Understanding Free and Complexed Enzyme Mechanisms and Factors Contributing to

Cell Wall Recalcitrance

Michael G. Resch SIMB SBFC Clearwater, FL 4/29/2014

NREL/PR-2700-60927 NREL is a national laboratory of the U.S. Department of Energy, Office of Energy Efficiency and Renewable Energy, operated by the Alliance for Sustainable Energy, LLC.

Acknowledgements

Task Leaders Gregg Beckham Michael Himmel Steve Decker TEM and SEM Imaging Bryon Donohoe Peter Ciesielski Intern Jennifer Nill

Clean Fractionation Rui Katahira Kellene McKinney Cellulose Allomorphs and XRD Ashutosh Mittal BAT Team Techno Economic Analysis Mary Biddy

Funding

Biomass Technologies Office (BETO) Office of Science

3

Economic Impact of Reducing Enzyme Loading

Humbird et al. 2011

4

Gilbert and Fontes, Ann. Rev. Biochem. 2010

Lynd et al. MMR 2002

“free” cellulases

“Free” vs. Complexed Enzymes

5

Different physical mechanisms of denconstruction

Cellulosomes “Free” Cellulases

Resch, M.G. et al. (2013) Energy and Environmental Science

6

Do Free and complexed enzymes degrade cellulose synergistically?

Avicel conversion at 10 mg/g

Resch, M.G. et al. (2013) Energy and Environmental Science

7

Enzyme Accessibility is Different

“Free” Enzymes Cellulosomes

• Cellulases are evenly distributed

• Cellulosomes are near the cell walls

Possible factors limiting cellulosome activity on DA pretreated biomass • Accessibility • Low koff compared to free cellulases • Binding to lignin

Resch, M.G. et al. (2013) Energy and Environmental Science

Clean Fractionation Pretreatment (Organosolve)

9

CEF

Solvent Temp (°C) Acid (mol/L) Lignin (%) Glucan (%) Xylan (%)

MIBK/Acetone/H2O 140 0.025 10.63 63.31 17.47 MIBK/Acetone/H2O 140 0.05 3.67 82.28 6.56 MIBK/Acetone/H2O 140 0.1 3.33 87.19 3.08

CEF Chemical Composition

10

How does Clean Fractionation affect biomass morphology?

140°C 0.1M

Katahira, R. et al (in review)

11

How does Clean Fractionation affect enzyme saccharification?

Resch, M. et al (in review)

12

Affect of PT Severity on Enzymatic Saccharification

10 mg/g enzyme loading, 72 hours

Resch, M. et al (in review)

13

Synergistic Combination of CTec2 and Cellulosomes

Resch, M. et al (in review)

14

Technoeconomic Results from CF Work

Mary Biddy (NREL)

15

SEM of Digested Cellulose Enriched Fraction

Resch, M. et al (in review)

16

TEM of Digested Cellulose Enriched Fraction

Resch, M. et al (in review)

17

Model of Enzymatic Deconstruction Mechanisms

Ciesielski, P., Resch, M. et al (in review)

Cellulose Allomorphs

19

Allomorph EH Study Objectives

Isolate cellulose allomorphs with identical crystallinity. How do complexed, free and a combination

perform on cellulose I, II and III? Does the crystallinity change over the course

of digestion? ? How does cellulose type effect the hydrolysis

mechanism with free, complexed and combination?

20

Crystal Structure Models

21

Cotton linters Allomorphs - XRD

10 20 30 402θ

Cellulose I

Cel I; CI - 62

PASC CL; CI - 44

PASC CL; CI - 34

Amorphous - Ball Milled - 6h

A

10 15 20 25 30 35 402θ

Cellulose II

Cel II @ 145C; CI - 57

Cel II @ 70C; CI - 49

Cel II @ 25C; CI - 40

Amorphous - Ball milled 6h

B

10 15 20 25 30 35 402θ

Cellulose III

Cel III @ 130C; CI - 60

Cel III @ 25C; CI - 49

Cel III @ -33C; CI - 32

Amorphous - Ball milled 6h

C

Allomorph Crystallinity Index (CrI)

Cellulose I 62 44 34

Cellulose II 57 49 40

Cellulose III 60 49 32 Ashutosh Mittal

22

Enzymatic Hydrolysis of Cellulose Allomorphs

23

XRD – 50% EH with Ctec2

10 15 20 25 30 35 40

Cellulose III - Ctec2

Cellulose III

Cel III Ctec2

10 15 20 25 30 35 40

Cellulose I - Ctec2

Cellulose I

Cel I Ctec2

10 15 20 25 30 35 40

Cellulose II - Ctec2

Cellulose II

Cel II Ctec2

No change in XRD of cellulose samples after 50% digestion with Ctec2

Ashutosh Mittal

24

XRD – 50% EH with Cellulosomes

10 15 20 25 30 35 40

Cellulose III - Cellulosomes

Cellulose III Cellulosomes

Cellulose III

10 15 20 25 30 35 40

Cellulose I - Cellulosomes

Cellulose I

Cellulose I Cellulosomes

10 15 20 25 30 35 40

Cellulose II - Cellulosomes

Cellulose II

Cellulose II Cellulosomes

XRD of cellulose II and III allomorphs change after 50% digestion with Cellulosomes • Change in the intensity of XRD from various crystal planes • Emergence of a new peak at 2θ = 24° Do cellulosomes attack crystalline regions selectively? Currently looking at the digestions of cellulose allomorphs with combination of Ctec2 and Cellulosomes

25

I

Free Complexed

II ? ?

III ? ?

26

Conclusions

• “Free” enzymes and cellulosomes have distinct mechanisms in cellulose and biomass deconstruction

• “Free” enzymes and cellulosomes are synergistic • Biomass composition and enzyme accessibility to cellulose ends affect

cellulosomes more than “free” enzymes • Combining properties of “free” and multi-domain enzymes in

commercial enzyme cocktails could lead to enzyme cost reductions

Acknowledgements

Task Leaders Gregg Beckham Michael Himmel Steve Decker TEM and SEM Imaging Bryon Donohoe Peter Ciesielski Intern Jennifer Nill

Clean Fractionation Rui Katahira Kellene McKinney Cellulose Allomorphs and XRD Ashutosh Mittal BAT Team Techno Economic Analysis Mary Biddy

Funding

Biomass Technologies Office (BETO) Office of Science

28

Clean Fractionation – Xylan Conversion

Resch, M. et al (in review)