Enzymatic Hydrolysis of Poplar Pretreated by Ammonia Fiber Explosion 

James F. Heidenreich, Tamika Bradshaw, Bruce E. Dale and Venkatesh Balan

BCRL, Department of Chemical Engineering and Materials Science, Michigan State University, E. Lansing, MI – 48824(www.everythingbiomass.org)

Hybrid Poplar advantages

• Relatively high productivity (18-26 dry Mg/(ha·year)

• Fast growing

• Widely available

• Pest-resistant

• Drought-resistant

• High density reduces cost of transportation

Populus nigra x Populus maximowiczii(Hardwood)

Comparing Biomasses

Glucan

Xylan Lignin

Poplar-II(High lignin)

43.8% 14.9% 29.1%

Poplar-I(Low

Lignin)45.1% 17.8% 21.3%

Corn Stover

37.7% 21.6% 18.6%

Switch-grass

33.5% 26.5% 18.1%

AFEX process Conditions:

Soaking/un soaking

Moisture in biomass ~50 to 70%

Temperature range 80-200 oC

BM : Ammonia loading 1: 0.6 -1: 2.0

Residence Time: 10 min.Hydrolysis Conditions

LAP009 (NREL Protocol)15ml reaction volume, 50 oC,1% BM

Enzymes: Spezyme CP and Multifect Xylanase 720 (Genencore)-Glucosidase (Novozyme)

0%

5%

10%

15%

20%

25%

30%

35%

80 90 100 110 120

72h

No soak - Xylan conversion

Temperature (oC)AFEX:1:1.2 biomass to NH3, 5 min.

0%

5%

10%

15%

20%

25%

80 90 100 110 120

72h

No Soak - Glucan conversion

Temperature (oC)

AFEX:1:1.2 biomass to NH3, 5 min.

0%

5%

10%

15%

20%

25%

80 90 100 110 120

72h 168h

Water soak - Glucan conversion

AFEX: 1:1.2 biomass to NH3, 10 min.

Temperature (oC)

0%

5%

10%

15%

20%

25%

30%

35%

80 90 100 110 120

72h 168h

Water soak - Xylan conversion

Temperature (oC)

AFEX: 1:1.2 biomass to NH3, 10 min.

Wet: Sample used immediately after AFEX, adjusted the pH to 4.8 by 1N HCl, followed by hydrolysis

Dry: Sampled dried in hood after AFEX overnight, adjusted the pH to 4.8 by 1N HCl, followed by hydrolysis

1AFEX: Doing AFEX reaction once

2AFEX: Doing AFEX reaction twice (consecutively)

Ammonium Hydroxide Soak- Glucan Conversion

0%

5%

10%

15%

20%

25%

wet dry wet dry wet dry wet dry

1 AFEX 2 AFEX 1 AFEX 2 AFEX

24 hour soak 168 hour soak

AFEX: 110°C, 1:1 biomass to NH3, 10 min

72h 168h

Water/

•Drying the sample AFEX results in high glucan Conversion

•Multiple AFEX does not improve the conversions

Other Strategy:

• Adding xylanase to increase xylose conversion and in turn to help glucan conversion

• To look for additives which can increase both glucan and xylan conversions

• Effect of oil addition during enzyme hydrolysis

0%

10%

20%

30%

40%

50%

noxylanase

11µLxylanase

noxylanase

11µLxylanase

noxylanase

11µLxylanase

noXylanase

Mineral Oil Corn Oil Tween noadditive

glucan xylan

Effect of Xylanase and additive after 168 hours of Hydrolysis

AFEX: 24h water soak, 120°C, 1:1.2 biomass to ammonia

Surfactant (Tween 80) 0.15g/g of dry biomass basis

AFEX Conditions

Mild conditions

Upto 120 oC, 1.0 : 1.2 Biomass to ammonia loading(pressure raise up to 300 psi)

Tough conditions

Upto 200 oC, 1.0 : 2.0 Biomass to ammonia loading(pressure raise up to 600 psi)

0%5%

10%15%20%

25%30%35%40%

120 140 160 180 200

72h 168h

Water soak - Glucan Conversion

Temperature (oC)

AFEX: 1:1 biomass to NH3, rinsed

Temperature and moisture conditions during AFEX

0%

2%

4%

6%

8%

10%

12%

1.50% 10% 30% 50% 70%

72h 168h

Moisture Content

AFEX: 1:0.8 biomass to NH3, 100 oC

Oven Dried - Glucan Conversion

High temperatureDuring AFEXImproves thepretreatment

Moisture has noEffect during AFEX pretreatment

Effect of Ammonia loading

0

10

20

30

40

50

60

1:1, C, X, A 1:1.5, C, X, A 1:2, C, X, A UT, C, A, X

168h

72h

Water soaked, AFEX (180 oC)

(Biomass to ammonia loading)

% G

luca

n C

on

vers

ion

Increasing ammonia Loading has some influence on pretreatment

0

10

20

30

40

50

1:1, C, X, A 1:1.5, C, X, A 1:2, C, X, A UT, C, A, X

168h

72h

Water soaked, AFEX (180 oC)

% X

yla

n c

on

ve

rsio

n

(Biomass to ammonia loading)

To Brief:

• Poplar I (high lignin) shows resistance to hydrolysis

• Soaking followed by AFEX does help to increase hydrolysis

• Varying moisture during AFEX does not influence hydrolysis but, increasing temperature during AFEX has some influence

• Increasing the ammonia loading shows some improvement in pretreatment

• Addition of Xylanase and additives like tween 80 along with cellulase does increase the hydrolysis

Some results on Poplar I (low lignin)

0

20

40

60

80

100168h

72h

Poplar (low lignin), soaked-AFEX

1:1 BM to ammonia 1:1.5 BM to ammonia

% G

luc

an

Co

nv

ers

ion

0

20

40

60

80

100168h

72h

Poplar (low lignin), unsoaked-AFEX

1:1 BM to ammonia 1:1.5 BM to ammonia

% G

luc

an

Co

nv

ers

ion

ESCA Studies

Higher the O/C ratioLesser the lignin on the surface of poplar

0

0.1

0.2

0.3

UntreatedPoplar

APR Poplar AFEX (120 oC) AFEX (160 oC)

O/C

ratio

ESCA Results on Poplar

0

0.01

0.02

0.03

Untreatedpoplar

ARP Poplar AFEX (120oC)

Gallic

Ferulic

% P

heno

lic E

quiv

alen

ts

Prussian Blue method

Conclusion:

AFEX conditionGlucan

conversionXylan

conversion

Poplar-II(High lignin)

1:5, 200 oC, 50% MC

50% 44%

Poplar-I(Low

Lignin)

1:1, 200 oC, 50% MC

83% 57%

Best AFEX conditions so for….

Wash stream after AFEX and before hydrolysis – to be analyzed

AcknowledgementAll the colleagues in my lab (BCRL Team)

CAFI-2 groupDr. Charles E. Wyman(Dartmouth College)Dr. Mark T. Holtzapple (Texas A & M University)Dr. Y. Y. Lee (Auburn University)Dr. Michael R. Ladisch (Purdue University)Dr. John N. Saddler (University of British Columbia)Dr. Richard Elander (National Renewable Research Laboratory)Dr. Mohammed Moniruzzaman (Genencor International)

United States Department of Agriculture

United States Department of Energy