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									Modified AFEX Pretreatment Conditions and enzymatic Hydrolysis for Poplar

Balan Venkatesh, Shishir Chundawat, Derek Marshall and Bruce E. Dale Department of Chemical Engineering and Material Science BCRL, Michigan State University
(www.everythingbiomass.org)

16th November, 2006
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Ammonia Fiber Expansion (AFEX) Pretreatment Process
Ammonia Recovery

Recycle Ammonia Biomass Reactor Heat

Gaseous Ammonia
Treated Biomass
Expansion

Conditions we wary:
• Temperature (70-180 oC) • Moisture content of biomass (40-80%) • Ammonia to biomass ratio(1:1-3:1) • Residence time (5-30 min.) More details in Understanding AFEX presentation 2 Friday, Nov. 17th 12. 50 p.m. (672b), HSF, continental 7

Conversions on Different Feed stock
Corn Stover (CS) Poplar (HLP) AFEX Conditions: 90 oC, 1:1 (BM:Ammonia), 5 min. residence time Enzyme Loading: 15 FPU cellulase, 64 pNPGU of b-glucosidase Reaction condition: 50oC, 90 rpm, 168 h
Component Glucan Xylan Arabinan Mannan Galactan Lignin Protein Ash Extractives CS 34.4 22.8 4.2 0.6 1.4 11.0 2.3 6.1 8.5 Poplar 43.8 14.5 0.61 3.94 1.02 29.12 nd 1.07 3.56

% Glucan/Xylan Conversion

100

Glucan
80 60 40 20 0 Untreated CS AFEX Untreated HLP AFEX

Xylan

HLP- High Lignin Poplar

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Conversions for Two batches of Poplar
100
% Glucan/Xylan conversion

Component

HLP

LLP

Glucan 80 60 40 20 0 Untreated AFEX treated HLP Untreated LLP AFEX treated Xylan

Glucan Xylan Arabinan Mannan Galactan Lignin Protein
Ash Extractives

43.8 14.5 0.61 3.94 1.02 29.12 nd
1.07 3.56

45.1 17.8 0.6 1.7 1.5 21.4 nd
0.8 3.4

HLP- High Lignin Poplar LLP- Low Lignin Poplar

AFEX Conditions: 100 oC, 1:1 (BM:Ammonia), 10 min. residence time
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Strategy:
 Low temperature pretreatment was not effective, hence went for

higher temperature  Modified conditions like soaking and autoclaving prior to AFEX  Tried different enzyme combinations and surfactant effect  Understand the degradation product formed during the pretreatment by LC-MS and LC-UV techniques.  High throughput AFEX (lesser heat transfer problem) for wide range of screening conditions

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Reaction conditions and Enzyme Loading
Unsoaked (US)/ AFEX 15/60 FPU C + 64 pNPGU bG and/or 3.1 mg X + 3.5 mg A 24h water soaked (WS) /AFEX 15/60 FPU C + 64 pNPGU bG and/or 3.1 mg X + 3.5 mg A Autoclaved (AC)/AFEX 15/60 FPU C + 64 pNPGU bG and/or 3.1 mg X + 3.5 mg A Combination of enzymes

15 FPU C + 64 pNPGU bG and/or X (3.1-31.0 mg) P (5.6-56 mg) F (1-10 mg) Substrate : HLP-High lignin poplar, LLP-low lignin poplar AFEX Condition : 100 oC/180 oC, 1:1 (BM:Ammonia) Enzyme Loading: mg of protein/g of glucan Abbreviation : C-Spezyme CP Cellulase, X-Multifect Xylanase, P-Pectinase, F- Depol 720 Feruloyl esterase, A-Additive (Tween 80) 6

To summarize……
60FPU of cellulase loading
100

% Glucan Conversion

80 60 40 20 0

100 oC 180 oC

72h Hydrolysis

US

WS

AC

US

WS

AC

High Lignin Poplar

Low Lignin Poplar

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Hydrogen bonding in cellulose

2D

3D

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Synergy Experiments on High Lignin Poplar (HLP)
%Glucan Conversion

40

160 US-HLP-180
30 20 10 0

168h 72h

C, bG X( 1 bG 00 ,X ) (1 C, b G 00) C, , X( b G 10 ,X ) C, b G (25 ) C, , X b G (5 , X 0) (1 00 P( ) bG 100 ,P ) (1 C, b G 00) ,P C, ( b G 10) ,P C, ( b G 25) ,P C, b G (50 ,P ) (1 00 F( ) 1 bG 00 ,F ) (1 C, b G 00) ,F C, b G (10 ) , C, F (2 5) bG ,F C, b G (50 AC , F ) (1 -L LP 00) AC , -H C, b LP G ,C ,b G

Enzyme Combinations 31.3 mg Cellulase (C) + 64 pNPGU bG (bG)/g of Glucan and/or Xylanase (X 10, 25, 50, 100) (3.1, 7.75, 15.5, 31.0 mg)/g of Glucan Pectinase (P10, 24, 50, 100) (5.6, 14, 28, 56 mg)/g of Glucan Feruloyl esterase (F 10, 25, 50, 100) (1, 2.5, 5, 10 mg)/g of Glucan 9

Overall Sugar Yield for HLP and LLP
100 100
25.7 18.1 14.9

% Sugar Yield % Sugar Yield % Sugar Yield

80 80 60 60

28.7

13.7 7.9

40 40 20 0 0
71.3 42.8 71.3 74.3

37.2

66.9

MSY MSY (LLP) (LLP)

AFEX AFEX AFEX AFEX (LLP-15) (LLP-60) (LLP-15) (LLP-60)

MSY MSY (HLP) (HLP)

AFEX AFEX AFEX AFEX (HLP-15) (HLP-60) (HLP-15) (HLP-60)

LLP – Low Lignin Poplar HLP – High Lignin Poplar MSY – Maximum Sugar Yield 15 - 15 FPU of cellulase 60 – 60 FPU of cellulase

AFEX Condition: 180 oC, 70 % moisture, 10 min. 1:1 (ammonia : Biomass) Sample Autoclaved prior to AFEX 168 h hydrolysis

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200

15

20

32

7 -0 6 -0 6 M G A F E X w a te r

AFEX treated HLP
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Representative Chromatogram
1, formic acid; 2, malonic acid; 3, lactic acid; 4, acetic acid; 5, maleic; 6, succinic; 7, fumaric; 8, t-aconitic acid; 9, levulinic Acid; 10, glutaric acid; 11, itaconic; 12, 2-hydroxy-2-methylbutyric acid; 13, gallic acid; 14, 5HMF; 15, 2-furoic acid; 16, furfural; 17, adipic acid; 18, 3,4-dihydroxylbenzoic acid; 19, 3,4-dihydroxybenzaldehyde; 5 020, 4-hydroxylbenzoic acid; 21, 2,5-dihydroxylbenzoic acid; 22, 4-hydroxybenzaldehyde; 23, vanillic acid; 24, homovanilic acid; 25, caffeic acid; 26, syringic acid; 27, 4-hydroxyacetophenone; 28, vanillin; 29, 4-hydroxycoumaric acid; 30, syringaldehyde; 31, benzoic acid; 32, ferulic acid; 33, sinapic acid; 34, 3-hydroxy-4-methoxycinnamic acid; 35, salicyclic acid; 36, 4-hydroxycoumarin; 37, o-toluic acid and 38, p-toluic acid.

150

mAU

100

3 25 19 30 27 22 23 2 1 4 5 6 8 10 11 12 14 16 17 18 21 26 29 34 38 31

50

0 0
25

24 10 20 30 40

re ten tio n tim e (m in )

7 -0 6 -0 6 M G U n w a te r
20

Untreated treated HLP

15

mAU

10

5 16 20 0 26 28 25

30 34 32 38

-5 0 10 20 30 40 50

The samples were analyzed by LCMS for qualitative purposes only.
reten tio n tim e (m in )

However, the concentrations determined using UV detection may exhibit a high bias due to 11 unidentified co-eluting components.

Comparing Degradation Products:
Type Acids Analyte AFEX-HLP g/100 g 0.19 2.30 0.66 0.00 1.40 0.00 0.01 0.03 0.01 0.01 0.01 0.02 0.02 0.01 0.13 AFEX-LLP g/100g 0.08 2.10 0.28 0.00 0.32 0.00 0.00 0.01 0.00 0.00 0.01 0.00 0.01 0.00 0.27

Formic acid *Lactic acid Acetic acid * Furans 5HMF 2-furoic acid furfural Phenols 3,4-dihydroxybenzaldehyde 4-hydroxylbenzoic acid 4-hydroxybenzaldehyde Vanillic acid Caffeic acid 4-hydroxyacetophenone Vanillin Benzoic acid Ferulic acid

* Unknown compounds co-elute along with lactic acid
Collaborative work with Dr. Kevin at Baylor Institute of Technology

Ref.:Dilute acid hydrolysate data from Chen et al., 2006

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High Throughput AFEX Unit

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Untreated and AFEX treated Poplar samples at different conditions
140 oC, 1:1 140 oC, 1:1.5 140 oC, 1:2 140 oC, 1:1 140 oC, 1:1.5

Untreated 140 oC, 1:2

160 oC, 1:1

160 oC, 1:1.5

160 oC, 1:2

160 oC, 1:1

160 oC, 1:1.5

160 oC, 1:2

180 oC, 1:1

180 oC, 1:1.5

180 oC, 1:2

180 oC, 1:1

180 oC, 1:1.5

180 oC, 1:2

Water un-soaked prior to AFEX

Water soaked prior to AFEX
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*All experiments done with 70% moisture and 30 minutes resident time

Some preliminary results………

72h Hydrolysis
100
% Glucan Conversion

80 60 40 20 0 US, 1:1 US, 1:1.5 15 FPU US, 1:2 US, 1:1 US, 1:1.5 60 FPU US, 1:2

Enzymatic hydrolysis: 50 oC, 90 rpm US- un-soaked High Lignin Poplar
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Conclusion:
 Combination

of autoclaving followed by AFEX gives better Glucan yield both for high and low lignin poplar

 Collective sugar yield for high and low lignin poplar was summarized for the best AFEX treated poplar

 At elevated temperature and in the presence of ammonia, some sugar degradation products like organic acid is formed, based on LC-MS/LC-UV studies
 Introduction to High through put AFEX (new tool to screen the different feed stock)

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Acknowledgement
• CAFI team for their valuable suggestions • Genencor for supplying their enzymes

• Dr. Kevin and Dr. Walsum for doing LCMS and LC-UV analysis • DOE for funding
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Biomass Conversion Research Lab at Michigan State University

Left to Right (Back): Shishir, Derek, Bryan, Ming Left to Right (Front): Elizabeth, Dr. Dale, Dr. Ven, Holly, Ben

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