a useful method for bioethanol feedstock with low production cost from 2nd generation, in particular utilization of cedar wood from forest thinning and sago waste from sago mills.

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AＣＴＩＶＩＴＩＥＳ Paper 1. Ina Winarni, Chihiro Oikawa, Tatsuhiko Yamada, Kiyohiko Igarashi, Keiichi

Koda, Yasumitsu Uraki : Improvement of enzymatic saccharification of unbleached cedar pulp with amphipathic lignin derivatives.

Bioresources : 8 (2)2195-2208 (2013). 2. Ina Winarni, Keiichi Koda, Pari, G, Waluyo, T.K, Yasumitsu Uraki : Enzymatic

saccharification of sago pulp from starch extraction waste using sago lignin-based amphipathic derivatives : under review.

Conference

1. Ina Winarni;Chihiro Oikawa;Keiichi Koda;Yasumitsu Uraki;Tatsuhiko Yamada： Improvement of cedar pulp with amphiphilic lignin derivatives,62rd Annual meeting of Japan Wood Research Society. Sapporo, March 2012. 2. Ina Winarni;Chihiro Oikawa;Tatsuhiko Yamada;Keiichi Koda;Yasumitsu Uraki：Improvement of cedar (Cryptomeria japonica) pulp with amphiphilic lignin derivatives as a cellulase aid-agent, International Conference of International Union of Forest Research Organization; Division 5, Lisbon-Portugal, July 2012. 3. Ina Winarni;Chihiro Oikawa;Tatsuhiko Yamada;Keiichi Koda;Yasumitsu Uraki：Amphiphilic lignin derivatives to improve enzymatic saccharification of lignocelluloses, CABS, Sapporo, August, 2012.

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Mesuring instrument： Biacore

Detect physical interactions between molecular (amount of adsorption and desorption)

BiacoreX

・・・CBH II ・・・lignin derivatives

Sensorchip

prism

Detector Light source

Flow derection

• Interaction of amphipathic lignin derivatives and PEG4000 with CBH II

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1 0 8 6 4 2 0 0

40

60

80

100

20

0

40

60

80

100

20

8 6 4 2 0

Reaction time (day) Reaction time (day)

Rel

ati

ve

cell

ula

se a

ctiv

ity

(%

)

(A) (B)

Filter paper PHA-pulp

Y. Uraki, N. Ishikawa, M. Nishida, Y. Sano (2001) J. Wood Sci. 47, 301.

Repeating hydrolysis of filter paper (A) and PHA-pulp(B) By cellulase and PE-AL – cellulase complex

Hydrolysis conditions: Substrate, 3 g; Cellulase, 240 mg; PE-AL, 0 wt.%(●) and 0.2 wt.%(●) based on 300 mL of buffer solution

How does PE-AL effect on cellulase?

remained epoxy group would reacts with other functional groups on cellulase to yield covalent bond.

Amphiphilic lignin derivative (PE-AL)

cellulase

Covalent bond ?

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The amount of enzyme required to liberate 1 μmol of glucose from filter paper/minute for 1 h

NREL/TP-510-42628(Ghose, 1987)

GC220 Limiting step Cellobiose to

glucose

Easy to prepare cellobiose (much endoglucanase)

Meicelase Limiting step

To produce cellobiose (less endoglucanase)

Cellobiose to glucose

• Why the complete recovery can be achieved ?

0.8 g/L0.6 g/L0.4 g/L0.2 g/L0.1 g/L

(A) (RU)250

200

150

100

50

0

-50 0 100 200 300 time(s)

67

0.8 g/L0.6 g/L0.4 g/L0.2 g/L0.1 g/L

(B)(RU)250

200

150

100

50

0

-50 0 100 200 300 400 500 time(s)

36

0.008 g/L0.006 g/L0.004 g/L0.002 g/L0.001 g/L

(C) (RU)250

200

150

100

50

0

-50 0 100 200 300 400 500 time(s)

21

(RU)250

200

150

100

50

0

-50 0 100 200 300 time(s)

1 g/L

(D)

33

PEGDE-AL EPEG-AL

PEG 4,000 DAEO-AL

Sensorgram the interaction between CBH II with amphipathic lignin derivatives and PEG4000

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H2O

N2

145℃

145℃

▲▼●

20 % KI

solution

Oil bath

Sample

HI solution

N2

Heater

Magnetic

Stirrer

To cleavage the ethylene oxide chain by the action of hydriodic acid (HI)

0

40

60

80

100

20

5 4 3 2 1

Reaction time (times)

Polyhydric alcohol

(PHA) pulp

Hydrolysis conditions: Substrate, 3 g; Cellulase, 240 mg; PEGDE-AL, 0 wt.% (●) and 0.2 wt.% (●) based on 300 mL of buffer solution

Y. Uraki, N. Ishikawa, M. Nishida, Y. Sano (2001) J. Wood Sci. 47, 301.

Eff

icie

ncy

of

sacc

ha

rifi

cati

on

(%

)

PEGDE-AL is an amphipathic lignin derivative prepared from acetic acid lignin(AL) and polyethylene glycol derivative

80%

14%

Repeated hydrolysis of and PHA-pulp by cellulase and PEGDE-AL-cellulase complex

(No additive)

(With PEGDE-AL)


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PE( hydrophilic )

stable

EPEG or DAEO

unstable

AL + AL +

EPEG-AL and DAEO-AL are more stable than PE-AL

hydrophilic hydrophobic

PE EPEG, DAEO

13

13


PEGDE-AL < EPEG-AL < DAEO-AL


20

30

40

50

60

70

80


Sur

face

tens

ion

(mN

/m)

10-6

10-4

10-2

10


20

30

40

50

60

70

80


Sur

face

tens

ion

(mN

/m)

10-6

10-4

10-2

10


20

30

40

50

60

70

80


Sur

face

tens

ion

(mN

/m)

10-6 10

-410

-21

0

DAEO-AL(6)


EPEG-AL(13,4) PEGDE-AL(13,1)

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The general understanding of cellulose degradation at present is that for efficient breakdown of crystalline cellulose, three types of enzymes are needed. They are endo glucanases (EGs, which cut cellulose chains randomly), cellobiohydrolases (CBHs, which cleave cellobiose from the cellulose chain ends) and b-glucosidases (which hydrolyze cellobiose and cellodextrins into glucose). Efficient cellulose degradation by microbes is achieved in two major ways. Production of a ‘soup’ of different enzymes that can work synergistically is one method. These different enzymes could have differential expression depending on the substrate, time and other stimuli. The second method is cellulosome activity. In this approach, different enzymes are found associated as one complex (the cellulosome) for fast breakdown of cellulose.

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Meicelase

Reduction of cellobiose production

GC 220 (Genencor)

Enzyme adsorption to substrate

Still remained endoglucanase activity (much production of

cellobiose)

No apparent reduction of FPU Reduction in FPU

Enzyme adsorption to substrate

In particular endo-glucanase occurred at 10 FPU

Documents

TEKNOLOGI BIOETANOL BERBASIS LIMBAH LIGNOSELULOSA … · TEKNOLOGI BIOETANOL BERBASIS LIMBAH LIGNOSELULOSA Ina Winarni Pusat Penelitian dan Pengembangan Keteknikan Kehutanan dan Pengolahan