Distinct Roles of Residual Xylan and Lignin in Limiting Enzymatic Hydrolysis of

Organosolv Pretreated Woody Biomass

Maobing Tu

Forest Products Laboratory

Auburn University

1

Tu Research Group: Carbohydrate-derived biofuels and bioproducts

Woody biomass

Pretreated biomass

Sugars

Biofuels

Pretreatment

Enzymatic hydrolysis

Fermentation

Hemicellulose Hydrolysate

Microbial and Catalytic conversion

Co-products (Lactic acid, Acrylic acid, Butanol, Xylonic acid) 2

Outline

• Background and Goals

• Effects of residual xylan/lignin on enzymatic

hydrolysis

• Effects of pectinase/xylanase supplementation

on enzymatic hydrolysis

• Ethyl xyloside production in SSF process

• Conclusion

3

Liquid Phase Rich in Hemicellulose

Background

Pretreatment

Biomass

Solid Phase Rich in Cellulose

Fermentation Fermentation

Enzymatic Hydrolysis

Biofuels

Enzymatic hydrolysis limiting factors Residual xylan and lignin Other factors (DP, crystallinity, pore size and accessible surface area) End product inhibition

4

• Goals

– Distinguish the different roles of residual xylan and lignin in enzymatic hydrolysis of pretreated woody biomass

– Improve enzymatic hydrolysis of woody biomass

• Hypotheses

– Residual xylan affects Initial hydrolysis rate

– Residual lignin affects Final hydrolysis yield

Goals

5

Methods

Organosolv Pretreatment (170 ◦C, 1.1% H2SO4 (w/w), 65%

ethanol (v/v))

Solid Fraction Liquid Fraction

Biomass Chemical Composition

Analysis (NREL)

SEM

Enzymatic Hydrolysis SSF

Langmuir Adsorption Isotherm

Loblolly pine and sweetgum

6

Langmuir Adsorption Isotherm

KC

KC

1

max

Ʈ: The surface concentration of adsorbed cellulase enzymes; Ʈmax: The surface concentration of protein at full coverage;

K: Langmuir constant; C: The free protein concentration in the bulk solution; R: The distribution coefficient

KR max

Methods

7

Compositions Untreated

loblolly pine (%)

Untreated

sweetgum (%)

OPLP substrate

(%)

OPSG substrate

(%)

Acetone extractives 1.64±0.08 0.99±0.09 8.53±0.19 8.47±0.21

Acid-insoluble lignin 28.48±0.04 23.56±0.28 18.38±0.17 8.16±0.03

Acid-soluble lignin 0.26±0.03 2.24±0.02 0.23±0.06 0.87±0.03

Glucan 41.33±0.49 41.19±0.73 63.32±1.24 69.80±1.78

Xylan 6.34±0.13 16.18±0.50 2.93±0.79 9.74±0.53

Galactan 2.16±0.09 1.87±0.32 NA NA

Arabinan 1.30±0.11 0.83±0.11 0.90±0.21 NA

Mannan 12.17±0.39 3.33±0.49 4.50±0.59 3.01±0.28

Total 93.69 90.20 98.79 100.05

Results and discussion

Table 1. Chemical composition of untreated biomass and pretreated biomass

OPLP: organosolv pretreated loblolly pine; OPSG: organosolv pretreated sweetgum.

8

SEM images of OPLP (left) and OPSG (right)

Lignin droplets

9

Effect of enzyme loading on the hydrolysis of glucan in OPLP and OPSG

0 24 48 72

0

25

50

75

100

0 24 48 72

0

25

50

75

100

Glu

co

se

yie

ld (

%)

Time (h)

OPLP + 20 FPU

OPLP + 10 FPU

OPSG + 20 FPU

OPSG + 10 FPU

1. Initial glucan hydrolysis rate (g L-1 h-1): OPLP >OPSG 1.0 vs 0.7 (10 FPU) 1.5 vs 1.2 (20 FPU)

2. Final glucan hydrolysis yield: OPSG>OPLP 89% vs 60% (10 FPU) 100% vs 81% (20 FPU)

3. Crossing point at 17 h

OPLP: organosolv pretreated loblolly pine; OPSG: organosolv pretreated sweetgum. 10

Effect of enzyme loading on the hydrolysis of xylan in OPLP and OPSG

1. Xylan hydrolysis (rate): OPSG>OPLP 0.14 vs 0.03

0.21 vs 0.05

2. Xylan hydrolysis (yield): OPSG>OPLP 87% vs 43%

100% vs 66%

0 24 48 72

0

25

50

75

100

0 24 48 72

0

25

50

75

100

Xy

los

e y

ield

(%

)

Time (h)

OPLP + 20 FPU

OPLP + 10 FPU

OPSG + 20 FPU

OPSG + 10 FPU

OPLP: organosolv pretreated loblolly pine; OPSG: organosolv pretreated sweetgum. 11

• Initial glucan hydrolysis rate: – OPLP (1.0 g· L -1·H-1)>OPSG (0.7 g· L -1·H-1) 10 FPU – OPLP (1.5 g· L -1·H-1)>OPSG (1.2 g· L -1·H-1) 20 FPU

• Final glucan hydrolysis yield: – OPSG (89%) > OPLP (60%) 10 FPU – OPSG (100%)>OPLP (81%) 20 FPU

• Xylan hydrolysis (initial rate and final yield): – OPSG>OPLP

• Question:

– Why the initial hydrolysis rate (glucan) was higher in OPLP than that in OPSG?

Results and discussion

12

Cellulase adsorption isotherms on OPLP and OPSG

Cellulases Ʈmax (mg/g) K(mL/mg) R(L/g)

Cellulases on OPLP 35.09 3.11 0.11

Cellulases on OPSG 60.19 1.31 0.08

Celluclast on EPLP* 87.69 3.48 0.31

Celluclast on SELP* 101.05 1.45 0.15

0.0 0.5 1.0 1.5 2.0

0

10

20

30

40

0.0 0.5 1.0 1.5 2.0

0

10

20

30

40

Adsorb

ed c

ellu

lases o

n s

ubstr

ate

s (

mg/g

)

Free cellulases in solution (mg/mL)

Cellulase adsorption on OPSG

Cellulase adsorption on OPLP

13

Correlation between distribution coefficient and initial hydrolysis rate

0.05 0.10 0.15 0.20 0.25 0.30 0.35

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4In

itial

hyd

roly

sis

rate

(g/

L/h)

Distribution coefficient R (L/g)

R2=0.9, initial hydrolysis rate is well correlated to the Distribution coefficient (R)

14

Effect of pectinase on enzymatic hydrolysis of OPLP and OPSG

0 24 48 72

0

25

50

75

100

0 24 48 72

0

25

50

75

100

Glu

co

se

yie

ld (

%)

Time (hr)

OPLP + Pectinase

OPLP

OPSG + Pectinase

OPSG

Pectinase addition (3.6

mg/g glucan)

1.Initial rate (g L-1 h-1) OPLP >OPSG 1.0 vs 0.7 (10 FPU) 1.0 vs 0.8 (+Pectinase)

2. Final yield 60% to 67% in OPLP

89% to 99.9% in OPSG

15

Effect of pectinase on enzymatic hydrolysis of OPLP and OPSG

0 24 48 72

0

25

50

75

100

0 24 48 72

0

25

50

75

100

Xy

los

e y

ield

(%

)

Time (h)

OPLP + Pectinase

OPLP

OPSG + Pectinase

OPSG

1. Xylose yield increased 43% to 48% in OPLP, 87% to 99.9% in OPSG

16

0 24 48 72

0

25

50

75

100

0 24 48 72

0

25

50

75

100

Glu

co

se

yie

ld (

%)

Time (hr)

OPLP 10 FPU + Xylanase

OPLP 10 FPU

OPSG 10 FPU + Xylanase

OPSG 10 FPU

Effect of xylanase on enzymatic hydrolysis of OPLP and OPSG

Xylanase addition (2.7

mg/g glucan)

1.Initial rate (g L-1 h-1): OPLP >OPSG 1.0 vs 0.7 (10 FPU) 1.0 vs 1.0 (+Xylanase)

2. Final yield 60% to 67% in OPLP

89% to 99.9% in OPSG

17

Effect of xylanase on enzymatic hydrolysis of OPLP and OPSG

0 24 48 72

0

25

50

75

100

0 24 48 72

0

25

50

75

100

Xy

los

e y

ield

(%

)

Time (hr)

OPLP 10 FPU + Xylanase

OPLP 10 FPU

OPSG 10 FPU + Xylanase

OPSG 10 FPU

1. Xylose yield increased 43% to 52% in OPLP

87% to 100% in OPSG

18

• Pectinase supplementation (3.6 mg/g glucan)

– Initial hydrolysis rate (OPSG) did not change much (0.7 vs 0.8)

– Initial hydrolysis rate (OPLP) kept the same (1.0)

– Final hydrolysis yield increased in both substrates • From 60% to 67% in OPLP; from 89% to 99.9% in OPSG

• Xylanase supplementation (2.7 mg/g glucan)

– Initial hydrolysis rate (OPSG) increased by 50% (0.7 vs 1.0)

– Initial hydrolysis rate (OPLP) kept the same (1.0)

– Final hydrolysis yield increased in both substrates • From 60% to 67% in OPLP; from 89% to 99.9% in OPSG

Results and discussion

19

Correlation between residual xylan/lignin and enzymatic hydrolysis

0 5 10

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

OPSG

EPHP

OPLP

EPLP

Initia

l h

ydro

lysis

rate

(g/L

/h)

Xylan (%)

8 10 12 14 16 18 20

75

80

85

90

95

100

105

110

OPLP

EPHP

EPLPOPSG

Fin

al h

yd

roly

sis

yie

ld (

%)

Lignin (%)

Organosolv pretreated lodgepole pine (EPLP), loblolly pine (OPLP), sweetgum (OPSG) and hybrid polar (EPHP).

20

Effects of enzyme loading on SSF

0 24 48 72 96

0.0

0.5

1.0

1.5

2.00 24 48 72 96

0.0

0.5

1.0

1.5

2.0

Xy

los

e c

on

ce

ntr

ati

on

(g

/L)

Time (hr)

OPLP 20 FPU

OPLP 10 FPU

OPSG 20 FPU

OPSG 10 FPU

0 24 48 72 96

0

2

4

6

8

10

0 24 48 72 96

0

2

4

6

8

10

Eth

an

ol

co

nc

en

tra

tio

n (

g/L

)

Time (hr)

OPLP 20 FPU

OPLP 10 FPU

OPSG 20 FPU

OPSG 10 FPU

1. Initial ethanol production rate (OPLP>OPSG): 0.5 vs 0.3 (10 FPU) 0.8 vs 0.6 (20 FPU)

2. Final ethanol yield: OPSG>OPLP 8.8 vs 7.8 (10 FPU), 10.3 vs 9.8 (20 FPU)

1. Decreased in OPSG after 48hr with 20 FPU loading ?

21

• Ethanol production – Initial ethanol production rate: OPLP>OPSG

– Final ethanol yield: OPSG>OPLP

– Same trend as glucan hydrolysis but the crossing point changed (58h)

• Xylose release – Decreased after 48hr with 20 FPU loading

– Potential enzyme-catalyzed reaction between xylose and ethanol

Results and discussion

22

Formation of ethyl xylopyranoside in the process of SSF

8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 21.0 min

0.00

0.25

0.50

0.75

1.00

uV(x100,000)

8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 21.0 min

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

uV(x100,000)

Chromatograms in SSF with 20FPU/g glucan (a)softwood; (b) hardwood

Ethyl xylopyranoside

Ethanol

glycerol Xylose

0 hr

2 hr

48 hr

96 hr

23

LC/MS analysis of potential reaction product between xylose and ethanol

OOH

OHOH

OH CH3CH2OH

OOCH2CH3

OHOH

OH H2Oenzyme

H

Xylopyranose Ethanol Ethyl xylopyranoside

as is

m/z 179 180

%

0

100

Sweet gum SSF 20FPU SSF96hr_092612_3 42 (1.712) Cm (39:120) 1: TOF MS ES+ 3.21e3 179.0912

179.0273

178.5125

178.0955

178.1452 178.4682

178.5703

178.9840 178.6145

180.0247

179.1429 179.9775 179.5088

179.1866 179.6135

179.8453 180.1507

24

• Interactions between xylan/lignin and cellulase affect the enzymatic hydrolysis of pretreated biomass – Residual xylan controls the initial hydrolysis rate

– Residual lignin controls the final hydrolysis yield

– Two-phase hydrolysis

• Xylanase supplementation increases initial hydrolysis rate by removing xylan

• Linear correlation between initial hydrolysis rate and distribution coefficient

• Potential enzyme-catalyzed production of ethyl xyloside

Conclusion

25

Acknowledgement

• AAES and OVPR at Auburn University

• Dr. Yonnie Wu (Chemistry and Biochemistry)

• Dr. Sushil Adhikari (Biosystems Engineering)

• Graduate students (Mi Li, Rui Xie, Lu Lu and Jing Li)

26

SEM images of enzymatic hydrolyzed OPLP and OPSG

Enzymatic hydrolyzed biomass OPLP(left) and OPSG (right) ) after 72 hr with 10 FPU 27