Microscopic Examination of Changes of Plant Cell Structure in Corn Stover
Due to Cellulase Activity and Hot Water Pretreatment
Meijuan Zeng1,2, Nathan S. Mosier1,2, Chia-Ping Huang5, Debra M. Sherman5, Joan Goetz1,4 and Michael R. Ladisch* 1,2,3
2 Department
of Agricultural and Biological Engineering
3
1 Laboratory of Renewable Resources Engineering
Weldon School of Biomedical Engineering 4 Department of Consumer Sciences and Retailing
Purdue University
5
Life Science Microscopy Facility
RESULTS
• Objectives: explore the changes of biomass
ultrastructure at nanoscale during pretreatment and
enzyme hydrolysis. Try to set a specific way to measure
enzyme activity on single observable plant cell walls,
since overall enzyme hydrolysis effect will result in cell
wall disruptions. This could serve as a basis for
understanding enzyme mechanisms at the nanoscale
level, making manipulating enzyme hydrolysis more
directly and efficiently.
3h (10.7% glucose yield)
72h (13.9% glucose yield)
~3 0µ
m
m
• Hypotheses: (1) small particle sizes of cellulosic
substrate are more readily hydrolyzed than large ones;
(2) pretreatment enlarges accessible and susceptible
surface area for enzyme treatment..
hollows
100 µm
100 µm
Coarse; plate-like cells
3
1
Break appears in cell walls,
leaving hollow areas
Pores enlarged
24h (12.5% glucose yield)
Longitudinal view (400×)
Longitudinal view (350×)
Honeycomb-like appearance to
cells as contents are digested
Reticular area apparent
Reticular area more prominent
Frayed edges as cell
structure unravels
(150×)
~5µm
(1) cross-sectional view;
(2) longitudinal view;
(3) non-specific orientation.
100 µm
100 µm
Longitudinal view (300×)
Pores appear
Rectangular cells having
smooth surfaces
NATIVE
100 µm
100 µm
Longitudinal view (250×)
Longitudinal view (250 ×)
53-75 µm Corn Stover
Top layer peeling off
Corn Stover
(different particle size)
Solid Liquid
HPLC
Spezyme 15 FPU/g glucan
Novozyme 40 IU/g glucan
Sample loading 2%
Spezyme + Novozyme hydrolyze
50oC for 3h, 24h, 72h, 168h**
Liquid
Solid
SEM
** enzyme treatment was different from NREL LAP009:
enzymes were added into pretreated slurry directly
Loops structures appear as
vascular cells degenerate
3h
Glucose Yield During Enzymatic Hydrolysis
(combined liquid and solids)
24h
425-710 micron
10 µm
(1)
72h
10 µm
100 µm
10 µm
168h
Shape distribution based on
particle orientation (80×)
(1) Cross-sectional view;
(2) Longitudinal view.
(500×)
10 µm
72h
100 µm
10 µm
10 µm
30
20
Particle at various orientations
10
3 h, (800×) ( 18.0%)*; 24 h, (700×) ( 20.7%)*;
3 h, (700×) (41.1%)*; 24 h, (550×) (51.6%)*;
0
72 h, (600×) ( 22.4%)*; 168 h, (200×) (21.1%)*.
72 h, (400×) (60.1%)*; 168 h, (600×) (58.1%)*.
CONCLUSIONS
*4.28% glucose from pretreatment
in 53-75 micron fraction
40
Particle at various orientations
* glucose yield
Pretreated corn stover
*3.82% glucose from pretreatment
in 425-710 micron fraction;
50
168h
10 µm
Reticular area (2000×)
53-75 micron
60
10 µm
Native corn stover
0
20
40
60
80
100
120
140
160
180
Time (h)
* glucose yield
WORK IN PROGRESS
ACKNOWLEDGEMENTS
water
Slurry
JEOL JSM-840
Scanning Electron Microscope
(JEOL USA Inc., Peabody, MA)
PRETREATED + ENZYME
NATIVE + ENZYME
24h
Longitudinal view (300×)
Reticular area enlarged
(4.3% glucose yield)
3h
100 µm
100 µm
Longitudinal view (200×)
Longitudinal view (200×)
Particle splitting
PRETREATED
SEM Images
Compositional
Analysis
water
loops
100 µm
1 mm
10 µm
Pretreat at 190oC, 15min
24h (52.7% glucose yield)
1 mm
Shape distribution based
on particle orientation
(20×)
(2)
Grinding and sieving
through a series of Fisher
Brand standard sieves
100 µm
Cross-sectional view (200×)
Longitudinal view (150×)
2
10 µm
100 µm
100 µm
Cross-sectional view (250×)
Cross-sectional view (250×)
• Research Challenges: Selecting representative samples
that are representative of cell wall changes during
enzyme treatment and relating these to enzyme activity.
EXPERIMENTAL PROCEDURE
PRETREATED + ENZYME
3h (40.2% glucose yield)
72h (59.6% glucose yield)
NATIVE + ENZYME
(3.8% glucose yield)
0µ
HYPOTHESES AND OBJECTIVES
PRETREATED
NATIVE
SEM Images
~1
• The present study reports the particle size effect on corn
stover pretreatment and enzyme hydrolysis, focusing on
microscopic observations of changes on particle surface.
This, combined with analyses of sugars released during
the process, gives insights on enzyme mechanisms at a
cellular level.
• Scanning electron microscopy (SEM) shows that both
liquid hot water pretreatment and enzyme treatment
induce pore formation on the surface of the corn stover.
Enzyme hydrolysis of pretreated stover results in greater
porosity and glucose conversion than enzyme hydrolysis
of stover that has not been pretreated.
• Analysis of glucose conversion during the process
suggests that 53-75 µm corn stover particles are more
susceptible to hydrolysis than 425 to 710 µm corn stover
particles. This difference is eliminated by liquid hot water
pretreatment at 190oC for 15min.
425-710 µm Corn Stover
Glucose yield (%)
ABSTRACT
• Ultrastructural changes occur for 425 to 710 µm and 53 to 75 µm corn stover particles: (1) similar during pretreatment and enzymatic hydrolysis; (2) large particles are degraded to small
particles gradually; (3) 53 to 75 µm size is more susceptible to enzyme hydrolysis than 425 to 710 µm-----pretreatment with water at 190oC, 15 min, eliminates this difference.
• Measure assessable surface area before and after
pretreatment.
• Enzyme action on native corn stover: (1) pore-digestion; (2) cut large particles into smaller pieces; (3) digest particle from it cross section along longitudinal dimension.
• Construct a cellular level model to quantify changes
during corn stover pretreatment or enzyme
hydrolysis.
• Enzyme hydrolysis of liquid hot water pretreated corn stover is connected to pore formation (during pretreatment) and enlargement (during hydrolysis).
Disruption of particles on pretreated corn stover during enzymatic hydrolysis is more significant than enzyme action on native corn stover since pretreatment generates more pores or
enlarges pore size, thus enhancing enzyme accessibility.
• The 3 to 4 times higher glucose yield upon enzymatic hydrolysis and SEM micrographs show enzyme hydrolysis of pretreated corn stover is related to the exposed plant cell wall surface
structures and/or enzyme accessible pores.
• Measure and minimize non-specific binding of
cellulases on biomass particles during enzyme
hydrolysis to improve overall enzyme activity.
• The material in this work was supported by USDAIFAFS Contract 00-52104-9663, US DOE Award
Number DE-FC36-01GO11075, and Agricultural
Research Programs at Purdue University.
• Rick Hendrickson and Linda Liu in LORRE for
helpful assistance with the experiments.
• Youngmi Kim, Tom Huang, Rebecca Davis, Winnie
Chen, Ryan Warner, Yulin Yu, Linda Liu, Rick
Hendrickson for suggestions and review of this work.