Enzymatic Hydrolysis

Introduction

The overarching goal of this work is to investigate high-solids
enzymatic cellulose hydrolysis for biomass conversion using corn stover
as the model feedstock. The immediate objectives are to pursue in-house
and subcontracted work to understand high-solids enzymatic-based
cellulose processing, which is expected to reduce both the risk and cost
of enzyme-based process technology. This subtask will have two distinct
but related efforts to 1) understand the rheology and mixing
characteristics of high-solid biomass slurries and 2) to understand the
performance of enzymatic cellulosic saccharification at high solids
loadings. High-solids saccharification work will advance efforts in the
Sugar Processing Integration (SPI) task to produce process relevant
residues (e.g., wastewater or combustion residue) for analysis and
testing, enabling that group to provide process relevant samples to
industry and groups.

Understanding rheology of biomass slurries is important for designing
equipment and predicting process performance and was suggested as
appropriate for investigation at the SPI stage gate interim review
meeting held in Sept. 2004. Initial efforts in this area will focus on
characterizing rheology of high-solid biomass slurries during enzymatic
saccharification. Some of this work will be subcontracted and will focus
on fundamental properties influencing slurry viscosity as a function of
the extent of saccharification and will complement in-house efforts to
characterize rheology and other related solid phase properties (particle
size, pore size distribution, etc.) that influence cellulose hydrolysis
kinetics. Complementing this effort will be work to understand the
mixing characteristics of high-solid biomass slurries during
saccharification. Achieving good enzyme distribution and uniform
temperature control with low mixing power input is one key to good
process performance at low cost. This work will also be subcontracted
and is expected to generate better information for estimating capital
and operating cost for high solids bioreactors.

The second effort to characterize the performance of high-solids
enzymatic cellulose saccharification will proceed along two avenues of
investigation. We will complete work started last year in the SPI task
to characterize cellulose saccharification of washed pretreated corn
stover using a new high-solids bioreactor. The goal is to demonstrate
cellulose conversion yields similar to that achieved in a shake flask,
which unlike stirred tank reactors doesn't suffer from heat transfer
limitations. Additionally, we began work to understand the effect
hydrolyzate liquor on cellulose conversion yields at high solids
loadings. The performance of the newly developed cellulases from the
enzyme manufactures will also be assessed during this work.

# Background

![](media/image1.wmf){width="4.375196850393701in"
height="3.530708661417323in"}

Early work in this area discovered that enzymatic cellulose conversion
yields obtained in shake flasks were much greater than those obtained in
stirred tank reactors (STR), even at relatively modest solids loadings
of 10%-15% (w/w). Figure 1 shows cellulose conversion for the two
reactor systems loaded at a 10% solid concentration with pretreated corn
stover (PCS). The PCS was produced by dilute sulfuric acid pretreatment
using a pilot-scale 1 ton/d pretreatment reactor (Schell et al. 2003).
These results are believed to reflect the fact that even modest solid
levels were hindering good fluid mixing and likely producing temperature
gradients within the STR system that reduced cellulose conversion
yields. Shake flasks do not experience these problems, since they are
incubated at a uniform temperature.

# Key Results

***Enzymatic Hydrolysis of High Solid Slurries***

Initial work explored enzymatic cellulose conversion of washed PCS in
shake flasks. Figure 2 shows 7-day cellulose conversion yields and
corresponding glucose concentrations as a function of insoluble solids
concentration at enzyme loadings of 20- or 40-mg protein/g cellulose.
The horizontal dashed line at 80% represents our target goal of 80%
cellulose conversion. This plot clearly illustrates the trade off
between cellulose conversion yield and glucose product concentration and
demonstrates how cellulose conversion decreases at higher solids
loadings, presumably because of increasing end-product inhibition. As
enzyme manufacturers further improve enzyme effectiveness, high
conversion yields will be achieved at more economical enzyme dosages.

More detailed investigations explored performance at high solids
loadings when it became apparent that end product and/or mass transfer
limitations were occurring. Mass transfer becomes a problem when the
rate of diffusion of reactants to the reaction site or rate of diffusion
of products away from reaction sites is slower than the reaction rate.
This could occur during high-solids enzymatic saccharification, since
there is little free water available to facilitate movement of enzymes
and sugars.

![](media/image2.wmf){width="4.625196850393701in"
height="3.7681102362204726in"}Shake flask results presented in Figure 3
suggest that mass transfer limitations may be reducing cellulose
conversion yields at high solids loadings (\>20%). This figure presents
glucose concentration during enzymatic cellulose hydrolysis as a
function of the initial insoluble solids concentration at constant time
points. At lower solids levels, glucose concentration increases nearly
linearly with solid loading as would be expected for a first order
reaction. But, glucose concentration decreases significantly after a
critical solids level is reached. If end product inhibition were the
only factor affecting performance at high solids level, than glucose
concentration should level off at a constant value. The observed
decrease in concentration suggests that mass transfer limitations are
significantly affecting performance at high solids levels.

However, the goal is to perform cellulose saccharification in
commercially relevant reactor systems, for example, this could be a STR
or some other reactor type. In previous work that will not be discussed
in detail in this paper, we demonstrated that a fed-batch approach could
also be used to achieve high effective solids concentrations (\~25%) in
STRs with cellulose conversion yields similar to that achieve in shake
flasks (see Figure 2). As an alternative approach, we designed a batch
high solids bioreactor (HSBR) that is simple to operate and can mix high
solid biomass slurries. The HSRB is pictured in Figure 4 and consist of
a cylindrical ![](media/image4.png){width="3.25in"
height="2.594488188976378in"}glass chamber (reactor section), in which
the interior surface is swept by a series of blades positioned along the
length of the reactor. The rotational speed of the blades can be
adjusted from very slow (2-5 rpm) to rather fast (50-60 rpm), but it is
the continual turnover and kneading of material by the blades that
promotes good mixing. An electric heating jacket surrounding the reactor
creates and maintains a uniform temperature environment. Both
temperature and pH are monitored and controlled by probes inserted
through the reactor side.

Figure 5 compares cellulose conversion yields achieved in the HSBR with
those achieved in shake flasks at two different enzyme loadings. This
side-by-side comparison reveals that cellulose conversion in the HSBR
was similar to or even better than that achieved in shake flask. This
promising result suggests that reactor systems can be designed for high
solids cellulose saccharification.

![](media/image5.wmf){width="4.125196850393701in"
height="3.245275590551181in"}Recently, we began working on whole slurry
hydrolysates instead of washed solids. However, insoluble solid
concentrations are much lower than can be achieved with washed solids
because PCS as produced is only 18% insoluble solids, and thus the upper
limit for solid concentration. Whole slurry and washed solids results
are compared in Figure 6. At the same level of insoluble solids,
cellulose conversion is lower for pretreated solids in the presence of
background liquor resulting in lower glucose production by enzymatic
hydrolysis. However, the absolute glucose level is greater because of
glucose initially present in the hydrolysate. The results for both
washed solids and whole slurries suggest that glucose inhibition at
modest insoluble solids loadings is not severely affecting conversion
yields. The decreasing yields as insoluble solids level increase from 5%
to 15% may be due to adsorption of enzyme on solids, which may be
further exacerbated by adsorption onto liquid phase components present
in the whole slurry.

![](media/image6.wmf){width="4.5in" height="3.589763779527559in"}

***Rheology and Mixing of High Solid Biomass Slurries***

High solids saccharification is difficult due to the challenging
rheological characteristics of high-solid biomass slurries that can
cause non-uniform heat and mass transfer. In addition, dynamic changes
in rheology and biomass properties occur as the cellulose structure is
broken down during enzymatic hydrolysis. The objective of this work is
to develop a fundamental understanding of the rheology of high solids
biomass slurries during enzymatic hydrolysis.

We recently investigated the rheological properties of a dilute acid
pretreated corn stover slurry at a high solid concentration in
preparation for work on enzymatically hydrolyzed slurries. Our results
show that pretreated corn stover slurries behave as yield stress fluids
that exhibit shear thinning properties. The rheological behavior of
these slurries can be described using a Hershel-Buckley (H-B) model and
the experimental and predicted behavior is shown in Figure 7. The H-B
model can be written as,

![](media/image7.wmf){width="3.6826771653543307in"
height="2.852755905511811in"}$\sigma = \sigma_{y} + {K\gamma}^{n}$

where σ is the shear stress (Pa), σ~y~ is the yield stress, K is the
consistency index, γ is the shear rate and n is the power law index.
Model parameters were determined by regression of experimental data. The
results demonstrate that viscosity increases with increasing insoluble
solid concentrations up to 30%. However, when insoluble solids
concentration increased beyond 30%, there was little change in the
rheological properties or slurry flow behavior (see Figure 7). Visually,
there is no continuous liquid phase above a concentration of 30%, which
is likely due to complete absorption of liquid by the biomass. This
indicates that flowability depends on the presence of a continuous
liquid phase.

# Future Work

***Near term (FY06-07)***

Our work on enzymatic hydrolysis has demonstrated that performance
limitations exist even at low solids concentrations, which may not be
entirely due to end-product inhibition. Furthermore, mass transfer
limitations are also apparent at high solids concentrations that further
reduce conversion yields. Figure 8 presents a simple gantt chart
outlining activities (tasks 2-6) for the next few years. The substrate
for this work will be dilute acid (DA) pretreated corn stover. We
propose to extend efforts to understand the impact of background sugars
and other inhibitors on cellulose conversion while testing both
commercial and advanced enzyme preparation being developed by the enzyme
manufacturers (tasks 2 and 6). Testing will be done with both Genencor
and Novozyme enzymes. Also two new subcontracted efforts are starting
shortly with the goals to advance understanding of rheology (task 3) and
mixing (task 4) of high solid biomass slurries, primarily to facilitate
efforts to design large-scale bioreactors for enzymatic cellulose
saccharification.

![](media/image8.png){width="6.904166666666667in"
height="1.5527777777777778in"}

During enzymatic hydrolysis, cellulose is solubilized leading to changes
in biomass structure that effect slurry rheology. Little cellulose
conversion is required before the volume of free liquid increases and
flowability and mixing characteristics of the slurry are improved.
Characterizing solid phase properties of cellulosic biomass during
saccharification and determining their impact on slurry rheology may
provide important insights into both reactor design and the choice of
process configurations (e.g., separate hydrolysis and fermentation
versus simultaneous saccharification and fermentation). We will perform
experiments to measure changes in the solid phase properties (e.g.,
composition, particle size distribution, free liquid volume, insoluble
solid concentration, pore size distribution) of biomass during enzymatic
hydrolysis and relate these changes to the kinetics of sugar release and
to slurry rheology (task 5)

*Longer Term*

Beginning in FY07, new pretreatment technologies may be developed that
perform better and are more cost effective than current pretreatment
technologies. But much of knowledge learned during this work on dilute
acid pretreated corn stover is still expected to be relevant.
Nevertheless, we expect new technologies will require additional
development efforts and testing. We propose to assess these issues
beginning in mid-FY07 (task 9), and then to initiate research activities
to address these issues (task 10). We will also continue to provide
feedback to the enzyme manufacturers that may help improve further
improve cellulase enzymes and we will test these enzymes under realistic
process condition when they become available (task 11).

The overall goal of this work is to demonstrate effective high-solids
enzymatic cellulose saccharification in economical reactor
configurations using the newest, most effective, lower-cost enzymes
being produced by the enzyme manufacturers. Insights gained from this
work will facilitate SPI's efforts to incorporate high-solids enzymatic
saccharification into pilot-scale process integration. Ultimately, the
efforts discussed above support our goal to provide process relevant
information to the emerging biorefinery industry.

# References 

Schell, D., J. Farmer, M. Newman, J. McMillan (2003) Dilute sulfuric
acid pretreatment of corn stover in a pilot-scale reactor: Investigation
of yields, kinetics, and enzymatic digestibility of solids. *App.
Biochem. Biotechnol.* **105-108**, 69-86
<http://devafdc.nrel.gov/bcfc/oraclefind3.cgi?7893>.