1. Fundamental Understanding of Biomass Pretreatment Technologies: The Case of Ammonia Fiber Expansion (AFEX) Venkatesh Balan, Shishir Chundawat, Bryan Bals and Bruce Dale Biomass Conversion Research Laboratory, Department of Chemical Engineering and Material Science Michigan State University E. Lansing, MI 48824 http:\\www.everythingbiomass.org AICHE 2006, 17 th Nov 2006 San Francisco

2. A. AFEX Pretreatment Process B. Changes in the biomass after pretreatment Mosier et al., Bioresour. Technol. 2005 Expansion

3. AFEX Treated Untreated Teymouri et al., Applied Biochem. Biotechnol., 2004 Fed-batch SSF on CS after 168 hrs CS- corn stover

4. Understanding the AFEX Process What kind of compounds are released during AFEX process and do they inhibit enzymes and microbes ? Techniques used –SEM (Scanning Electron Microscope) –LSCM (Laser Scanning Confocal Microscope) using Safranin Fluorescent dye –ESCA (Electron Spectroscopy for Chemical Analysis) –Water Washing to remove degradation compounds Phenolic Estimation (Prussian Blue Method) LC-MS/UV analysis of wash stream –Enzymatic Hydrolysis (cellulase, beta-glucosidase)

5. (Drawn by G. Tambour, Forstliche, Fakultat, Gottingen) Schematic view of a woody plant cell wall at four different magnifications

6. LSCM Corn Cob Fiber Cell Cross Section UntreatedAFEX treated Secondary Cell Walls rich in lignin Safranin dye binds to lignin-like compounds smeared on the surface 50  ms

7. Corn Cob Granule SEM Imaging Untreated 100x AFEX treated 100x

8. ESCA Surface Characterization

9. Washing Protocol UCS: Untreated Corn Stover ACS: AFEX Corn Stover Chundawat et. al., Biotech. Bioeng., 2006 (in press) AFEX and untreated Wash streams

10. SG – Switch grass WS – Wheat Straw RS – Rice Straw CS – Corn stover Prussian Blue Analysis Bagasse

11. Degradation Products During Dilute Acid Pretreatment Palmqvist et. al., Bioresource Technol. 2000

12. * * Unknown compounds co-elute along with lactic acid Collaborative work with Dr. Kevin at Baylor Institute of Technology Dilute acid hydrolysate data from Chen et al., 2006 Comparing Degradation Products: AFEX vs Dilute Acid Pretreatment

13. Inhibition of Enzymes at varying Wash Stream Concentrations 24h 48h 168h (A)Low Concentration of Wash Stream (equivalent to 1% glucan loading) (B)High Concentration of Wash Stream (equivalent to 10% glucan loading) (A)(B) (A) (B) Enzymes: 15 FPU Cellulase (Genencor), 64 p-NPGU  - Glucosidase (Novozyme) Biomass Substrate: 1% avicel loading Wash Streams (WS): AFEX & Untreated (Unt) Corn Stover

14. 72 hrs hydrolysis data 15 FPU of Cellulase Low Conc (A) Medium Conc (B) High Conc (C) Inhibitory effect of some AFEX degradation products LA Lactic acid AA Acetic acid CA Cafeic acid V Vanillin BA Benzoic acid FA Ferulic acid BD 4-hydroxybenzaldehyde CouA p-coumaric acid Combined – mixture of all (A)Low Concentration of Wash Stream (equiv to 1% glucan loading) (B)Medium Concentration of Wash Stream component (equiv to 3.3% glucan loading) (C)High Concentration of Wash Stream (equiv to 10% glucan loading) A B C

15. Source: http://141.150.157.117:8080/prokPUB/chaphtm/297/03_00.htm Lignin-Hemicellulose Complex

16. Oligosaccharide and monosaccharide componentsin AFEX wash stream Corn syrup oligosaccharide standard Acid hydrolyzed AFEX CS wash stream AFEX CS wash stream (Biorad Aminex 42-A Column Chromatograms) What do we know about AFEX wash stream Oligosaccharides ? High degree of polymerization (> 10), similar for AFEX treated grasses (Corn stover, rice straw, bagasse, switchgrass) Arabinoxylan based oligosaccharides, for corn stover atleast Need to study if AFEX is cleaving very specific LCC (lignin- hemicellulose complex) bonds…

17.  AFEX is a novel alkali based pretreatment, however, a fundamental understanding of the process is still lacking  ESCA, LSCM, SEM indicate lignin-hemicellulose degradation, solubilization and deposition on biomass surface  Liquid ammonia also cleaves hemicellulose to oligosaccharides, at specific cleavage sites possibly  Degradation products released during AFEX process: Short chain Organic Acids (acetic, lactic), Phenolic and Aromatic acids inhibit enzymes to a certain degree.  Future Studies: Wash Stream analysis for other biomasses, Effect of degradation components on microbes Summary

18. 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

19. Acknowledgements CAFI Team (for valuable suggestions) Dr Shirley Owens, MSU (LSCM) Dr Per Askeland, MSU (ESCA) Dr Kevin Chambliss & Group, Baylor University (LC-UV/MS) Genencor International (for Enzymes)

20. References Effect of particle size based separation of milled corn stover on AFEX pretreatment and enzymatic digestibility (p n/a) Shishir P. S. Chundawat, Balan Venkatesh, Bruce E. Dale (In Press, J Biotech Bioeng) Understanding AFEX I: Inhibitory Effect of Ammonia Fiber Explosion (AFEX) Pretreatment of Corn Stover on Enzymatic Hydrolysis. Shishir PS Chundawat, Balan Venkatesh, Bryan D Bals, Shou-Feng Chen, Christopher Becker, C Kevin Chambliss, G Peter van Walsum, Bruce E Dale (Manuscript Submitted) High-performance liquid chromatography method for simultaneous determination of aliphatic acid, aromatic acid and neutral degradation products in biomass pretreatment hydrolysates. Chen SF, Mowery RA, Castleberry VA, van Walsum GP, Chambliss CK. 2006. J Chromatogr A. 1104:54-61