Fundamental Understanding of

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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, 17th Nov 2006 San Francisco
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A. AFEX Pretreatment Process

Expansion
B. Changes in the biomass after pretreatment
Mosier et al., Bioresour. Technol. 2005
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AFEX Treated
Untreated
CS- corn stover
Fed-batch SSF on CS after 168 hrs
Teymouri et al., Applied Biochem. Biotechnol.,
2004
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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)

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Schematic view of a woody plant cell wall at
four different magnifications
(Drawn by G. Tambour, Forstliche, Fakultat,
Gottingen)
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LSCM Corn Cob Fiber Cell Cross Section
50 mms
50 mms
Untreated
AFEX treated
Secondary Cell Walls rich in lignin
Safranin dye binds to lignin-like compounds
smeared on the surface
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Corn Cob Granule SEM Imaging
Untreated 100x
AFEX treated 100x
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ESCA Surface Characterization
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Washing Protocol

• UCS Untreated Corn Stover
• ACS AFEX Corn Stover

AFEX and untreated Wash streams
Chundawat et. al., Biotech. Bioeng., 2006 (in
press)
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Prussian Blue Analysis
Bagasse
SG Switch grass WS Wheat Straw RS
Rice Straw CS Corn stover
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Degradation Products During Dilute Acid
Pretreatment
Palmqvist et. al., Bioresource Technol. 2000
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Comparing Degradation Products AFEX vs Dilute
Acid Pretreatment

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
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Inhibition of Enzymes at varying Wash Stream
Concentrations
Enzymes 15 FPU Cellulase (Genencor), 64 p-NPGU
b-Glucosidase (Novozyme) Biomass Substrate 1
avicel loading Wash Streams (WS) AFEX
Untreated (Unt) Corn Stover
168h
48h
24h
(A)
(B)
(A)
(B)

• Low Concentration of Wash Stream (equivalent to
  1 glucan loading)
• High Concentration of Wash Stream (equivalent to
  10 glucan loading)

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Inhibitory effect of some AFEX degradation
products
72 hrs hydrolysis data 15 FPU of
Cellulase Low Conc (A) Medium
Conc (B) High Conc (C)
A
B
C
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

• Low Concentration of Wash Stream (equiv to 1
  glucan loading)
• Medium Concentration of Wash Stream component
  (equiv to 3.3 glucan loading)
• High Concentration of Wash Stream (equiv to 10
  glucan loading)

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Lignin-Hemicellulose Complex
Source http//141.150.157.1178080/prokPUB/chapht
m/297/03_00.htm
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Oligosaccharide and monosaccharide componentsin
AFEX wash stream

• What do we know about AFEX wash stream
  Oligosaccharides?
• High degree of polymerization (gt 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

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Summary

• 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

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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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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)

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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. 110454-61

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