**INTEGRATION OF LEADING BIOMASS PRETREATMENT TECHNOLOGIES WITH
ENZYMATIC DIGESTION AND HYDROLYZATE FERMENTATION**

Charles E. Wyman, Dartmouth College

Bruce Dale, Michigan State University

Richard Elander, National Renewable Energy Laboratory

Mark T. Holtzapple, Texas A&M University

Michael R. Ladisch, Purdue University

Y.Y. Lee, Auburn University

Mohammed Moniruzzaman, Genencor International

John N. Saddler, University of British Columbia

Funded by U.S. Department of Energy and Natural Resources Canada

# Goals and Objectives

The goal of the project is to develop and compare leading biomass
pretreatment technologies coupled with fermentation and enzymatic
digestion. We also seek to better understand interactions among
pretreatment, fermentation, and enzymatic hydrolysis to gain insight
that will facilitate selection and commercialization of cellulosic
technologies and lead to step change cost reductions. Another goal is to
train and educate students in biomass technologies. Corn stover will be
used to tie to previous research funded by the USDA IFAFS Program, but
the team will focus primarily on poplar, a leading woody energy crop,
coupled with fermentation and enzymatic hydrolysis. It is also proposed
that the team work closely with Genencor International, a leading
manufacturer of cellulase and other enzymes, to apply commercial and new
state-of-the-art enzyme formulations in this research. Although the
fermentation portion focuses on ethanol, the results should be valuable
to making other products.

The objectives of the proposed project are as follows:

1.  *Cellulosic biomass will be pretreated by leading technologies to
    improve cellulose accessibility to enzymes.*

2.  *Conditioning methods will be developed as needed to maximize
    fermentation yields by a recombinant yeast, the cause of inhibition
    will be determined, and fermentations will be modeled.*

3.  *Cellulose and hemicellulose in pretreated biomass will be
    enzymatically hydrolyzed, as appropriate, and models will be
    developed to understand the relationship between pretreated biomass
    features, advanced enzyme characteristics, and enzymatic digestion
    results.*

4.  *Capital and operating costs will be estimated for each integrated
    pretreatment, hydrolysis, and fermentation system and used to direct
    research.*

5.  *The proposal team will work with an Industrial and Agricultural
    Advisory Board and others to evaluate these findings; identify
    opportunities to utilize the pretreatment technologies; define
    opportunities to improve coupled pretreatment, fermentation, and
    enzymatic hydrolysis; and disseminate the results widely.*

# Proposed Research Approach

Biomass conversion experts from Dartmouth College; Auburn, Michigan
State, Purdue, and Texas A&M Universities; the National Renewable Energy
Laboratory (NREL); the University of British Columbia; and Genencor
International will collaborate on this project. The first 7 are members
of a Biomass Consortium on Applied Fundamentals and Innovation (CAFI)
formed in early 2000, and the first 6 are completing coordinated
research through USDA IFAFS support to develop comparative data and
economics for pretreatment of corn stover by leading technologies. That
project is also improving the understanding of these pretreatments and
establishes analytical procedures as well. It is proposed to expand this
approach to hardwood, an important feedstock class with different
characteristics than stover that NREL will provide from a maintained
source. However, our focus will shift toward understanding in more depth
the effects of leading pretreatments on fermentation and enzymatic
hydrolysis and applying this insight to improve pretreatment. Dartmouth
College will lead the overall project that would be conducted in
elements corresponding to each project objective. Each element will be
led by a team member but will use other team members to take advantages
of their talents.

**Element 1: Pretreat cellulosic biomass by leading technologies.**
Michigan State University (MSU) will be responsible for this element of
dilute acid, SO~2~, controlled pH, AFEX, ARP, flowthrough, and lime
pretreatment. All participants already have the experimental systems and
procedures needed in place. For the first, NREL will provide liquid
hydrolyzate and pretreated cellulose samples from their facilities based
on extensive experience with poplar. They will also apply their new
technology to prepare hot water washed dilute acid treated cellulose
with improved digestibility. The University of British Columbia will
provide pretreated solids and hydrolyzate for poplar pretreated with
SO~2~ in their steam gun at conditions they defined before. Purdue will
make hydrolyzates and pretreated solids by their controlled pH
pretreatment method.

Because AFEX has not been applied extensively to poplar, MSU will
require more time to define conditions to maximize both cellulose
digestibility and total hemicellulose and cellulose sugar recovery.
Based on results from the USDA IFAFS funded CAFI project with corn
stover, the approximate AFEX treatment ranges expected include
temperatures of 60-110 ^0^C, sample moisture content of 50-150% on a dry
weight basis, ammonia to dry sample loadings of 0.5 -- 1.5 kg ammonia/kg
dry sample, and times of 5-15 minutes, but research is needed to
determine what conditions will work for poplar. Models will be developed
to relate AFEX performance to these variables. Based on prior results,
AFEX is expected to remove little lignin or hemicellulose.

Auburn University has shown through USDA IFAFS support that lignin
removal by their ARP process improves enzymatic hydrolysis of corn
stover, and comparing the digestibility of cellulose from ARP and AFEX
pretreated solids will provide insight into the effects of exposure of
lignin to ammonia versus lignin removal by ammonia on enzymatic
hydrolysis. Auburn will also evaluate whether their new approach to
reducing ammonia and water use with corn stover through IFAFS funding
will work as well for hardwood. In addition, kinetic models will be
developed to describe hemicellulose and lignin removal.

Dartmouth has applied flowthrough pretreatment to remove up to 70% of
lignin and all hemicellulose without addition of any chemicals or use of
very dilute sulfuric acid (\<0.1%). Although known approaches use too
much water to be economically viable, flowthrough recovers virtually all
hemicellulose sugars and produces highly digestible cellulose. The
latter is thus a low lignin substrate for comparison to lignin removal
with ammonia via ARP. Finally, lime has also been shown to remove large
amounts of lignin, and Texas A&M will treat poplar wood with lime to
provide a substrate for comparison to lignin removal via ARP and flow.

MSU's responsibility will include monitoring the application of
procedures applied in previous CAFI research to insure accurate mass
balance closure. Auburn University will assist MSU by insuring all
analytical work to determine solids carbohydrate content (glucan, xylan,
arbinan, mannan, and galactan), Klason lignin, extractables, ash in
solids, and sugars and soluble lignin in liquids is performed according
to established or modified protocols. These analyses will include
differentiating hydrolytic from reversion and decompositions reactions
and reactions with lignin for dilute acid and flowthrough pretreatments.

**Element 2: Develop conditioning methods and ferment hydrolyzates with
a recombinant fermentative organism.** Purdue University will lead this
element to develop and understand both conditioning and fermentations of
pretreatment hydrolyzates. First, they will apply additional analyses to
identify total dissolved solids, organic acids, furfural, and
5-hydroxymethyl furfural (HMF) in liquid streams from pretreatments and
enzymatic hydrolysis of pretreated solids based on NREL laboratory
analytical procedures (LAPs). However, some modifications will be made
such as using a HPX-87H instead of a HPX-87C column to concurrently
determine acetic acid released from hemicellulose and sugar degradation
products formed in pretreatment. Although not all biological inhibitors
have been identified, this information will help understand how known
inhibitors change with pretreatment approach.

Purdue will develop conditioning methods as needed to improve
fermentability to ethanol using their genetically engineered
*Saccharomyces cerevisiae* strain (17). Overliming, widely used to
prepare dilute acid hydrolyzates, will be used as a baseline. However,
based on Purdue's experience, polymeric, hydrophobic resins will be
tested, and their adsorption and desorption properties determined over
varying furfural and HMF concentrations at temperatures of 20 to 80°C
and contact times up to equilibrium. Analytical liquid chromatography
data will be used to determine kinetics and Langmuir constants for
continuous chromatographic adsorption models. Model predictions will be
confirmed using large scale continuous chromatographic columns for
on-off removal and regeneration that will also provide conditioned
liquid for fermentation tests. In contrast to other pretreatments, it is
expected the AFEX material will require no conditioning.

Genetically engineered *Saccharomyces* strain 424A(LNH-ST) will be used
to ferment conditioned and unconditioned hydrolyzates in 300 ml baffled
Erlenmeyer flasks in Dr. Ho' s laboratory at Purdue (17). The yeast will
be pre-grown in 100 ml YEPD (1% yeast extra, 1% peptone, and 2% glucose)
and transferred into 100 ml of hydrolyzate supplemented with 10 ml 10 %
yeast extract. Flasks will be sealed with Saran wrap to allow
fermentation under largely anaerobic conditions, and samples taken every
6 hrs. Glucose, xylose, ethanol, and xylitol concentrations will be
analyzed by HPLC to determine how the presence and concentration of
inhibitors affects fermentation. The conditioning approach will be
modified as possible to improve ethanol yields. Data from successful
fermentations will be used to develop models to relate rates and yields
of fermentation to inhibitor concentrations and to the types and
concentrations of sugars. Such models will be valuable in understanding
how well we can account for inhibition and in applying and improving
fermentation and conditioning technologies.

**Element 3: Characterize and model relationship between pretreated
solids features and advanced enzyme properties and apply to improve
pretreatments and enzymes**. Dartmouth College will lead this portion of
the project with help from Texas A&M. First, pretreated solids from
Element 1 will be hydrolyzed with cellulase and hemicellulase to measure
changes in hydrolysis performance with pretreatment conditions and
mixtures of these two enzymes with the goal of solubilizing over 90% of
residual cellulose and hemicellulose in pretreated solids to sugars.
Genencor, a major supplier and developer of cellulase and hemicellulase,
will review team results and supply commercial and advanced formulations
to improve performance. Use of cellulase loadings of 15 and 60 FPU/g
cellulose in straight hydrolysis and SSF fermentations will be applied
per NREL LAP methods. Results will also be continuously supplied to
researchers in Element 1 to modify pretreatment conditions to improve
results.

Texas A&M will lead characterization of solids at optimum or near
optimum overall sugar recovery for pretreatment and enzymatic hydrolysis
for each pretreatment to determine how crystallinity, acetylation,
hemicellulose and lignin removal, degree of polymerization of cellulose,
and other features vary among pretreatments and conditions. The ability
of a model they developed previously to relate enzymatic digestibility
to these features will be evaluated and modified as needed to more
accurately describe performance. The results will help us better
understand the effect of pretreated substrate features on digestibility.

Dartmouth College will lead research on understanding the relationship
of cellulase action to balance of activities and interactions with
pretreated biomass. Results from the initial digestion and substrate
characterization portions of this Element will help narrow the effort
from many possible choices to pretreated substrates that promise better
insight. Dartmouth will work with Genencor to measure how performance is
altered with changes in the levels and sources of endoglucanase,
exoglucanase, and β-glucosidase for the different pretreatments to
determine whether pretreatment type affects optimal activity balances.
Research will also be directed at measuring cellulase adsorption by
pretreated solids from different pretreatments using established methods
practiced by team members and others, and data will be developed on
adsorption of cellulose on washed lignin recovered after enzymatic
digestion to determine whether pretreatment affects non-productive
cellulase binding (8). Work will be included on whether treating
residual lignin with ammonia affects cellulase adsorption, as mentioned
briefly in the literature. Kinetic models will be developed based on
hydrolysis data, enzyme adsorption, and pretreated solids features to
aid in understanding pretreatment/cellulase interactions.

The University of British Columbia (UBC) will lead research that targets
breakdown of residual hemicellulose in solids following pretreatment
using hemicellulases supplied by Genencor. This research is particularly
important for AFEX, ARP, and lime pretreatments that leave a large
fraction of residual hemicellulose. Thus, Michigan State, Auburn, and
Texas A&M will work closely with UBC. However, enzymatic release of
sugars from residual hemicellulose and oligomers following dilute acid
and SO~2~ pretreatment could allow use of less severe conditions and
improve overall hemicellulose sugar yields, and UBC will coordinate this
research with NREL as well. Hemicellulase activities, loadings,
substrate features, and performance will be related, and fundamental
models will be developed to relate performance to substrate and enzyme
features. This research will be integrated with that on cellulase to
determine if cellulase and hemicellulase actions are interrelated or
independent.

**Element 4: Estimate capital and operating costs for each integrated
pretreatment, hydrolysis, and fermentation system and use to guide
project.** NREL will be responsible for a comparative economic analysis
of leading pretreatment options based on technical results from the
proposed work. NREL process engineers have developed baseline models for
conversion of biomass to ethanol for each pretreatment through DOE
funding for the existing USDA IFAFS funded CAFI project. In cooperation
with the CAFI team, NREL will modify and refine these ASPEN based
material and energy balance models based on hardwood results from the
proposed project. In addition, capital and operating costs will be
estimated based on NREL's extensive cost database. These models will
focus on making ethanol from biomass, but most of the operations, and
therefore implications, are applicable to biological production of other
products. The result will provide insight on how each pretreatment
technology affects costs when integrated into a representative process.
This approach will also help us identify important technical issues that
impact cost and could impede applications and provide valuable insight
that will allow us to better target our research as new results are
produced.

**Element 5: Work with Industrial and Agricultural Advisory Board and
others in execution, dissemination, application, and advancement of
integrated pretreatment systems.** Dartmouth College will be responsible
for assembling an Advisory Board for the project. This portion will
build off the Advisory Board assembled for the USDA IFAFS funded project
that has representatives from 16 companies including existing large
processing companies (e.g., Dupont and Cargill), start up companies
(Cargill Dow and Applied Carbochemicals), agriculture organizations
(e.g., National Corn Growers Association), consultants (e.g., CEA), and
enzyme companies (e.g., Genencor and Novozymes). This combination of
advisors will provide diverse perspectives on the interests of industry
and agriculture that should help us better focus research.

We propose to continue conference calls among the research team twice a
month in which each team member on a rotating basis presents informal
written reports provided via email to the team, and these reports are
forwarded on to the Advisory Board to keep them apprised of progress. We
also plan to continue our current practice of meeting with the Advisory
Board in May and November of each year in conjunction with the Annual
Symposium on Biotechnology for Fuels and Chemicals and National AIChE
meeting, respectively, to review progress and seek suggestions on
promising directions and opportunities. These interactions plus email
correspondence also facilitate management of the project to include
tracking progress and costs.

An important goal is to disseminate our results widely to facilitate
commercial decisions and further research, and the team will work
closely with the Advisory Board and others to identify audiences with
interest in applying the technical and economic results and approaches
to contact such parties. For example, consideration will be given to
sending letters to each party identified to notify them of the project,
the participants, and the approach being taken. Such a letter could
provide information on contacting the team for data and analysis. The
team will also prepare a final report summarizing comparative data,
economics, and modeling results to serve as a basis for several
presentations and peer reviewed papers in the literature.

# Project Timetable

The proposed project is ambitious but achievable because of extensive
team experience with cellulosics, the ability to build from technical
and modeling platforms in USDA IFAFS project, experience with leading
teams, the assignment of responsibilities, and use of results to adjust
to capitalize on opportunities. The research is planned according to the
following milestones assuming a start on April 1, 2004 with the number
following E referring to the proposed work element. Also in parentheses
are initials of the institution performing the research for that
element, although others may perform some of the research with
AU=Auburn, DC=Dartmouth, MSU=Michigan State, NREL=National Renewable
Energy Laboratory, PU=Purdue, TAMU=Texas A&M, UBC=University of British
Columbia. Furthermore, MSU is responsible for coordinating E1 research,
PU for E2, DC for E3 and E5, and NREL for E4.

**Ongoing:**

-   Meet with Agricultural and Industrial Advisory Board to review
    progress, gain feedback, and adjust plans (E5: All) May and November
    2004 to 2006.

-   Hold twice a month conference calls with each team member reporting
    on a rotating basis and distribute informal reports prepared for
    calls to Advisory Board (E5: All) 2/month over project life.

**Year 1:**

-   Provide corn stover hydrolyzates and solids pretreated by dilute
    acid at pilot scale at conditions established in past (E1: NREL).
    September 2004.

-   Provide poplar hydrolyzates and solids pretreated by dilute acid at
    bench scale at conditions established in past (E1: NREL). October
    2004.

-   Provide poplar hydrolyzates and solids pretreated by dilute acid at
    pilot scale at conditions established in past (E1: NREL). December
    2004.

-   Define pretreatment conditions to maximize recovery of sugars from
    hemicellulose and cellulose from corn stover via SO~2~ pretreatment
    and provide corn stover solids and hydrolyzates pretreated by sulfur
    dioxide at these conditions for Elements 2 and 3 (E1: UBC). March
    2005.

-   Determine how cellulase and hemicellulase loadings impact glucose
    release from corn stover and poplar from dilute acid pretreatment
    (E3: DC/UBC) March 2005.

**Year 2:**

-   Define conditioning and fermentation methods to ferment hydrolyzates
    from dilute acid and SO~2~ pretreatment at high yields with
    genetically modified yeast (E2: PU). April 2005.

-   Apply controlled pH pretreatment to poplar, define pretreatment
    conditions to maximize total sugar recovery, and supply pretreated
    hydrolyzate and solids (E1: PU). May 2005.

-   Determine effect of cellulase and hemicellulase loadings on sugar
    yields from hemicellulose and cellulose for sulfur dioxide treated
    corn stover (E3: DC/UBC). June 2005.

-   Define pretreatment conditions to maximize recovery of sugars from
    hemicellulose and cellulose from poplar via SO~2~ pretreatment and
    provide materials prepared at these conditions for Elements 2 and 3
    (E1: UBC). August 2005.

-   Define pretreatment conditions to maximize recovery of sugars from
    hemicellulose and cellulose from poplar via AFEX technology and
    provide materials prepared at these conditions for Elements 2 and 3
    (E1: MSU). September 2005.

-   Define ARP pretreatment conditions for maximum sugar recovery from
    poplar and provide hydrolyzate and solids at these conditions to
    Elements 2 and 3 (E1: AU). October 2005.

-   Define conditioning, hydrolysis, and fermentation methods to realize
    high yields of ethanol from hemicellulose for controlled pH
    pretreated poplar (E2 and E3: PU/DC/UBC). November 2005.

-   Determine effect of cellulase and hemicellulase loadings on sugar
    yields from hemicellulose and cellulose for controlled pH treated
    solids and define best conditions (E3: DC/UBC). January 2006.

-   Apply flowthrough to prepare poplar hydrolyzate and solids samples
    that facilitate comparison to other pretreatments with similar
    hemicellulose and lignin removal (E1: DC). March 2006.

**Year 3:**

-   Pretreat poplar with lime and provide pretreated solids at
    conditions found to maximize digestibility of cellulose (E1: TAMU).
    April 2006.

-   Develop economic models for all pretreatments based on initial data,
    and review with team to adjust approach and clarify additional data
    needs (E4: NREL). June 2006.

-   Define conditioning, hydrolysis, and fermentation methods to realize
    high yields of ethanol from hemicellulose for AFEX and ARP
    pretreated poplar (E2 and E3: PU/DC/UBC). November 2006.

-   Determine effect of cellulase and hemicellulase loadings on sugar
    yields from hemicellulose and cellulose for AFEX, ARP, and lime
    treated solids to define best conditions (E3: DC/UBC). December
    2006.

-   Complete data and models to relate digestibility of cellulose and
    hemicellulose to chemical and physical characteristics of pretreated
    biomass (E3: DC/TAMU/UBC). January 2007.

-   Define conditioning, hydrolysis, and fermentation methods to realize
    high yields of ethanol from hemicellulose for lime pretreated poplar
    (E2 and E3: PU/DC/UBC). February 2007.

-   Develop kinetic models to predict performance of hydrolyzate
    fermentations using genetically engineered yeast including effects
    of inhibitors on performance (E2: PU). February 2007.

-   Complete data and models to relate cellulose digestion to enzyme
    activities, enzyme adsorption by lignin, and effects of pretreatment
    chemicals on lignin adsorption (E3: DC). February 2007.

-   Complete data and models to relate digestion of hemicellulose to
    hemicellulase activities and adsorption by lignin (E3: UBC).
    February 2007.

-   Develop models to relate the performance of AFEX and ARP
    pretreatments to concentration of ammonia, temperature, water
    concentration, and time (E1: MSU/AU). March 2007.

-   Finalize economic models and compare pretreatments for poplar based
    on data produced in project (E4: NREL). March 2007.

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-   Prepare report summarizing comparisons in technical performance and
    economics for pretreatment, fermentation, and enzymatic hydrolysis
    and documenting models to describe these operations (E5: All). March
    2007.

# CAFI Project Advisory Board

> Quang Nguyen, Abengoa Bioenergy
>
> Mat Peabody, formerly Applied CarboChemicals
>
> Gary Welch, Aventinerei
>
> Greg Luli, BC International
>
> Paris Tsobanakis, Cargill
>
> James Hettenhaus, CEA
>
> Lyman Thompson, Chevron-Texaco
>
> Kevin Gray, Diversa
>
> Paul Roessler, Dow
>
> Susan M. Hennessey, DuPont
>
> Michael Knauf, Genencor
>
> Don Johnson, GPC (Retired)
>
> Dale Monceaux, Katzen Engineers
>
> Kendall Pye, Lignol
>
> Farzaneh Teymouri, MBI
>
> Richard Glass, National Corn Growers Association
>
> Bill Cruickshank, Natural Resources Canada
>
> Robert Wooley, Nature Works
>
> Joel Cherry, Novozymes
>
> Ron Reinsfelder, Shell
>
> Carl Miller, Syngenta
>
> Carmela Bailey, USDA
>
> Don Riemenschneider, USDA

The following table summarizes key yield data developed through a prior
CAFI project funded by the USDA Initiative for Future Agriculture and
Food Systems Program and the DOE Office of the Biomass Program.

  --------------- ----------- ----------- ----------- --------- ------- ----------- ----------- ----------- -----------
  Yields of                                                                                                 
  xylose and                                                                                                
  glucose for                                                                                               
  pretreatment of                                                                                           
  corn stover by                                                                                            
  leading options                                                                                           
  studied through                                                                                           
  USDA IFAFS and                                                                                            
  DOE OBP support                                                                                           
  following                                                                                                 
  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     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     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                                                                                              

  Controlled pH   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                        34.6/29.3   34.6/29.3             59.8    59.8                    94.4/89.1   94.4/89.1

  ARP             17.8/0      15.5        33.3/15.5   0         56.1    56.1        17.8/0      71.6        89.4/71.6

  Lime            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
  --------------- ----------- ----------- ----------- --------- ------- ----------- ----------- ----------- -----------