> **COMPARATIVE SUGAR RECOVERY DATA FROM LABORATORY SCALE APPLICATION OF
> LEADING PRETREATMENT TECHNOLOGIES TO CORN STOVER**

Charles E. Wyman^1^, Bruce E. Dale^2^, Richard T. Elander^3^, Mark
Holtzapple^4^, Michael R. Ladisch^5^, YY Lee^6^

1\. Dartmouth College, Hanover, New Hampshire 03755; 2. Michigan State
University, East Lansing, Michigan 48824; 3. National Renewable Energy
Laboratory, Golden, Colorado 80401; 4. Texas A&M University, College
Station, Texas 77843; 5. Purdue University, West Lafayette, Indiana
47907; 6. Auburn University, Auburn, Alabama 36849

**ABSTRACT**

Biological processing of cellulosic biomass to fuels and chemicals would
open up major new agricultural markets and provide powerful societal
benefits, but pretreatment operations essential to economically viable
yields have a major impact on costs and performance of the entire
system. However, little comparative data is available on promising
pretreatments. To aid in selecting appropriate systems, leading
pretreatments based on ammonia explosion, aqueous ammonia recycle,
autocatalyzed hydrolysis, controlled pH, dilute acid, flowthrough, and
lime were evaluated in a coordinated laboratory program using a single
source of corn stover, the same cellulase enzyme, shared analytical
methods, and common data interpretation approaches to make meaningful
comparisons possible for the first time. Each pretreatment made it
possible to subsequently achieve high yields of glucose from cellulose
by cellulase enzymes, and the cellulase formulations used were effective
in solubilizing residual xylan left in the solids after each
pretreatment. Thus, overall sugar yields from hemicellulose and
cellulose in the coupled pretreatment and enzymatic hydrolysis
operations were high for all of the pretreatments with corn stover. In
addition, high pH methods were found to offer promise in reducing
cellulase use provided hemicellulase activity can be enhanced. However,
the substantial differences in sugar release patterns in the
pretreatment and enzymatic hydrolysis operations have important
implications for the choice of process, enzymes, and fermentative
organisms.

**INTRODUCTION**

As mentioned in the introductory paper for this special issue, this
project was a multi-institutional effort funded by the US Department of
Agriculture Initiative for Future Agriculture and Food Systems Program
to develop comparative technical and economic information on
pretreatment of cellulosic biomass by leading options using a single
source of corn stover, a shared supply of cellulase enzyme, identical
analytical methods, and identical approaches to data analysis.
Comparative data of this nature are sorely needed to aid in selection of
pretreatment options for commercial uses but are unfortunately lacking.
The pretreatments investigated and corresponding lead investigators and
institutions for this study were 1) aqueous ammonia recycle pretreatment
by YY Lee at Auburn University, 2) water only and dilute acid hydrolysis
by co-current and flowthrough systems by Charles Wyman at Dartmouth
College, 3) ammonia fiber explosion (AFEX) by Bruce Dale at Michigan
State University, 4) controlled pH pretreatment at Michael Ladisch of
Purdue University, and 5) lime pretreatment at Mark Holtzapple of Texas
A&M University. In addition, feedstock and enzyme supply, other
logistical support, and economic analyses were provided through Richard
Elander of the National Renewable Energy Laboratory (NREL) made possible
by funding from the Office of the Biomass Program of the US Department
of Energy. These participants are all members of a Biomass Refining
Consortium for Applied Fundamentals and Innovation (CAFI) that has the
mission of developing a fundamental understanding of biomass hydrolysis
that will facilitate commercialization, accelerating the development of
next generation technologies that dramatically reduce the cost of sugars
from cellulosic biomass, and training future engineers, scientists, and
managers.

This paper presents a brief overview of the project and the approach
applied to define sugar yields from hemicellulose and cellulose. Then,
key xylose and glucose yield results are presented for each technology
for the operations of pretreatment and subsequent enzymatic hydrolysis.
Overall, the goal is to provide a single source of comparative
information that will assist readers in understanding the unique
features and performances of leading options for releasing sugars from
cellulosic biomass. References are given to the source of the data
summarized for the reader to obtain more detailed information on each
system, if desired.

**BACKGROUND**

The tasks undertaken in this project were to apply leading pretreatment
technologies to 1) prepare corn stover for conversion to products, 2)
characterize resulting fluid and solid streams, 3) close material and
energy balances for each pretreatment process, 4) determine cellulose
digestibility and liquid fraction fermentability/toxicity, and 5)
compare performance of pretreatment technologies on a consistent basis.
The project period was from late 2000 to late 2003, and the emphasis of
the project was on research quality to be sure the data would be useful
to potential practitioners. A single source of corn stover provided by
NREL through BioMass AgriProducts from a source in Harlan IA was washed
and dried in small commercial operation and knife milled to pass ¼ inch
round screen prior to distribution to the participants. The composition
of this feedstock, as measured by NREL, was 36.1% glucan, 21.4% xylan,
3.5% arabinan, 2.5% galactan, 1.8% mannan, 17.2% lignin, 7.1% ash, 4.0%
protein, 3.6% uronic acid, 3.2% acetyl, and 1.2% non-structural sugars
on a dry weight basis.

**Calculation of sugar yields**

Material balances were closed for each pretreatment system by measuring
the composition and total mass of each liquid and solid stream leaving
pretreatment and converting this data to amounts of sugars produced, as
illustrated by an example for AFEX in Figure 1. The procedures applied
were as described in the Introduction paper to this special volume.
Yields were then calculated based on the xylose and glucose available in
the corn stover fed to the systems. Thus, based on the material
composition used for this study and the appropriate increase in mass
with hydrolysis, at most about 40.1 mass units of glucose and 24.3 mass
units of xylose could be produced from 100 mass units of corn stover
feed giving a total sugar potential of 64.4 units per 100 units of bone
dry feed. It is important to note that although accurate analysis and
measurements are quite simple in concept, they are challenging for
biomass, and extensive time and care are essential to obtain meaningful
information.

The consideration of sugar yields must account for the fact that the
overall system consists of two stages for each pretreatment system
evaluated and that not all of the sugar is released as monomers. In
Stage 1, biomass is pretreated to open up the structure of the residual
solids and facilitate access by enzymes to obtain high yields. Enzymes
are then added to the pretreated solids in Stage 2. For many operations,
some amounts of glucose and xylose are released in Stage 1 and recovered
in the liquid stream. Furthermore, although cellulase enzyme is added to
Stage 2, it has enough xylanase activity to hydrolyze a substantial
portion of the xylan, and both xylose and glucose are typically found in
the liquid streams from Stage 2. Thus, yields of each sugar are reported
for each stage as appropriate. Because some of the pretreatments produce
sugar oligomers as well as monomers in Stage 1, the yields of each sugar
from each stage are further differentiated to reflect this information.

Comparison of the amount of each sugar monomer or oligomer produced to
the maximum potential amount for that sugar would give the percent yield
of each. However, it is important to recognize that corn stover, along
with most other forms of cellulosic biomass, are richer in glucose than
xylose, and as a result, yields of glucose have a greater impact than
those of xylose. Thus, sugar yields were defined in this project by
dividing the amount of xylose or glucose or the sum of the two by the
maximum potential amount of both sugars to better reflect the relative
contribution to overall sugar production. On this basis, the maximum
xylose yield is calculated as 24.3/64.4 or 37.7%, the maximum glucose
yield is 40.1/64.4 or 62.3%, and the maximum amount of total xylose and
glucose is 100%.

In the case of fermentation to ethanol, the definition of yields applied
here is appropriate in principle because the theoretical yields are the
same from all sugars and the yields of sugars reflect reasonably closely
the amount of ethanol that could be obtained. Thus, consideration of
total sugars released will closely approximate the total amount of
ethanol that could be produced. However, some organisms suffer from
diauxic effects that reduce or prevent fermentation of low
concentrations of xylose or other sugars that exist in solutions
containing greater concentrations of glucose. Furthermore, although some
organisms can ferment sugar oligomers, others do not, making conversion
of the oligomers released during pretreatment more challenging or
perhaps impossible at reasonable cost in such cases. For these reasons,
the utility of each sugar type can vary with the process chosen, and
total sugar yields may not be equally relevant for all applications.
However, the breakdown of data by stage and sugar type should allow the
user to assess the yield of sugars appropriate for the intended
application. In addition, caution is urged in use of this data in that
it may be possible to adjust pretreatment and hydrolysis conditions
somewhat to achieve higher yields of selected sugars from those reported
here for maximum total yields.

**COMPARATIVE RESULTS**

Details for each of the pretreatment systems applied in this project are
provided in other papers in this volume, and this section will focus on
summarizing data on a common basis to facilitate comparisons. However,
it is important to realize that the use of somewhat different enzymes
and other process specifics could alter the results for some of the
systems from those reported.

**Distinguishing features**

Several observations are important to note in terms of the different
effects of pretreatment on the characteristics of the pretreated
biomass. First, pretreatments at lower pH by dilute acid, controlled pH,
and flowthrough all gave a liquid fraction containing most of the sugars
from hemicellulose and a solid residue that contained most of the
cellulose from the original corn stover. Furthermore, little lignin was
removed by dilute acid or controlled-pH pretreatments, whereas the
flowthrough or partial flow operations removed as much as 75% of the
lignin in addition to hemicellulose. On the other hand, the high-pH
technologies based on lime and liquid ammonia recycle removed lignin and
left a solid residue containing most of the cellulose and hemicellulose.
Although AFEX is also a high-pH process, it did not generate free liquid
and did not separate the lignin or hemicellulose from the cellulose.
Rather, a solid material was left that looked very much like the
original substrate with the cellulose and hemicellulose well preserved
and essentially 100% of the feedstock recovered as dry matter.

**Xylose sugar yields**

Tables 1 and 2 document the yields of xylose from hemicellulose for
enzyme loadings of 60 and 15 FPU/g glucose in the original corn stover,
respectively. The pretreatment technologies are listed in order of
increasing pH, and reasonably high xylose yields were achieved for all
systems. Most of the xylose was released in pretreatment, Stage 1, for
Dilute Acid, Flowthrough, Partial Flow, and Controlled pH pretreatment.
Furthermore, most of the xylose was released as monomers for other than
the flowthrough systems. On the other hand, the high-pH pretreatments by
ARP and lime released a large percentage of xylose sugars in the second
stage, with about half being solubilized in the second stage for ARP and
two thirds for lime. Essentially all of the xylose was released in the
second stage for AFEX.

**Glucose sugar yields**

Tables 1 and 2 summarize glucose yields for cellulase loadings of 60 and
15 FPU/g glucose in the original corn stover, respectively. All of the
pretreatments considered resulted in a small fraction of the total
glucose being released in Stage 1 with most solubilized in Stage 2.
Nonetheless, total glucose yields were close to the maximum possible of
62.3% for all pretreatments at the higher enzyme loading and dropped
only slightly when cellulase use was cut by 75%. Thus, all pretreatments
were effective in making cellulose accessible to enzymes. The glucose
released was predominately as monomers, demonstrating virtually complete
hydrolysis by cellulase. The data show slightly higher yields for lime,
ARP, and flowthrough technologies that remove substantial amounts of
lignin. However, the fact that AFEX achieved slightly higher yields even
though no lignin was removed suggests that lignin removal is not
essential to enhance the digestibility of corn stover cellulose.
Understanding the causes of these differences could suggest new
approaches to enhanced cellulose digestion.

**Total sugar yields**

The total sugar yields are also shown in Tables 1 and 2. All of the
pretreatments realized overall sugar yields of around 90% at high enzyme
loadings, with some advantage being apparent for systems using flowing
water either alone (flowthrough) or with ammonia (ARP), apparently due
to lignin removal and greater recovery of hemicellulose. However, yields
for the other technologies were not substantially lower. Furthermore,
when cellulase loadings were lowered substantially to 15 FPU/g, all of
the yields were still similar. Thus, all pretreatments achieved high
total sugar yields.

**Conditions for maximum yields**

Table 3 summarizes the key conditions employed to maximize the yields to
those reported in Tables 1 and 2. Most of the temperatures were in the
range of 160 to 200^o^C. However, AFEX used only 90^o^C whereas lime
applied 55^o^C. Thus, high temperatures are not essential to achieving
good sugar recovery. Except for the 4-week period applied for lime, all
pretreatments were run for between 5 and 24 minutes, making it possible
to conduct most of them in relatively small vessels. On the other hand,
a novel approach such as pretreatment in a pile would be needed to
handle the large volumes of biomass for lime pretreatment. Chemical
demands are as listed.

**CONCLUSIONS**

This project took a major step toward addressing a compelling need for
comparative data on the performance of leading pretreatment
technologies, and the information gathered through this first
coordinated evaluation of biomass pretreatment and subsequent enzymatic
hydrolysis supports several important conclusions. As expected, dilute
acid, neutral pH, and water-only pretreatments solubilized mostly
hemicellulose whereas addition of lime or percolation with ammonia
removed mostly lignin. On the other hand, when ammonia was released at
the end of the pretreatment process via AFEX, neither lignin nor
hemicellulose was physically removed from cellulose and other
components. When water was pushed through biomass in a flowthrough mode,
virtually all of the hemicellulose and up to about 75% of the lignin
were removed with or without addition of very dilute sulfuric acid.
Thus, different pretreatments can affect biomass in very different ways.

Although removal of hemicellulose and lignin differed, high yields of
glucose were achieved by enzymatically hydrolyzing the remaining solids
for all of these pretreatments. The digestibility was somewhat better
for higher pH and flowthrough methods, likely due to the removal of
lignin that can interfere with the accessibility of cellulase to
cellulose and non-productively adsorb lignin. However, the AFEX process
achieved excellent enzymatic digestion at low enzyme loadings even
though essentially no lignin or hemicellulose was removed. This
difference suggests that ammonia affects lignin and possibly
hemicellulose differently than other additives, reducing the ability of
lignin to adsorb enzyme and/or to impede its access to cellulose.

An interesting observation was that the enzyme formulation used
contained enough xylanase activity to solubilize a large portion of the
xylan remaining for the high-pH pretreatments. Thus, although AFEX,
lime, and ammonia percolation approaches produced moderate to low yields
of xylose during the pretreatment step itself, the enzymes hydrolyzed
much of the residual xylan in the pretreated solids, and a substantial
fraction of the xylan could be recovered from the residual solids over
the two stages for the high pH methods. Much less xylan remained in the
pretreated solids at optimum conditions that prevented excessive xylose
degradation for pretreatments at lower pH, but that some of that left in
the solids was still broken down to xylose by enzymes, noticeably
enhancing yields. Thus, increasing hemicellulase activity promises
improved yields for all methods and could have particularly important
benefits for neutral or alkaline pretreatment.

A related factor is the release of oligomers during pretreatment and
their implications for process design. At one extreme, dilute sulfuric
acid pretreatment released almost all of the xylose as monomers, and
these sugars are readily fermented by many organisms. On the other hand,
pretreatments by methods such as flowthrough or controlled-pH
technologies gave high relative amounts of oligomers, which are not as
easily utilized and may require additional steps such as post hydrolysis
with acids or enzymes to break the oligomers into monomeric species.
Consequently, these differences in the pattern of oligomer release must
be considered in selecting a pretreatment system.

Overall, the results show interesting tradeoffs among the pretreatments
evaluated. The enzymatic digestibility is similar for all methods, but
those at high (alkaline) pH offer somewhat lower enzyme loadings for a
given yield due to lignin removal. However, AFEX seems to particularly
benefit in this regard even though little lignin is removed.
Furthermore, although some pretreatments release primarily xylose during
pretreatment and primarily glucose in subsequent enzymatic hydrolysis,
other pretreatments release mixtures containing appreciable
concentrations of both xylose and glucose sugars during enzymatic
hydrolysis. Because most organisms preferentially use glucose instead of
xylose or other sugars, fermenting this sugar mixture in the enzymatic
hydrolyzate can present important challenges. Thus, the choice of
pretreatment technology is not simple and must consider the sugar
release patterns and solids concentrations for each pretreatment as well
as their compatibility with the process, enzymes, and fermentative
organisms.

It is also important to keep in mind that the data reported here pertain
specifically to corn stover. Similar performance may be expected for
other agricultural residues such as wheat straw or rice straw and
possibly for herbaceous crops such as switchgrass. However, such
interpretations are extrapolations and cannot be substantiated without
data. Furthermore, it would be unrealistic to imply that similar results
would be achieved with hardwoods such as poplar, and much different
results would almost certainly be observed for softwoods. In fact,
experiments of the type reported here for corn stover are needed with
each of these feedstocks before any meaningful conclusions can be drawn
on the relative merits of these different pretreatment technologies for
application to multiple crops. On that basis, another project built
around a team augmented from that reporting here is in progress to
develop comparative data on how each pretreatment performs with poplar
wood. In addition, this project will examine in more depth how leading
pretreatment technologies impact hydrolyzate conditioning needs for
effective hydrolyzate fermentation by recombinant organisms, cellulase
effectiveness, the effect of hemicellulase addition on performance, the
relationship of enzyme formulation to performance, and substrate
features and their effect on digestibility.

> **ACKNOWLEDGMENTS**

We would particularly like to acknowledge the support of the Initiative
for Future Agricultural and Food Systems (IFAFS) Program of the United
States Department of Agriculture for funding this CAFI research project
through Contract 00-52104-9663. The IFAFS Program filled a vital
national need for supporting collaborative research projects of this
nature that would not be possible through most conventional programs,
and the authors regret that the IFAFS Program has not been funded to
continue its unique and valuable mission. We would also like to thank
the United States Department of Energy Office of the Biomass Program for
funding the National Renewable Energy Laboratory to provide controlled
sources of feedstock and enzymes and provide economic analyses along
with other invaluable services vital to the successful execution of this
project.

**REFERENCES**

1\. Lloyd TA, Wyman CE. 2005. "Total Sugar Yields for Pretreatment by
Hemicellulose Hydrolysis Coupled with Enzymatic Hydrolysis of the
Remaining Solids," *Bioresource Technology*, this volume.

2\. Liu C, Wyman CE. 2005. \"Partial Flow of Compressed-Hot Water
Through Corn Stover to Enhance Hemicellulose Sugar Recovery and
Enzymatic Digestibility of Cellulose," *Bioresource Technology*, this
volume.

3\. Mosier N, Hendrickson R, Ho N, Sedlak M, Ladisch MR. 2005.
"Optimization of pH Controlled Liquid Hot Water Pretreatment of Corn
Stover," *Bioresource Technology*, this volume.

4\. Teymouri, F. Laureano-Perez, L., Alizadeh, H and Dale, BE. 2004.
"Ammonia Fiber Explosion Treatment of Corn Stover" *Applied Biochemistry
and Biotechnology* **113-116**: 951-963.

5\. Kim TH, Lee YY, Kim JS, Sunwoo CS. 2005. "Pretreatment of Corn
Stover by Low-Liquid Ammonia Percolation Process," *Biotechnology and
Bioprocess Engineering*, submitted.

6\. Kim TH, Lee YY. 2003. "Pretreatment of Corn Stover by Soaking in
Aqueous Ammonia," AIChE Annual Meeting, San Francisco, CA. November.

7\. Kim S, Holtzapple MT. 2005. "Lime Pretreatment and Enzymatic
Hydrolysis of Corn Stover," *Bioresource Technology*, this volume.

![](media/image1.wmf){width="4.995833333333334in"
height="3.745138888888889in"}

Figure 1. Example of the material balance approach when applied to the
ammonia fiber explosion (AFEX) process. As shown, yields of about 59.8%
for glucose, 29.3% for xylose, and 89.1% for total glucose plus xylose
were obtained based on the maximum potential production of 64.4 lbs of
total sugars per 100 lbs of dry corn stover.

  ----------------- ----------- --------- ----------- --------- ------- ----------- ----------- ----------- -----------
  Table 1. Yields                                                                                           
  of xylose and                                                                                             
  glucose for each                                                                                          
  pretreatment                                                                                              
  system studied                                                                                            
  followed by                                                                                               
  enzymatic                                                                                                 
  hydrolysis with a                                                                                         
  loading of 60                                                                                             
  FPU/g glucan in                                                                                           
  the original corn                                                                                         
  stover. Stage 1                                                                                           
  refers to                                                                                                 
  pretreatment and                                                                                          
  Stage 2 refers to                                                                                         
  the enzymatic                                                                                             
  digestion of the                                                                                          
  solids produced                                                                                           
  in pretreatment.                                                                                          
  The first value                                                                                           
  reported in each                                                                                          
  column is for                                                                                             
  total sugars                                                                                              
  released into                                                                                             
  solution, and the                                                                                         
  second is for                                                                                             
  just the monomers                                                                                         
  released. A                                                                                               
  single value                                                                                              
  indicates release                                                                                         
  of only monomers.                                                                                         
  Yields are                                                                                                
  defined based on                                                                                          
  the maximum                                                                                               
  potential sugars                                                                                          
  released from the                                                                                         
  corn stover used                                                                                          
  of 64.4 g per 100                                                                                         
  grams of dry                                                                                              
  solids with the                                                                                           
  maximum potential                                                                                         
  xylose being                                                                                              
  37.7% and the                                                                                             
  maximum potential                                                                                         
  yield of glucose                                                                                          
  being 62.3% on                                                                                            
  this basis. ND =                                                                                          
  not determined.                                                                                           

  Pretreatment      Xylose                            Glucose                       Total                   
  system            yields                            yields                        sugars                  

                    Stage 1     Stage 2   Total       Stage 1   Stage 2 Total       Stage 1     Stage 2     Combined
                                          xylose                        glucose                             total

  Dilute acid^1^    32.1/27.5   3.2       35.3/30.7   3.9       53.3    57.2        36.0/31.4   56.5        92.5/87.9

  Flowthrough^2^    36.3/1.7    0.8/0.7   37.1/2.4    4.5/4.4   57.0    61.5/61.4   40.8/6.1    57.8/57.7   98.6/63.8

  Partial flow      31.5/2.8    \-        \-          4.3/4.2   \-      \-          \-          \-          \-
  pretreatment^2^                                                                                           

  Controlled pH^3^  21.8/0.9    8.9       30.7        3.5/0.2   54.7    58.2        25.3/1.1    63.6        88.9

  AFEX^4^                       ND/30.2   ND/30.2               61.8    61.8                    ND/92.0     ND/92.0

  ARP^5,6^          17.8/0      17.0      34.8/17.0   0         59.4    59.4        17.8/0      76.4        94.2/76.4

  Lime^7^           9.2/0.3     20.2      29.4/20.5   1.0/0.3   59.5    60.5/59.8   10.2/0.6    79.7        89.9/80.3
  ----------------- ----------- --------- ----------- --------- ------- ----------- ----------- ----------- -----------

  ----------------- ----------- ----------- ----------- --------- ------- ----------- ----------- ----------- -----------
  Table 2. Yields                                                                                             
  of xylose and                                                                                               
  glucose for each                                                                                            
  pretreatment                                                                                                
  system studied                                                                                              
  followed by                                                                                                 
  enzymatic                                                                                                   
  hydrolysis with a                                                                                           
  loading of 15                                                                                               
  FPU/g glucan in                                                                                             
  the original corn                                                                                           
  stover. Stage 1                                                                                             
  refers to                                                                                                   
  pretreatment and                                                                                            
  Stage 2 refers to                                                                                           
  the enzymatic                                                                                               
  digestion of the                                                                                            
  solids produced                                                                                             
  in pretreatment.                                                                                            
  The first value                                                                                             
  reported in each                                                                                            
  column is for                                                                                               
  total sugars                                                                                                
  released into                                                                                               
  solution and the                                                                                            
  second is for                                                                                               
  just the monomers                                                                                           
  released. A                                                                                                 
  single value                                                                                                
  indicates release                                                                                           
  of only monomers.                                                                                           
  Yields are                                                                                                  
  defined based on                                                                                            
  the maximum                                                                                                 
  potential sugars                                                                                            
  released from the                                                                                           
  corn stover used                                                                                            
  of 64.4 g per 100                                                                                           
  grams of dry                                                                                                
  solids with the                                                                                             
  maximum potential                                                                                           
  xylose being                                                                                                
  37.7% and the                                                                                               
  maximum potential                                                                                           
  yield of glucose                                                                                            
  being 62.3% on                                                                                              
  this basis.                                                                                                 

  Pretreatment      Xylose                              Glucose                       Total                   
  system            yields                              yields                        sugars                  

                    Stage 1     Stage 2     Total       Stage 1   Stage 2 Total       Stage 1     Stage 2     Combined
                                            xylose                        glucose                             

  Dilute acid^1^    32.1/31.2   3.2         35.3        3.9       53.2    57.1        36.0/35.1   56.4        92.4/91.5

  Flowthrough^2^    36.3/1.7    0.6/0.5     36.9/2.2    4.5/4.4   55.2    59.7/59.6   40.8/6.1    55.8/55.7   96.6/61.8

  Partial flow      31.5/2.8    2.6/2.4     34.1/5.2    4.3/4.2   51.2    55.5/55.4   35.8/7.0    53.8/53.6   89.6/60.6
  pretreatment^2^                                                                                             

  Controlled pH^3^  21.8/0.9    9.0         30.8        3.5/0.2   52.9    56.4        25.3/1.1    61.9        87.2

  AFEX^4^                       34.6/29.3   34.6/29.3             59.8    59.8                    94.4/89.1   94.4/89.1

  ARP^5,6^          17.8/0      15.5        33.3/15.5   0         56.1    56.1        17.8/0      71.6        89.4/71.6

  Lime^7^           9.2/0.3     19.6        28.8/19.9   1.0/0.3   57.0    58.0/57.3   10.2/0.6    76.6        86.8/77.2
  ----------------- ----------- ----------- ----------- --------- ------- ----------- ----------- ----------- -----------

+-----------+----------+-------+---------+-------+-------------------+
| Table 3.  |          |       |         |       |                   |
| Reaction  |          |       |         |       |                   |
| c         |          |       |         |       |                   |
| onditions |          |       |         |       |                   |
| employed  |          |       |         |       |                   |
| to obtain |          |       |         |       |                   |
| maximum   |          |       |         |       |                   |
| total     |          |       |         |       |                   |
| sugar     |          |       |         |       |                   |
| yields    |          |       |         |       |                   |
| from corn |          |       |         |       |                   |
| stover    |          |       |         |       |                   |
| for each  |          |       |         |       |                   |
| pre       |          |       |         |       |                   |
| treatment |          |       |         |       |                   |
| system.   |          |       |         |       |                   |
+-----------+----------+-------+---------+-------+-------------------+
| Pre       | Temp     | Rea   | C       | Pe    | Other notes       |
| treatment | erature, | ction | hemical | rcent |                   |
| system    | ^o^C     | time, | agent   | che   |                   |
|           |          | mi    | used    | mical |                   |
|           |          | nutes |         | used  |                   |
+-----------+----------+-------+---------+-------+-------------------+
| Dilute    | 160      | 20    | S       | 0.49  | 25% solids        |
| acid^1^   |          |       | ulfuric |       | concentration     |
|           |          |       | acid    |       | during run in     |
|           |          |       |         |       | batch tubes       |
+-----------+----------+-------+---------+-------+-------------------+
| Flowt     | 200      | 24    | none    | 0     | Continuously flow |
| hrough^2^ |          |       |         |       | just hot water at |
|           |          |       |         |       | 10mL/min for      |
|           |          |       |         |       | 24minutes         |
+-----------+----------+-------+---------+-------+-------------------+
| Partial   | 200      | 24    | none    | 0     | Flow hot water at |
| flow      |          |       |         |       | 10mL/min from 4-8 |
| pretre    |          |       |         |       | minutes, batch    |
| atment^2^ |          |       |         |       | otherwise         |
+-----------+----------+-------+---------+-------+-------------------+
| C         | 190      | 15    | none    | 0     | 16% corn residue  |
| ontrolled |          |       |         |       | slurry in water   |
| pH^3^     |          |       |         |       |                   |
+-----------+----------+-------+---------+-------+-------------------+
| AFEX^4^   | 90       | 5     | An      | 100   | 62.5% solids in   |
|           |          |       | hydrous |       | reactor           |
|           |          |       | ammonia |       |                   |
|           |          |       |         |       | (60% moisture dry |
|           |          |       |         |       | weight basis), 5  |
|           |          |       |         |       | minutes at        |
|           |          |       |         |       | temperature       |
+-----------+----------+-------+---------+-------+-------------------+
| ARP^5,6^  | 170      | 10    | ammonia | 15    | Flow aqueous      |
|           |          |       |         |       | ammonia at 5      |
|           |          |       |         |       | mL/min without    |
|           |          |       |         |       | presoaking        |
+-----------+----------+-------+---------+-------+-------------------+
| Lime^7^   | 55       | 4     | lime    | 0.08  | Purged with air.  |
|           |          | weeks |         | g     |                   |
|           |          |       |         | CaO/g |                   |
|           |          |       |         | bi    |                   |
|           |          |       |         | omass |                   |
+-----------+----------+-------+---------+-------+-------------------+