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 UniversityE. Lansing, MI 48824

http:\\www.everythingbiomass.org

AICHE 2006, 17th Nov 2006San Francisco

A. AFEX Pretreatment Process

B. Changes in the biomass after pretreatment

Mosier et al., Bioresour. Technol. 2005

Expansion

AFEX Treated Untreated

Teymouri et al., Applied Biochem. Biotechnol., 2004

Fed-batch SSF on CS after 168 hrs

CS- corn stover

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)

(Drawn by G. Tambour, Forstliche, Fakultat, Gottingen)

Schematic view of a woody plant cell wall at four different magnifications

LSCM Corn Cob Fiber Cell Cross Section

Untreated AFEX treated

Secondary Cell Walls rich in lignin

Safranin dye binds to lignin-like compounds smeared on the surface

50 ms 50 ms

Corn Cob Granule SEM Imaging

Untreated 100x AFEX treated 100x

ESCA Surface CharacterizationDrop in O/C ratio after AFEX

0

0.1

0.2

0.3

0.4

0.5

0.6

Unt

reat

ed

AFE

X

AFE

X

Unt

reat

ed

AFE

X

Unt

reat

ed

AFE

X

Unt

reat

ed

AFE

X

Unt

reat

ed

AFE

X

Poplar Poplar120

Poplar 160

Corn Stover Switchgrass Wheat Straw Rice straw

O/C ratio

Washing Protocol

Biomass (untreated/AFEX)(UCS-143.8g, ACS-145.5g)

DI Water10ml/gm BM

Biomass Wash Slurry(15 min. soaking)

Washed Solids UCS-94%, ACS-87%

Wash Liquid

Fine solids<0.1%

Supernatant

Freeze Dried Soluble ExtractUCS-6%, ACS-13%

9000 rpm

Lyophilization

Filtration

Biomass (untreated/AFEX)(UCS-143.8g, ACS-145.5g)

DI Water10ml/gm BM

Biomass Wash Slurry(15 min. soaking)

Washed Solids UCS-94%, ACS-87%

Wash Liquid

Fine solids<0.1%

Supernatant

Freeze Dried Soluble ExtractUCS-6%, ACS-13%

9000 rpm

Lyophilization

Filtration

• UCS: Untreated Corn Stover

•ACS: AFEX Corn Stover

Chundawat et. al., Biotech. Bioeng., 2006(in press)

AFEX and untreated Wash streams

SG – Switch grass WS – Wheat StrawRS – Rice Straw CS – Corn stover

Prussian Blue Analysis

Bagasse

Degradation Products During Dilute Acid Pretreatment

Palmqvist et. al., Bioresource Technol. 2000

Potentially Inhibitory Compounds

Untreated CS

(mg/l)

AFEX CS

(mg/l)

Acid Pretreatment Hydrolyzate*

(mg/l)

Concentration Fold lower than acid

Formic 13 59 128.88 2.18Lactic 17 2560 3693.28 1.44Acetic 77 570 1537.28 2.70

Levulinic 0 present 174.18HMF 0 0 88.40

Furfural 0 0 1796.883,4-dihydroxybenzaldehyde 0.85 1.1 9.11 8.28

4-hydroxybenzoic acid present present 2.904-hydroxybenzaldehyde 0.9 553 9.27 0.02

vanillic acid 4 7.5 5.71 0.76syringic acid 1.55 1.7 6.73 3.96

vanillin 0.6 12 9.12 0.76syringaldehyde 0.72 1.2 14.38 11.98

4-hydroxy-3-methoxycinnamic acid 6 17.9 28.00 1.563-hydroxy-4-methoxycinnamic acid 0 3.49 6.22 1.78

*

* Unknown compounds co-elute along with lactic acid

Collaborative work with Dr. Kevin at Baylor Institute of TechnologyDilute acid hydrolysate data from Chen et al., 2006

Comparing Degradation Products: AFEX vs Dilute Acid Pretreatment

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

72 hrs hydrolysis data 15 FPU of Cellulase Low Conc (A) Medium Conc (B) High Conc (C)

Inhibitory effect of some AFEX degradation products

0

20

40

60

80

100

LA AA CA V BA FA BD CouA Combined

% of Control

LA Lactic acidAA Acetic acidCA Cafeic acidV VanillinBA Benzoic acidFA Ferulic acidBD 4-hydroxybenzaldehyde CouA p-coumaric acidCombined – 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

Source: http://141.150.157.117:8080/prokPUB/chaphtm/297/03_00.htm

Lignin-Hemicellulose Complex

Oligosaccharide and monosaccharide componentsin AFEX wash stream

Retention time (mins)

DP=2

Arabinose

Glucose

Xylose

Higher Degree of Polymerization Oligomers(DP 2 to 10+)

Oligomeric Components

i

ii

iii

Retention time (mins)

DP=2

Arabinose

Glucose

Xylose

Higher Degree of Polymerization Oligomers(DP 2 to 10+)

Oligomeric Components

i

ii

iii

Corn syrup oligosaccharide standard

Acid hydrolyzed AFEXCS 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…

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

Biomass Conversion Research Lab at Michigan State University

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

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)

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