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Modified AFEX Pret reatment 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) 16 th November, 2006

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slide1

Modified AFEX PretreatmentConditions

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

slide2

Ammonia Fiber Expansion (AFEX) Pretreatment Process

Ammonia Recovery

Gaseous

Recycle

Ammonia

Ammonia

Treated

Biomass

Heat

Biomass

Expansion

Reactor

  • 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

Friday, Nov. 17th 12. 50 p.m. (672b), HSF, continental 7

slide3

Component

CS

Poplar

Glucan

34.4

43.8

Xylan

22.8

14.5

Arabinan

4.2

0.61

Mannan

0.6

3.94

Galactan

1.4

1.02

Lignin

11.0

29.12

Protein

2.3

nd

Ash

6.1

1.07

Extractives

8.5

3.56

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

HLP- High Lignin Poplar

slide4

Component

HLP

LLP

Glucan

43.8

45.1

Xylan

14.5

17.8

Arabinan

0.61

0.6

Mannan

3.94

1.7

Galactan

1.02

1.5

Lignin

29.12

21.4

Protein

nd

nd

Ash

1.07

0.8

Extractives

3.56

3.4

Conversions for Two batches of Poplar

HLP- High Lignin Poplar

LLP- Low Lignin Poplar

AFEX Conditions: 100 oC, 1:1 (BM:Ammonia), 10 min. residence time

slide5

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

slide6

Reaction conditions and Enzyme Loading

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)

slide7

To summarize……

60FPU of cellulase loading

slide8

3D

Hydrogen bonding in cellulose

2D

slide9

Synergy Experiments on High Lignin Poplar (HLP)

160

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

slide10

Overall Sugar Yield for HLP and LLP

25.7

28.7

18.1

14.9

13.7

7.9

71.3

42.8

71.3

74.3

37.2

66.9

AFEX Condition:

180 oC, 70 % moisture,

10 min. 1:1 (ammonia : Biomass)

Sample Autoclaved prior to AFEX

168 h hydrolysis

LLP – Low Lignin Poplar

HLP – High Lignin Poplar

MSY – Maximum Sugar Yield

15 - 15 FPU of cellulase

60 – 60 FPU of cellulase

slide11

Representative

Chromatogram

AFEX treated HLP

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;

20, 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.

Untreated treated HLP

The samples were analyzed by LC-MS for qualitative purposes only.

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

slide12

Comparing Degradation Products:

*

* 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

slide14

Untreated and AFEX treated Poplar

samples at different conditions

Untreated

140 oC, 1:1

140 oC, 1:1.5

140 oC, 1:2

140 oC, 1:1

140 oC, 1:1.5

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

*All experiments done with 70% moisture and 30 minutes resident time

slide15

Some preliminary results………

72h Hydrolysis

Enzymatic hydrolysis: 50 oC, 90 rpm

US- un-soaked High Lignin Poplar

slide16

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)

acknowledgement
Acknowledgement
  • CAFI team for their valuable suggestions
  • Genencor for supplying their enzymes
  • Dr. Kevin and Dr. Walsum for doing LC-MS and LC-UV analysis
  • DOE for funding
slide18

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