Optimization of pH Controlled Liquid Hot Water Pretreatment of Corn Stover
Nathan Mosier1,2, Richard Hendrickson1, Nancy Ho1, Miroslav Sedlak1, Michael R. Ladisch1,2,3
1
2
Laboratory of Renewable Resources Engineering
Department of Agricultural and Biological Engineering
3
Department of Biomedical Engineering
LORRE
500 Central Dr.
Purdue University
West Lafayette, IN 47907
Phone: 765-494-7022
Fax: 765-494-7023
Email: ladisch@purdue.edu
Abstract
Controlled pH, liquid hot water pretreatment of corn stover has been optimized for
enzyme digestibility with respect to processing temperature and time.
This processing
technology does not require the addition of chemicals such as sulfuric acid, lime, or ammonia
that add cost to the process because these chemicals must be neutralized or recovered in addition
to the significant expense of the chemicals themselves. Second, an optimized controlled pH,
liquid hot water pretreatment process maximizes the solubilization of the hemicellulose fraction
as liquid soluble oligosaccharides while minimizing the formation of monomeric sugars. The
optimized conditions for controlled pH, liquid hot water pretreatment of a 16% slurry of corn
stover in water was found to be 190°C for 15 minutes. At the optimal conditions, 90% of the
cellulose was hydrolyzed to glucose by 15 FPU of cellulase per gram of glucan. When the
resulting pretreated slurry, in undiluted form, was hydrolyzed by 11 FPU of cellulase per gram of
glucan, a hydrolyzate containing 32.5 g/L glucose and 18 g/L xylose was formed. Both the
xylose and the glucose in this undiluted hydrolyzate were shown to be fermented by recombinant
yeast 424A(LNH-ST) to ethanol at 88% of theoretical yield.
Keywords: fuel ethanol, liquid hot water pretreatment, lignocellulose, bioprocessings, biomass
2
Introduction
The controlled pH, liquid hot water process for pretreating plant materials has several
advantages. Foremost, this processing technology does not require the addition of chemicals
such as sulfuric acid, lime, or ammonia that add cost to the process. These chemicals must be
neutralized or recovered thus adding to the expense of the chemicals themselves. Second, an
optimized controlled pH, liquid hot water pretreatment process will maximize the solubilization
of the hemicellulose fraction as liquid soluble oligosaccharides while minimizing the formation
of monomeric sugars (Weil et al., 1997, 1998a, 1998b; Bobleter, 1994; Mok and Antal, 1992).
The minimization of complete hydrolysis to monosaccharides minimizes the subsequent
degradation of these sugars to various aldehydes (Weil et al., 1998b; Ladisch et al., 1998; Beery
et al., 2000) during pretreatment. The formation of these degradation products lower the yield of
fermentable sugars obtained from the pretreated material and more importantly these degradation
products are inhibitory to yeast and bacterial fermentations in concentrations as low as 0.25%
w/w (Zaldivar et al., 1999; Delgenes 1996; Palmqvist et al., 1999).
Materials and Methods
Corn Stover
Corn stover was obtained from the National Renewable Energy Laboratory (NREL) from
a large batch grown and harvested near Harlan, IA. NREL washed, dried, and knife-milled the
stover to pass a ¼ inch (6.4 mm) screen. All collaborating laboratories in the Biomass Refining
Consortium for Applied Fundamentals and Innovation (CAFI) evaluated their pretreatment
technology using this common batch of corn stover. The ¼ inch (6.4 mm) stover was used for
the pretreatment optimization. Part of the stover received from NREL was milled in a laboratory
3
mill to pass 40 mesh screen. This ground stover was used for compositional analyses and for the
CAFI enzyme digestibility assays described below.
Cellulase Preparations
Allotments of Spezyme CP (Genencor, Palo Alto, CA) from a single production batch
were obtained and stored by NREL for the CAFI research. NREL also performed regular
enzyme activity assays to monitor the stability of enzyme over time. Aliquots of the enzyme
enzyme were given to each CAFI collaborator. Enzyme activity (FPU/mL of liquid) were
determined by NREL and all loadings listed below are based upon this standard activity
measurement. Celluclast 1.5 L and Novozyme 188 (Novozyme, Franklinton, NC) were obtained
from Sigma-Aldrich (St. Louis, MO). The cellulase activity for a 1:1 blend of Celluclast 1.5 L
and Novozyme 188 were determined at Purdue using the NREL standard cellulase activity assay
for filter paper units (Adney and Baker 1996) and was found to be 51 FPU per mL of mixture.
Liquid Hot Water Pretreatment
Figure 1 illustrates the processing scheme for continuous production scale pH controlled
liquid hot water pretreatment of corn stover using a plug-flow, coil reactor. However, the
optimization of the pretreatment parameters (temperature and time) required the use of
laboratory scale simulated plug-flow reactors constructed from 316 stainless steal tubes heated in
a fluidized sandbath to control the reaction time and temperature. Stainless steel tubes having a
length of 4.5 inches, an outside diameter of 1.0 inch and a measured volume of 45 mL were used
to construct the reactors. Swagelok® 316 stainless steel caps were fitted onto each end of the
tubes to maintain reactor pressure and to prevent the vaporization of the liquid during
pretreatment. The working volume of each tube was 33.75 mL which allows a 25% head space
above the sample for expansion during heating. Each reactor tube contained 5.18g (wet) of ¼
4
inch (6.4 mm) corn stover (11-13% MC) and 28.57 mL of de-ionized water resulting in a 16%
solids slurry. A Tecam® SBL-1 fluidized sandbath was used for heat-up and temperature control
of the reactors (Cole-Parmer, Vernon Hills, IL).
Heat-up times were measured using
thermocouple-fitted reactors filled with water. The heat-up time was found to be 5 minutes for
all temperatures examined, thus a pretreatment time of 15 minutes at 190°C required a total time
of 20 minutes. Cool-down was achieved by quenching the reactors in room-temperature water
which caused the internal tube temperatures to drop below 100°C in less than 30 seconds.
Compositional Analyses
Corn stover composition was determined by NREL standard analytical procedures.
All
calculations of yield in this paper use the composition as determined by NREL for this common
batch of stover. The carbohydrate content (glucan and xylan) of the pretreated corn stover was
determined at Purdue University following the sulfuric acid hydrolysis and HPLC analysis
protocol developed by NREL (Ruiz and Ehrman, 1996a). Pretreatment liquid was analyzed by
the NREL LAP-014 method (Ruiz and Ehrman, 1996b) to measure the soluble monosaccharides
and oligosaccharides.
Optimization of Pretreatment Parameters
The single criterion used to determine the optimal pretreatment parameters was the
maximization of enzyme digestibility of the pretreated material. In order to determine the effect
of pretreatment on the enzyme digestibility of the corn stover, the pretreated stover was analyzed
using the NREL LAP 009 (Brown and Torget, 1996) enzyme digestibility procedure with the
changes noted below. The entire contents (liquid and solid) of one pretreatment tube described
above for each condition were hydrolyzed using a cellulase loading of 30 FPU/g glucan. This
was accomplished by emptying the entire content of three replicate reaction tubes into a 250 mL
5
polypropylene bottle following pretreatment.
To the pretreated slurry was added 1 mL of
sodium azide (1 mg/mL) to prevent microbial growth and a 1-to-1 mixture of Celluclast 1.5 L
and Novozyme 188 (Novozyme, Franklinton, NC) at a loading of 30 FPU/g glucan. Deionized
water was then added to bring the total liquid volume to 100 mL. This diluted the pretreated
slurry by approximately 1/3rd. Hydrolysis was carried out in a New Brunswick Scientific model
G24 Environmental Incubator Shaker (Edison, NJ) at 50C and 250 rpm for 48 hours. The
hydrolyzate was analyzed by HPLC in triplicate utilizing a HPX-87H organic acid column (BioRad Laboratories, Hercules, CA). Soluble xylan yields are reported as a total of monomeric
xylose and xylobiose.
Measurement of solublization of the corn stover by pretreatment or pretreatment followed
by hydrolysis was determined by filtering the contents of one pretreatment reactor from each
treatment condition through Whatman #1 filter paper in a Buchner funnel. The retained solids
were washed with 500 mL of 80°C deionized water and dried at 105°C for 4 hours before
weighing. Solublization was calculated by difference of final dry mass of undissolved solids
from initial dry mass of stover loaded into the reactor tube.
Cellulase Digestibility Comparison to Other Pretreatment Methods
For comparison between pH controlled liquid hot water pretreatment and other
pretreatment processes evaluated in the Biomass Refining Consortium for Applied Fundamentals
and Innovation (CAFI), corn stover ground to pass a 40 mesh screen was pretreated at the
optimal conditions derived from the above experimental procedure in 50 tube reactors. The
contents of the reactor tubes were filtered using Whatman #1 filter paper and washed with hot
deionized water as described above. The washed, pretreated corn stover was then pooled and
thoroughly mixed. This pretreated corn stover was tested at three enzyme loadings (60, 15, and
6
7.5 FPU/g glucan) of the common batch of Spezyme CP (Genencor, Palo Alto, CA) using a
standardized digestibility procedure developed for the CAFI project based upon the NREL LAP
009 – Enzyme Digestibility (Brown and Torget, 1996). The digestibility of the pretreated stover
for each enzyme loading was measured in triplicate. For each replicate, wet, pretreated stover
was weighed into a 250 mL polypropylene bottle. The mass weighed into the bottles contained 1
g of glucan as determined by compositional analysis of the pretreated solids.
Into the
polypropylene bottle was also added the appropriate volume of enzyme, 1 mL of sodium azide (1
mg/mL), pH 4.8 citrate buffer, and distilled water as described in the NREL LAP-009 (Brown
and Torget, 1996). Hydrolysis was carried out in a New Brunswick Scientific model G24
Environmental Incubator Shaker (Edison, NJ) at 50C and 250 rpm for 7 days. Samples were
then analyzed for cellobiose, glucose, xylobiose, and xylose by HPLC in triplicate utilizing a
HPX-87H organic acid column (Bio-Rad Laboratories, Hercules, CA). Yields of glucan were
calculated from the HPLC measured glucose and cellobiose plus the glucose measured in the
pretreatment liquid as described above.
Yields of xylan were calculated from the HPLC
measured xylose and xylobiose plus the xylose measured in the pretreatment liquid as described
above.
Hydrolyzate Fermentability
The fermentability of the pretreated slurry after cellulase hydrolysis was also tested. The entire
contents (both liquid and solids) of three reactor tubes after pretreatment at the optimized conditions
were emptied into a single 1 L autoclavable flask. This slurry was then hydrolyzed with 11 FPU/g
glucan cellulase (1:1 mixture of Celluclast 1.5L and Novozyme 188) at 50°C for 4 days. The resulting
hydrolysate was then fermented at 37C for 56 hours using the glucose and xylose fermenting
recombinant yeast 424A(LNH-ST) (Ho et al., 1999; Sedlak et al., 2003). For ethanol production, 8 mL
7
of seed culture was used to inoculate 100 mL YEPD (YEP plus 2% glucose) in a 300 mL baffled
Erlenmeyer flask equipped with a sidearm. The cultures were incubated in a shaker at 30 C and 200
rpm and grown aerobically overnight (O.D. 350-400 KU). The yeast was harvested by centrifugation (J21 Beckman) at 5000 rpm for 5 min at room temperature. The supernatant was discarded and the cells
were transferred into a 300 mL baffled Erlenmeyer flask containing 100 mL of hydrolysate
supplemented with 10 mL of 10 % yeast extract. The initial cell mass concentration prior to
fermentation in each experiment was 8.5 - 9 g dry weight/L. The flasks were then sealed with Saran
wrap to allow fermentation to be carried out under largely anaerobic conditions. The cultures were
placed in shaker and incubated at 37 C. At regular intervals one mL samples of the fermentation
mixture was removed for monitoring the fermentation. Fermentation samples were analyzed by HPLC
using a BioRad HPX-87H column under similar conditions described previously for the corn stover
compositional analysis procedure. This HPLC column was able to resolve glucose, xylose, glycerol, and
ethanol as well as organic acids in the fermentation broth samples.
Scanning Electron Microscopy of Stover Surface Characteristics
Samples of corn stover before and after pretreatment were mounted on stubs and sputter-coated
with AuPd prior to imaging with a JEOL JSM-840 scanning electron microscope (SEM) using 5kV
accelerating voltage. Digital images were captured using 1280x960 resolution and 160 second dwell
time.
Results and Discussion
Controlled pH Liquid Hot Water Pretreatment Optimization for Corn Stover
Figure 2 a) illustrates that up to 40% of the corn stover was dissolved during pretreatment
over the range of temperatures and times tested. When the pretreated material is then hydrolyzed
for 48 hours with 30 FPU/g glucan, the dissolution approaches 60% in the best case (Figure 2 b).
8
The yields of fermentable sugars from the dissolution of the corn stover are illustrated in Figure
3. Nearly 80% of the cellulose is converted to fermentable glucose in 48 hours by 30 FPU/g
glucan in the best case as shown in Figure 3 a).
While the cellulase enzyme preparations used to hydrolyze the pretreated stover had little
xylanase activity, the hydrolyzate contained significant amounts of monomeric xylose as
indicated in Figure 3 b). Table 1 shows the yields of the various structural sugars as soluble
monomeric and oligomeric sugars generated by pretreatment at 190°C for 15 minutes. Very
little of the structural sugars have been hydrolyzed to monomeric form. Nearly 40% of the xylan
is solublized as oligomers through pretreatment, as measured by 4% sulfuric acid hydrolysis of
the pretreatment liquid (NREL LAP-014). These results suggest that the addition of xylanase to
the enzyme blend may significantly increase the yield of monomeric xylose from pretreated corn
stover. There is also substantial amount of glucan recovered in the pretreatment liquid as
monomeric glucose (5.7%). The xylose yields are less than 70%, which indicates that some
degradation may be occurring during pretreatment, especially at the higher temperature (190°C
and especially 200°C) treatments. This is not surprising since the hemicellulose fraction is more
labile than the cellulose. The difference in result between pretreatment at 200°C for 5 minutes
and 190°C for 15 minutes can not be defined as being statistically different; however the lower
pretreatment temperature (190°C) was selected as preferable since the operational pressures are
lower. These results are consistent with the data from Varga, Reczey, and Zacchi (2004) using
steam pretreatment of corn stover with 2% sulfuric acid where the results between 190°C and
200°C are similar in terms of digestibility, but lower sugar yields result from the 200°C
pretreated stover. Future research should address optimization of hemicellulose removal in order
9
to maximize xylose yields by subsequent enzymatic hydrolysis of the soluble oligosaccharides
and the remaining insoluble hemicellulose.
The conditions for maximum enzyme digestibility (190°C, 15 minutes and 200°C, 5
minutes) correlate to the maximum yields of hemicellulose after hydrolysis (Figure 3 b).
However, the major difference between the results from these pretreatment conditions is the
solubilization of the stover by pretreatment before enzyme hydrolysis. Approximately 30% of
the solids are dissolved by pretreatment at 200°C for 5 minutes while approximately 35% of the
solids are dissolved by pretreatment at 190°C for 15 minutes. There is also a correspondence
between decreasing enzyme digestibility and glucose yields for the 200°C pretreatments as
processing time increases (Figure 3 a) and the decreasing yields of non-degraded xylose (Figure
3 b). Liquid samples from pretreatment at 200°C for longer times contained decreased amounts
of xylose or soluble xylans while concurrently containing increased levels of the xylose
degradation product furfural. These results suggest that the mechanism of pretreatment is largely
a function of the removal of hemicellulose by partial hydrolysis to soluble oligosaccharides
while limiting the formation and degradation of xylose to furfural and insoluble furfural-derived
polymers. While the results of enzyme hydrolysis at a loading of 30 FPU/g glucan are similar
for both 200°C – 5 minutes and 190°C – 15 minutes, it is not clear that this is true for lower
enzyme loading levels. Due to the high cost of cellulolytic enzymes, the effect of lower enzyme
loadings is of interest.
Comparison of Standardized Enzyme Loadings on Digestibility of Pretreated Corn Stover
Corn stover was pretreated at the optimal conditions of 190°C for 15 minutes using the
batch tube reactors described above for digestion using the previously described standard for
pretreatment technology comparison. The difference in results between 60 and 15 FPU/g of
10
glucan are not statistically significant at a 95% confidence level (Figure 4). In both cases,
approximately 90% of the cellulose was converted to glucose and approximately 80% of the
xylan was converted to monomeric xylose. When the cellulase loading was reduced to 7.5
FPU/g of glucan, the glucose yield dropped to approximately 75%. However, the xylose yield
only dropped to approximately 75% from the 80% yields derived from the higher cellulase
loading. These results indicate that corn stover pretreated at 190°C for 15 minutes is highly
digestible with similar results at enzyme loadings above 60 FPU/g glucan. While there is a
significant loss of yield at 7.5 FPU/g glucan cellulose loading, additional research is required to
optimize the economics of pretreatment processing in terms of energy input, enzyme usage, and
glucose yield.
Fermentation of Pretreated and Hydrolyzed Corn Stover
The entire contents of several tube reactors (liquid and solids) were pooled and
hydrolyzed using a cellulase loading of 11 FPU/g glucan.
Unlike the diluted material,
hydrolysate from undiluted pretreatment slurry gave fermentable sugar yields sufficiently high
enough to allow the fermentation of the liquid to ethanol without the addition of other sugars or
media.
In addition, the pretreatment liquid was not removed or conditioned prior to the
introduction of the yeast in order to determine the effect, if any, toxins produced during
pretreatment would have on fermentation. Figure 5 illustrates the concentrations of glucose,
xylose, ethanol, and glycerol over the course of fermentation by the xylose-fermenting yeast,
424A(LNH-ST). The glucose is rapidly converted to ethanol (< 6 hours). The xylose ferments
much more slowly, reaching minimum fermentable xylose concentrations after 55 hours of
fermentation. The pH of the hydrolysate was 4.5 which also negatively affected the rate of
xylose fermentation since a pH of 5.5 is optimal for converting xylose to ethanol. The ethanol
11
yields from this fermentation are approximately 88% of theoretical for both glucose and xylose
and the pretreatment liquid was non-toxic to the fermentation.
While the final ethanol concentration is low, data show that sugars produced from corn
stover can be efficiently fermented to ethanol. In order to make the fermentation process and
subsequent ethanol distillation economically viable, the pretreatment and saccharification
process must be integrated into a whole-plant process. This systems engineering approach that
may include staged fermentations and recycling of processing liquid is the next step in
developing the technology that will allow the economic use of corn stover as a feedstock for biofuel production.
Overall mass balance
Dry matter recoveries and compositional analyses of solids and liquids after the
pretreatment step, the hydrolysis step, and the fermentation step were used to develop a mass
balance around the process. The other soluble mass in the hydrolyzate liquid was determined by
difference. The results from the 15 FPU/g glucan loading are shown in Figure 6. Approximately
91% of the glucan (cellulose) is hydrolyzed to monomeric glucose and approximately 82% of the
xylan is converted to monomeric xylose through pretreatment and cellulase hydrolysis. The
mass listed for glucose and xylose include water added through hydrolysis cause the mass
outputs to sum to greater than 100 lb. Using the experimentally derived fermentation efficiency
of 88% of theoretical, 100 lb (45 kg) of corn stover will generate 25 lb (11 kg) of ethanol.
Physical Surface Changes to Corn Stover Due to Pretreatment
Besides affecting the chemical composition of the corn stover, pretreatment also affected
the physical appearance of the corn stover at the microscopic level. Figure 7 is a scanning
electron micrograph (SEM) of corn stover at 1000 x magnification. The center of the picture is
12
dominated by vascular structures from the plan stem exposed by the milling of the whole corn
stover. The surface of the stover that is undamaged by the grinding is more-or-less smooth and
contiguous.
Figure 8 a) is an SEM of corn stover after pretreatment at 190°C (374°F) in water for 15
minutes.
The smooth, contiguous surface of untreated stover has been perforated by the
pretreatment processes. These pores may increase the enzyme-accessible surface area which
increases the enzyme digestibility of the corn stover. Future research is required to measure
changes in surface area caused by pretreatment. At 8000 x magnification, Figure 8 b), the pores
created by pretreatment can be seen in greater detail.
These oblong pores measure
approximately 4.5m by 1m in size.
Conclusions
The optimized conditions for controlled pH, liquid hot water pretreatment of a 16% slurry
of corn stover in water was found to be 190°C for 15 minutes. At the optimal conditions, 90% of
the cellulose was hydrolyzed to glucose by 15 FPU of cellulase per gram of glucan. When the
resulting pretreated slurry was hydrolyzed by 11 FPU of cellulase per gram of glucan, a
hydrolyzate containing 32.5 g/L glucose and 18 g/l xylose was formed. This hydrolyzate was
shown to be directly fermented by recombinant yeast able to convert both glucose and xylose to
ethanol at 88% of theoretical yields.
Acknowledgements
The material in this work was supported by USDA IFAFS Grant 00-52104-9663. We thank Deb
Sherman, manager of the microscopy center in Purdue’s School of Agriculture, and Dr. Wilfred
Vermerris and Youngmi Kim for their internal review of this paper and helpful suggestions.
13
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Procedure. National Renewable Energy Laboratory, Golden, CO.
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effects of acetic acid, furfural, and p-hydroxybenzoic acid on growth and ethanol productivity of
yeasts. Biotechnol. Bioeng. 63 (1), 46-55.
Ruiz, R., Ehrman, T. 1996a. Determination of carbohydrates in biomass by high performance
liquid chromatography. LAP-002 NREL Analytical Procedure. National Renewable Energy
Laboratory, Golden, CO.
Ruiz, R., Ehrman, T. 1996b. Dilute acid hydrolysis procedure for determination of total sugars
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Sedlak, M., Edenberg, H.J., Ho N.W.Y. 2003. DNA microarray analysis of the expression of
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Varga, E., Reczey, K., Zacchi, G. 2004. Optimization of steam pretreatment of corn stover to
enhance enzymatic digestibility. Appl. Biochem. Biotech. 113-116, 509-523.
Weil, J. R., Sarikaya, A., Rau, S-L., Goetz, J., Ladisch, C. M., Brewer, M., Hendrickson, R.,
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15
List of Tables
Table 1. Concentration and yields of structural carbohydrates as soluble monomeric and
oligomeric sugars generated by pretreatment of corn stover (¼ inch).
16
Table 1.
g/L
0.16
3.14
0.91
19.13
0.55
2.11
glucose
glucan
xylose + galactose
xylan + galactan
arabinose
arabinan
17
% yield
0.26%
5.71%
1.83%
43.59%
7.66%
33.51%
List of Figures
Figure 1.
Schematic of production scale continuous, controlled pH, liquid hot water
pretreatment of corn stover.
Figure 2.
Solubilization of corn stover (¼ inch) by a) pretreatment, b) pretreatment
followed by enzyme hydrolysis (50°C, 48 hrs.).
Figure 3.
Yields by enzyme hydrolysis (50°C, pH 4.8, 48 hrs) of pretreated corn stover (¼
inch) a) glucose, b) xylose.
Figure 4.
Effect of cellulase (Spezyme) loading on digestibility of pretreated (190°C, 15
min.) corn stover (40 mesh). Error bars represent 95% confidence intervals.
Figure 5.
Fermentation of pretreated (190°C, 15 min.) and hydrolyzed corn stover (¼ inch)
by recombinant yeast 424A(LNH-ST).
Figure 6.
Mass balance for pretreatment, hydrolysis (15 FPU/g glucan), and fermentation
for corn stover (40 mesh).
Figure 7.
Scanning electron micrograph (SEM) of corn stover (¼ inch) at 1000 x
magnification.
Figure 8.
SEM of pretreated (190°C, 15 min.) corn stover (¼ inch) at a) 1000 x
magnification and b) 8000 x magnification.
18
Saccharification
Stover
Heat
Recovery
Steam
Trim Heat
Water
Figure 1
19
Plug Flow
Reactor Coil
Solubilization by Pretreatment (%)
a)
Solubilization by Pretreatment
and Enzyme Hydrolysis(%)
b)
80%
200°C
190°C
180°C
170°C
70%
60%
50%
40%
30%
20%
10%
0%
80% 0
5
10
15
20
Pretreatment Time (min.)
25
70%
60%
50%
40%
30%
200°C
190°C
180°C
170°C
20%
10%
0%
0
5
10
15
20
Pretreatment Time (min.)
Figure 2.
20
25
Cellulase Hydrolysis of Pretreated Corn
Stover, Glucose Yield (%)
a)
100
90
80
70
60
50
40
200 C
190 C
180 C
170 C
30
20
10
0
b)
Cellulase Hydrolysis of Pretreated Corn
Stover, Xylose/Galactose Yield (%)
0
5
10
15
20
25
20
25
Pretreatment Time (min)
100
200 C
190 C
180 C
170 C
90
80
70
60
50
40
30
20
10
0
0
5
10
15
Pretreatment Time (min)
Figure 3.
21
100%
Glucose
Xylose
Cellulase Loading (FPU/g glucan)
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
60
15
Digestion Yield (%)
Figure 4.
22
7.5
35
Glucose
Xylose
Glycerol
Ethanol
Concentration (g/L)
30
25
20
15
10
5
0
0
10
20
30
Time (hrs.)
Figure 5.
23
40
50
60
Water
620 lb
Controlled pH
Stover
Liquid Hot Water
100 lb (dry basis)
Treated 62.8 lb undissolved solids
36.1 lb glucan
Stover 37.2 lb dissolved solids
21.4 lb xylan
Slurry 620 lb water
Cellulase Enzyme Hydrolysis
Fermentation
2209 FPU per
Hydrolyzate
lb stover (30 FPU/ml)
Liquid 36.32 lb glucose
19.89 lb xylose
Cellobiase Enzyme
5891 IU per lb
6.81 lb other
Residual 43.0 lb
stover (309 IU/ml)
Solids
Ethanol 25.2 lb
90.54% total glucan conversion (raw stover basis)
81.80% total xylan conversion (raw stover basis)
88% of theoretical ethanol yield from glucose +
xylose
Figure 6.
24
10 m
Figure 7.
25
a)
10 m
b)
1 m
~ 4.5 m
1 m
Figure 8.
26