Pretreatment and Enzymatic Hydrolysis Task: Exploratory Pretreatment
subtask

Stage Gate Interim Review

9 June 2005

# Introduction

The Exploratory Pretreatment subtask began four years ago as part of the
enzyme technology research at NREL. Originally envisioned as a mechanism
to build knowledge and materials for non-cellulase enzymatic digestion
of biomass, it evolved as a means to evaluate non-dilute acid
pretreatments in terms of enzymatic digestibility. This evaluation has
primarily focused on CAFI-generated materials, but has expanded to
include other, non-traditional pretreatments, as well as the exploration
of the physical ultrastructural effects of pretreatments and enzymatic
digestion. The focus on pretreatment has enabled this subtask to serve
as bridge between the Pretreatment and Enzymology research teams at
NREL. The recent effort to understand the effect of pretreatment and
enzyme digestion on cell wall architecture has fostered a significant
tie to the Plant Cell Wall Deconstruction subtask in the Target
Conversion Research task. This cross-task collaboration is best
exemplified in the progress towards NREL's first joint milestone for the
Office of Biomass Program, "*Cell wall component susceptibility to
pretreatment chemistries and enzymatic degradation*" on schedule for
submission on Septemebr 30^th^ 2005.

As the subtask has evolved, several branches of research have diverged
from the main focus of non-cellulase digestion of biomass. These areas
focus on how various pretreatments affect the architecture of the plant
cell wall and how these changes, in turn, influence the ability of
enzyme catalysts to infiltrate and degrade the plant cell wall. The
relationships between these varied research paths are linked together in
the various pretreatment chemistries being investigated (Error:
Reference source not found). The two primary areas of focus are enzyme
assisted pretreatment and catalyst transport. The data warehouse is a
secondary function and is being led by the Targeted Conversion Research
task's Biomass Surface Characterization Management subtask.

The ultimate goal of the subtask is to enable lower severity
pretreatments in order to drive the cost of bioethanol down. In the
pretreatment arena, the current dilute-acid model imparts extensive
capital costs due to corrosivity concerns in reactor metallurgy. Other
pretreatment processes are less corrosive, but require catalyst recovery
or regeneration, large downstream processing capacity, or other issues
which impart elevated capital construction costs (Table 1). In order to
reduce the capital cost, lower severity pretreatment conditions can be
incorporated, but these invariably result in biomass that is less
digestible, typically because of high lignin and/or hemicellulose levels
remaining in the solids. To realize the cost savings of lower severity
pretreatment, improvements in biomass digestibility need to be realized.
Since these non-dilute acid methods typically result in lower
hemicellulose hydrolysis compared to dilute-acid methods, the addition
of hydrolytic non-cellulase enzymes, *i.e.* hemicellulases and accessory
enzymes, is one potential means of enhancing biomass digestibility in
these samples.

All of the information we have concerning biomass composition has been
derived from chemical composition data, i.e. one gram of glucose
measured in the hydrolysis liquor means there must be have 0.9 g of
cellulose digested. Similar conversions are used for the other sugars,
xylose from xylan for example, but all of this data is derived and
secondary. In order to evaluate physical barriers (read polysaccharide
fiber/lignopolysaccharide matrix components), we developed a plan to
directly examine cell wall components. With the help of the DOE OBP, the
sugar platform purchased several high-resolution imaging instruments
(eSEM, TEM, NSOM, AFM and confocal microscope) which NREL located in a
brand new facility. Used by numerous tasks both in and outside of the
sugar platform, these instruments, collectively known as the Biomass
Surface Characterization Laboratory, are allowing researchers to "see"
plant cell wall biopolymers at Angstrom resolution and form new ideas
about which components are important barriers to biomass conversion. Of
course, the real issue with using imaging to solve conversion problems
lies in data acquisition and interpretation. Sample preparation is
critical to maintaining relavent structural information while image
analysis must be based on multiple images compared to stringent and
relavent control samples. These issues are being addressed by the Plant
Cell Wall Deconstruction subtask in the Targeted Conversion Research
task under a D Milestone, "*Develop and validate surface analysis
methodologies for plant cell wall*" due July 30, 2005 and will be
covered in more detail in the TCR Stage Gate review.

The BSCL capabilities have triggered many new ideas in the minds of NREL
researchers. The Exploratory Pretreatment subtask has begun exploration
of which barriers affect the movement of catalysts, both pretreatment
and enzymatic, throughout the biomass and the ability of these catalysts
to enter into the plant cell wall. The early part of these transport
studies have focused on bulk movement in large pieces of corn stover.
Work on determining barriers to getting the catalysts into the interior
of the cell wall matrix will begin later this year and continue in out
years. Minimizing catalyst loading by targeting specific areas of the
biomass will enable lower catalyst loadings, leading to lowered
pretreatment and enzyme costs.

The somewhat diverse research paths in the Exploratory Pretreatment
subtask have generated an as yet incomplete dataset regarding the
relationships between pretreatment catalyst transport through biomass
and into cell wall interstices, the effect of different pretreatment
chemistries on structural barriers, and the effectiveness of
biomass-specific enzyme cocktails on digestibility and economics.
Further research will continue to make the connections between these
data sets and provide a much clearer and more detailed understanding of
biomass recalcitrance and the potential for different pretreatment
chemistries and enzyme activities to remove these barriers.

# Materials and Methods

## Feedstock

The biomass used in this work has been corn stover, either tub ground,
milled, or hand collected and sectioned. Intact corn stover was used in
transport studies in order to evaluate the "worst case" scenario; stover
that was largely intact and not subjected to size reduction of physical
structural damage. Pretreated feedstocks have emphasized AFEX material,
as it is the only CAFI feedstock we have directly received outside of
the CAFI subtask. The remaining CAFI materials are scheduled for
delivery later this year and will be examined as they are received.
Additional pretreated corn stover has been generated at NREL, either by
alkaline peroxide (1% H~2~O2, pH 11.5 (NaOH), 4 h at room temperature)
or high solids pretreatment in the vertical digester (30% solids, 2%
H~2~SO~4~, 190^o^C, 1 min residence time)

## Enzymes

Spezyme CP cellulase from Genencor International was used as the
"standard" cellulase in all experiments on enzyme digestibility.
Novozyme 188 from Novozymes Biotech was used as a standard β-glucosidase
in all work. Accessory enzymes currently being evaluated include
xylanases and pectinases. Xylanases were obtained from GCI and
Novozymes. The primary focus has been on the endoxylanase XynA from
*Thermomyces lanuginosus*. Although we had isolated a strain of *T.
lanuginosus* a decade ago from a Yellowstone National Park hot spring
and have been producing wild-type XynA in house for a number of years,
the commercial availability of this enzyme as a recombinant product from
Novozymes through Sigma Aldrich enabled us to obtain larger quantities
with much less effort. Additional commercial xylanases (GCI Multifect
xylanase and Novozymes 50030), cellulases (Novozymes 50013 and 50029),
and pectinases (USB and GCI Multifect pectinase) have been obtained and
used for several experiments, but complete datasets have not yet been
filled.

Additional activities are highly desired, but have proven difficult to
obtain. Specific enzyme activities of interest include, but are not
limited to, ferulic acid esterase, arabinofuranosidase, xyloglucanase,
β-xylosidase, laccase, lignin and manganese peroxididase, and
β-glucanases. Some of these are available commercially, but either as
crude preps requiring purification to remove interfering activities or
as high purity reagents in exceedingly small amounts. Potential sources
include university researchers currently expressing fungal glycosyl
hydrolases as recombinant products in yeast, but these collaborations
have yet to be formalized and the expression systems have been geared
towards throughput, quantity, and ease of purification and not
necessarily chosen for retained specificity and/or hydrolytic fidelity.
We are continuing discussions with these folks, but a plan to proceed is
pretty unclear at this point.

## Assays

Due to limitations on available biomass and enzymes, HPLC analysis time,
and the high number of permutations involved in carrying out enzyme
complementation studies (especially as we get into \>2 enzyme systems),
we have set up initial screening assays using microtiter plates
containing biomass and enzymes which are sealed and incubated for the
appropriate incubation time and temperature. The resulting sugars are
determined by DNS assay. Surface plots are generated and the transition
points determined (Figure 2). Points around these areas are tested more
extensively by standard 10 mL enzyme digestion assays. We have
demonstrated 5.3% standard deviation across all wells of a 96 well
plate, with a 95% confidence interval of about 1.1%. The water loss from
these samples averages at about 2% per day of incubation at 50^o^C,
however internal glucose standard wells can account for most if not all
of this.

## Analytical 

Sugars from pretreatment hydrolysis and enzymatic digestion were
analyzed by HPLC on a Shodex lead column. Reducing sugars were
determined by the DNS assay. Compositional analysis for cellulose,
xylan, lignin, and other components were carried out by standard NREL
LAP protocols.

# Results and Discussion

## Enzyme Assisted Pretreatment

In contrast to dilute acid, the CAFI alternative pretreatments cover a
range of product compositions, from those resulting in little or no
change in composition to pretreatments approaching dilute acid levels of
hemicellulose removal (Table 2). As several of these conditions retain a
high fraction of the hemicellulose in the solids, additional enzymes can
benefit these pretreatments.

Control experiments using unpretreated corn stover have been carried out
with multiple cominations of cellulase, xylanase, and pectinase.
Addition of these non-celulase enzymes always increases sugar release,
but the overall digestibility is low, as expected for unpretreated
biomass (Table 3).

The ability of xylanase to enhance the release of sugars from biomass by
cellulase has been demonstrated. Ammonium Fiber Explosion
(AFEX)-pretreated biomass has been shown to be more digestible by
Spezyme when xylanase is added (Table 4). These data, taken at 60 h of
digestion, show the dependence of additional sugar release on the
severity of the pretreatment. Lower severity showed higher percent
increases compared to sugars released from unpretreated corn stover, but
lower overall yield. These results indicate a severity-dependent
physical change in the material, either a physical disruption allowing
better enzyme access or a chemical change such as an alteration in
lignin structure or a rearrangement of hemicellulose components. The
most severely pretreated stover (AFEX3) showed a significantly greater
susceptibility to cellulase digestion compared to lower severity stover
(AFEX1 and 2). AFEX 1 and 2, however, indicated a much higher degree of
enhanced cellulase digestion when xylanase was added, especially when
compared to raw stover. Although this was an expected and to some extent
obvious result, the greater susceptibility to enzymatic enhancement of
digestion indicates a potential route to maintenance of yield and
digestibility with reduced pretreatment severity.

We are currently examining the effects of pectinase on sugar release
from pretreated corn stover, including AFEX, dilute acid, and alkaline
peroxide pretreatments. Initial data shows minor increases in sugar
release from AFEX and alkaline peroxide when pectinase is added,
however, the amount of reducing ends increases disproportionately to the
free sugar release. This suggests that the pectinases may be endo-acting
and generating free ends that are not resulting in detectable free
sugars. The pectinase/cellulase treatment of dilute acid corn stover
shows slightly greater increase in sugar release compared to cellulase
alone.

## Catalyst Transport

The work on catalyst transport can be divided into two sections,
movement of liquid through the interstices of the bulk biomass and
movement of catalyst from the bulk liquid into the cell wall itself. The
bulk transport work was presented as a poster at the 27^th^ Symposium on
Biotechnology for Fuels and Chemicals in Denver, May 2005 and has been
submitted to Applied Biochemistry and Biotechnology. Although most of
the high-resolution imaging analysis is being carried out by the
Targeted Conversion Research task's Plant Cell Wall Deconstruction
subtask, Exploratory Pretreatment has completed two milestones this year
regarding this work. The first, "*Develop methodologies for progressive
pretreatment and enzymatic hydrolysis of "identical" stover anatomical
sections*" was submitted in February and detailed the construction of
immobilized pipe reactors for selectively pretreating corn stover
internode sections and pieces in preparation for image analysis. The
second, "*Generate progressively pretreated and enzymatically hydrolyzed
samples for ultrastructure analysis by Targeted Research Project*"
expanded on the first and supplied the initial materials to PCWD for
imaging and analysis.

Movement of liquid throughout the biomass is dependent on several
things, including the available porosity of the biomass, the size of the
molecules being translocated, and the affinity of the catalysts for the
biomass. Corn stover anatomy imparts several potential transport routes,
such as vascular tissue, apoplastic space, cell wall pits, and fissures
caused by senescence, as well as several barriers (Figure 3). In our
work on bulk transport, we have used Direct Blue I dye as a catalyst
analogue. Although initial work used capillary action (the old food
coloring and celery experiment), we rapidly realized that much of the
biomass in intact internodes was blocked from liquid access. Several
rounds of experimentation demonstrated that the primary impediment to
liquid movement was entrained air in the biomass. Without a place to go
and a system to facilitate its removal, trapped air prevented liquid
from saturating the biomass. This was exceedingly obvious in intact
internodes, where the rind prevented any type of radial air escape or
dye infusion.

Understanding how plant structure affects catalyst transport is key to
overcoming a number of pretreatment bottlenecks traditionally addressed
through costly process modifications. Using the stem internode as a
simplified model system, we have observed dye transport over a range of
impregnation conditions. Through light microscopy and subsequent image
analysis, we have observed a number of possible barriers and pathways
for the transport of catalyst through the internode matrix.

### Effect of temperature and pressure on dye uptake and distribution

Dye impregnation was very slow at room temperature and atmospheric
pressure (Figure 4. Lateral sections of internodes impregnated with
Direct Blue I at atmospheric pressure and elevated temperatures: (A)
50oC for 2 hours, (B) 70oC for 2 hours, and (C) 90oC for 2 hoursFigure
4). Even after 24 hours submerged in dye solution, a saturation of only
20-25% was achieved, wit 7dye movemen 8indicating that dye entered
laterally through segments ends and not through the rind. Increasing the
temperature of the dye solution did not significantly improve dye uptake
into the internodes. After 2 hours of exposure to dye solution at higher
temperatures, the saturation was only \~25% and dye movement was
restricted to internode ends (Figure 5, B

Saturation of 25-40% of the void space was achieved immediately (\<30
sec) when segments were impregnated under 22.5 inches Hg vacuum (A).
Under this condition, dye moved throughout the entire internode length
and microscopic observation of cross sections showed the presence of dye
within and along the vascular bundles, as well as throughout the entire
apoplastic continuum. After the initial rapid saturation, further
increase in moisture content in the internodes occurred at a much slower
rate, and a long period under vacuum was required t\>12h) to achieve
100% saturation. At lower vacuum strengths, more time was required to
achieve greater than 25% saturation. In cases where internodes were less
than 25% saturated, uniform dye distribution was not achieved.

Preheating of dry internodes and rapidly submerging them in dye solution
was studied as another method of dye transport into internodes (Figure
7). 3Saturations levels of 20-30% could be achieved by preheating pieces
above 10 ºC for only 5 mis and longer heating times did not appear to
significantly enhance dye uptake. Examination of lateral sections showed
dye movement throughout the internode length at these saturation level1.
Closer examination of cross sections showed that dye was present in most
of the apoplastic spacew While dye was present both within and around
the vascular bundles near the ends, it was present only in the apoplast
around the vascular bundles in the middle of the pieces.

The apparent inability of liquid to complete saturate the internode
without extended time at high vacuum suggests the idea of entrained air
being a primary impediment. The ability of brief exposure to vacuum to
distribute dye throughout the tissue, but only filling \~25% of the
potential void spaces indicates that part of the biomass is not being
saturated. Since we can see the dye in the apoplast and in the vascular
bundle cells, the most likely unsaturated section is the parenchyma cell
interior. These cells appear white and undyed under these impregnation
conditions. The effect of heating the air inside the stover and then
allowing it to cool and contract in the presence of dye is analogous to
brief vacuum exposure. The results shows dye saturation at the same
approximate level, but also present only in the apoplast and the
vascular bundles, supporting the idea that air is a primary barrier.

# 

# Our experiments with Direct Blue I revealed a number of pathways and barriers to the transport of dye into the model corn internode system. In general, the primary entry points were at the internode ends. Dye movement from this point occurred via four main pathways: 1) through the intercellular space surrounding parenchyma cells (the apoplastic continuum), 2) through vascular bundles, 3) along the parenchyma-vascular bundle interface, and 4) along the inner rind-pith interface. Radial movement of dye into the internode through the rind was not observed, suggesting that the waxy outer surface of the rind acts as a barrier to penetration (just as nature intended). The mechanisms driving fluid into these pathways in dry corn stems are most likely to be capillary action, varying pressure differentials or a combination of both. The primary barriers to movements were presented by the waxy outer surface of the rind and the air trapped within individual cells.

Dye transport was slow and uniform distribution was difficult to achieve
when impregnation was conducted at atmospheric pressure. Quick and
uniform distribution of dye was easily achieved when internodes were
preheated prior to impregnation or impregnated under high vacuum. In
both cases uniform distribution throughout most of the observed pathways
mentioned above occurred when the internode void space was 25 to 35%
saturated. Presumably the remainder of unsaturated volume was the
interior void space of the bulk of the parenchyma cells. We speculate
that dye may move to the interior of cells immediately surrounding the
vascular bundles but have questioned whether transport occurs into the
middle lamella and cell wall layers when the apoplast is fully
saturated. If catalyst from the apoplast can be absorbed into the cell
walls, then catalyst could be in contact with each hydrolyzable site
without the need to saturate the entire parenchyma intracellular space.
Complete impregnation of cell walls with catalyst could be achieved with
a moisture content of only \~30%. If our speculations are correct then
one could theoretically achieve catalyst contact with 100% of the cell
wall surfaces by saturating only 30% of the internode void volume. This
could, in turn, increase the solids content in pretreatment reactors up
to \~70%, significantly higher than current solid loadings of \~30-50%
*(1,* 2). Higher solids content during pretreatment would result in
lower use of process water, reduction in volume of catalyst, and higher
sugar concentrations; all of which would reduce downstream processing
costs.

# Conclusions

# 

The enzyme assisted pretreatment work is progressing, albeit slowly. We
have had some successes using commercial xylanases and limited success
with commercial pectinases, however, lack of good commercial sources of
clean accessory enzymes is hampering our ability to evaluate other
enzyme combinations. The CAFI pretreated material has been slow to be
delivered to NREL, but is expected soon. At a minimum, we expect to be
able to evaluate the various CAFI feedstocks using xylanase and
pectinase enzymes by the end of the year. We are continuing to negotiate
with commercial enzyme vendors for access to additional enzymes and have
also explored potential collaborations with academic researchers
expressing fungal glycosyl hydrolases in heterologous hosts, but have
concerns about the expression systems being used. We also have proposed
collaborating with the Targeted Conversion Research task to produce a
few of these enzymes in-house through heterologous expression. Despite
the concern that this may be time consuming, our experience with fungal
cellulase expression supports a relatively simple production of a few
enzymes could be accomplished with minor resource allocation.

The primary observed pathways for catalyst transport through our simple
corn internode model were: 1) through the vascular bundles, 2) along the
vascular bundle-parenchyma cell interface, 3) throughout the apoplastic
continuum, and 4) along the rind-parenchyma interface. Transport via all
routes was initiated at the open ends of the internodes, while virtually
no transport occurred through the impermeable rind exterior. Air trapped
inside cells, particularly the parenchyma, proved to be a difficult
barrier, blocking access to the interior cell wall surfaces. Dye
transport was slow and a uniform distribution was difficult to achieve
when impregnation was conducted at atmospheric pressure. Quick and
uniform distribution of dye was easily achieved when internodes were
preheated prior to impregnation or impregnated under high vacuum. In
both cases uniform distribution throughout most of the observed pathways
mentioned above occurred when the internode void space was 25 to 35%
saturated. We have speculated the possibility that dye may move to the
interior of cells surrounding the vascular bundles through pit membranes
and have questioned whether transport occurs into the middle lamella
when the apoplast is fully saturated. Currently, more experimental work
is necessary to bring light to these issues, but if our speculations are
correct then one could theoretically achieve catalyst contact 100% of
the cell wall biomass by saturating only 25% of the internode void
volume.

# REFERENCES

1\. Schell, D. J., Farmer, J., Newman, M. and McMillan, J. D., (2003),
Applied Biochemistry and Biotechnology, 105/108, 69-85.

2\. Tucker, M. P., Kim, K. H., Newman, M. M. and Nguyen, Q. A., (2003),
Applied Biochemistry and Biotechnology 105, 165-177.

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