twenty-fifth symposium on biotechnology for fuels and chemicals

Applied
Biochemistry
and Biotechnology
Applied
Biochemistry
and Biotechnology
Volumes 113–116 (12 Issues), Spring 2004, ISSN: 0273–2289
Executive Editor: Ashok Mulchandani
Biotechnology
for Fuels and Chemicals
The Twenty-Fifth Symposium
Editors
Mark Finkelstein
James D. McMillan
Brian H. Davison
Barbara Evans
Biotechnology
for Fuels and Chemicals
The Twenty-Fifth Symposium
Editors
Mark Finkelstein
James D. McMillan
Brian H. Davison
Barbara Evans
Humana Press • Totowa, New Jersey
Biotechnology for Fuels and Chemicals
The Twenty-Fifth Symposium
Presented as Volumes 113–116
of
Applied Biochemistry and Biotechnology
Proceedings of the Twenty-Fifth Symposium

on Biotechnology for Fuels and Chemicals
Held May 4–7, 2003, in Breckenridge, CO
Sponsored by
US Department of Energy’s Office of the Biomass Program (DOE-OBP)
National Renewable Energy Laboratory (NREL)
Oak Ridge National Laboratory (ORNL)
Argonne National Laboratory (ANL)
Idaho National Engineering and Environment Laboratory (INEEL)
Pacific Northwest National Laboratory (PNNL)
US Department of Agriculture (USDA)
Alltech
Archer Daniels Midland (ADM)
BBI International
Biotechnology Industrial Organization (BIO)
Breckenridge Brewery
Cargill, Inc.
Cargill Dow, LLC
Coors Brewing Company
Corn Refiners Association (CRA)
E. I. du Pont de Nemours & Co., Inc. (DuPont)
Genencor International
Iogen Corporation
Katzen International
Natural Resources Canada
Novozymes Biotech
Proctor and Gamble
Syngenta
Tate and Lyle
Tembec Industries
Editors

Mark Finkelstein and James D. McMillan
National Renewable Energy Laboratory
Brian H. Davison and Barbara Evans
Oak Ridge National Laboratory
Applied Biochemistry and Biotechnology
Volumes 113–116, Complete, Spring 2004
Copyright © 2004 Humana Press Inc.
All Rights Reserved.
No part of this publication may be reproduced or transmitted in any
form or by any means, electronic or mechanical, including photocopy,
recording, or any information storage and retrieval system, without
permission in writing from the copyright owner.
Applied Biochemistry and Biotechnology is abstracted or indexed
regularly in Chemical Abstracts, Biological Abstracts, Current Contents, Science
Citation Index, Excerpta Medica, Index Medicus, and appropriate related
compendia.
iii
Introduction to the Proceedings
of the Twenty-Fifth Symposium
on Biotechnology for Fuels and Chemicals
MARK FINKELSTEIN
National Renewable Energy Laboratory
BRIAN H. DAVISON
Oak Ridge National Laboratory
The 25
th
Symposium on Biotechnology for Fuels and Chemicals
was held in Breckenridge, CO, May 4–7, 2003. Over 450 attendees helped
underscore the growing importance of bioenergy and a biobased economy
during this special Silver Anniversary Symposium. Forty states and

twenty-three countries were represented at the Symposium, with over
40% of the attendees from industry, almost 40% from universities and
colleges, and the remainder from government agencies and laboratories.
The robust participation and sponsorship by industry highlights the rel-
evancy and importance of this Symposium.
While improving the economics of producing fuels and chemicals is
vital to many industrial sectors, the ability to do so in a sustainable and
environmentally responsible manner is becoming increasingly important.
The program for the 25
th
Symposium on Biotechnology for Fuels and
Chemicals was designed to deliver the latest research breakthroughs and
results in biotechnology that stimulate such improvements. The technical
focus of Symposium was evident at each session, as evolutionary as well
as revolutionary research findings were revealed. The eight technical ses-
sions contained 52 oral presentations and we had 272 poster presentations.
Whether one represented the industrial, academic, or government sector,
each was invited to participate in this stimulating exchange of information
and ideas. Valuable opportunities for productive interactions with col-
leagues, both from a national and international perspective, were evident
throughout the meeting.
iv
Introduction
With the 25
th
Symposium, we continued the tradition of providing an
informal, congenial atmosphere that our participants found conducive to
discussing technical program topics. This year’s topics included:
Session 1A Feedstock Supply, Logistics, Processing, and Composition
Session 1B Enzyme Catalysis and Engineering

Session 2 Microbial Catalysis and Engineering
Session 3 Bioprocessing including Separations
Session 4 Biotechnology for Fuels and Chemicals—Past, Present, and Future
Session 5 Biobased Industrial Chemicals
Session 6A Biomass Pretreatment and Hydrolysis
Session 6B Plant Biotechnology and Feedstock Genomics
Special Topics A Microbial Pentose Metabolism
Special Topics B International Bioenergy Agency Bioethanol Meeting
A summary of these sessions is included at the beginning of each
section of the Proceedings. The technical core of the Symposium remains
the creation, manipulation, and practical use of new biocatalysts to pro-
duce useful fuels and chemicals. This was shown in both fundamental
science discoveries as well as progress reports from commercialization
efforts. However, we have continued to expand interest into the bio-
feedstock (plant genomics, production, and pretreatment). While the pro-
duction of sugars from biomass and their subsequent bioconversion into
chemicals remains the dominant thrust, areas such as gasification and
direct production in plants were presented. The use of molecular biology
and genomics to provide new biocatalysts as well as understanding the
fundamentals of the microbes, enzymes, and biomass has increased the
likely achievement of both near and long-term commercialization goals.
This year Dr. J. Craig Venter, Genome Sequencer, Entrepreneur, and
Chief Executive Officer augmented our technical program with a spell-
binding after dinner presentation entitled “Genomic Approaches to the
Environment.” This touched on his latest efforts in metagenomics and in
constructing a minimal genome.
Charles D. Scott Award Presentation
For the past 10 symposia we have recognized an individual who has
distinguished him or herself in the application of biotechnology towards
the production of fuels and chemicals. This award acknowledges contribu-

tions to the field as a whole or this symposium, with particular attention to
innovation in fundamental and applied biotechnology, insight into
bioprocessing fundamentals, or commitment to facilitate commercializa-
tion of products from renewable resources. The award is named in honor
of Dr. Charles D. Scott, the founder of this symposium and its chair for the
first 10 years.
Dr. Tom Jeffries received this year’s award. Dr. Jeffries is a microbiolo-
gist whose research focuses on using plant matter to make the fuel ethanol.
He directs the Institute for Microbial and Biochemical Technology at the
USDA Forest Service Forest Products Laboratory in Madison, WI, and is a
Introduction
v
professor in the Bacteriology and Food Science departments at the Univer-
sity of Wisconsin, Madison. His recent research has aimed at modifying
yeast DNA to increase the amount of ethanol produced when the yeast
metabolizes certain components of wood. He was also one of four attendees
who attended the inaugural Symposium 25 years ago (along with Bob
Tanner, Y.Y. Lee, and Chuck Scott).
Session Chairpersons
Session 1A: Feedstock Supply, Logistics, Processing, and Composition.
Topics encompassed production and collection issues (availability, farm-
ing, silviculture, harvesting, densification, consistency and storage).
Chair: Jim Hettenhaus, E-mail:
Co-Chair: David Morris, E-mail:
Session 1B: Enzyme Catalysis and Engineering.
Speakers focused on how to identify, modify, develop, and cost-effectively
produce the use of enzymes to carry out a myriad of biological/chemical
transformations.
Chair: Mike Himmel, E-mail:
Co-Chair: David Wilson, E-mail:

Session 2: Microbial Catalysis and Engineering.
This session focused on finding new and developing existing microorgan-
isms for improved performance to produce fuels and chemicals.
Chair: Tom Jeffries, E-mail:
Co-Chair: Lee Lynd, E-mail:
Session 3: Bioprocessing, Including Separations.
This session covered reactor configurations and the integration of engineer-
ing with microbiology, biochemistry, and chemistry to produce fuels and
chemicals.
Chair: Dale Monceaux, E-mail:
Co-Chair: David Short, E-mail:
Session 4: Biotechnology for Fuels and Chemicals—
Past, Present, and Future.
This session gazed into the crystal ball while occasionally looking into the
rear-view mirror to review the past 25 years.
Chair: Chuck Scott, E-mail:
Co-Chair: Charles Wyman, E-mail:
Session 5: Biobased Industrial Chemicals.
The production of chemicals and other value-added bioproducts from bio-
logical rather than petrochemical uses was discussed at this session.
Advances in process integration, demonstration, economics, and commer-
cialization were highlighted.
Chair: Doug Cameron, E-mail:
Co-Chair: Marion Bradford, Tate & Lyle, Retired
vi
Introduction
Session 6A: Biomass Pretreatment and Hydrolysis.
Novel reactor configurations as well as new process approaches to pretreat
and saccharify biomass was the focus of this session.
Chair: Y. Y. Lee, E-mail:

Co-Chair: Bruce Dale, E-mail:
Session 6B: Plant Biotech and Feedstock Genetics.
The sustainable success of Bioenergy and Bioproducts requires new inte-
grated approaches. The potential impact of transgenic, genetic, and genomic-
based modifications to the archi-tectural, compositional, or metabolic func-
tions of plants was discussed in relation to an enhanced renewable base.
Chair: Jim McLaren, E-mail:
Co-Chair: Steve Thomas, E-mail:
Special Topics: Pentose Metabolism.
Microorganisms capable of converting biomass pentose sugars to fuels and
chemicals are essential for robust biomass-based processes. This session dis-
cussed recent technological developments and understanding that will
allow for the construction of superior microorganisms.
Chair: Barbel Hahn-Hagerdal, Lund University, Sweden
Co-Chair: Neville Pamment, University of Melbourne, Australia
Special Topics: IEA Task 39 Bioethanol
Chair: Jack Saddler, E-mail:
Co-Chair: David Gregg, E-mail:
Organizing Committee
Mark Finkelstein, Conference Chair, National Renewable Energy
Laboratory, Golden, CO
Brian Davison, Conference Co-Chair, Oak Ridge National Labora-
tory, Oak Ridge, TN
William Apel, Idaho National Engineering and Environmental
Laboratory, Idaho Falls, ID
Doug Cameron, Cargill, Minneapolis, MN
Tom Jeffries, USDA, Forest Service, Madison, WI
James Lee, Oak Ridge National Laboratory, Oak Ridge, TN
Lee Lynd, Dartmouth College, Hanover, NH
James McMillan, National Renewable Energy Laboratory, Golden, CO

Amy Mannheim, US Department of Energy, Washington, DC
Dale Monceaux, Katzen International, Cincinnati, OH
Jack Saddler, University of British Columbia, Vancouver, British
Columbia, Canada
Sharon Shoemaker, University of California, Davis, CA
David Short, DuPont, Newark, DE
Jim Spaeth, US Department of Energy, Golden, CO
Introduction
vii
Jeff Tolan, Iogen Corporation, Ottawa, Ontario, Canada
Nancy Watlington, Oak Ridge National Laboratory, Oak Ridge, TN
Liz Willson, National Renewable Energy Laboratory, Golden, CO
Charles E. Wyman, Dartmouth College, Hanover, NH
Guido Zacchi, Lund University, Lund, Sweden
Gisella M. Zanin, State University of Maringa, Maringa, Parana,
Brazil
Acknowledgments
The continued success of the symposium is due to the many partici-
pants, organizers, and sponsors, but is also a success and pleasure due to
the diligent and creative staff. In particular, Liz Willson of NREL and
Nancy Watlington of ORNL, provided organization, advice, persistence,
and unfailing good humor. Howard Brown of NREL provided communi-
cations and website acumen, Ivilina Thornton provided the overall con-
ference coordination, and the overall assistance from Lyn Lumberg of
NREL is gratefully acknowledged.
The National Renewable Energy Laboratory is operated for the US
Department of Energy by Midwest Research Institute and Battelle under
contract DE-AC36-99GO10337.
Oak Ridge National Laboratory is operated for the US Department of
Energy by UT-Battelle, LLC under contract DE-AC05-00OR22725.

A contractor of the US Government has authored the submitted manu-
scripts. Accordingly the US Government retains a non-exclusive, royalty-
free license to publish or reproduce the published forms of this contribution,
or allow others to do so for US Government purposes.
Other Proceedings in this Series
1. Proceedings of the First Symposium on Biotechnology in Energy Produc-
tion and Conservation (1978), Biotechnol. Bioeng. Symp. 8.
2. Proceedings of the Second Symposium on Biotechnology in Energy Pro-
duction and Conservation (1980), Biotechnol. Bioeng. Symp. 10.
3. Proceedings of the Third Symposium on Biotechnology in Energy Produc-
tion and Conservation (1981), Biotechnol. Bioeng. Symp. 11.
4. Proceedings of the Fourth Symposium on Biotechnology in Energy Pro-
duction and Conservation (1982), Biotechnol. Bioeng. Symp. 12.
5. Proceedings of the Fifth Symposium on Biotechnology for Fuels and Chemi-
cals (1983), Biotechnol. Bioeng. Symp. 13.
6. Proceedings of the Sixth Symposium on Biotechnology for Fuels and Chemi-
cals (1984), Biotechnol. Bioeng. Symp. 14.
7. Proceedings of the Seventh Symposium on Biotechnology for Fuels and
Chemicals (1985), Biotechnol. Bioeng. Symp. 15.
8. Proceedings of the Eighth Symposium on Biotechnology for Fuels and
Chemicals (1986), Biotechnol. Bioeng. Symp. 16.
9. Proceedings of the Ninth Symposium on Biotechnology for Fuels and
Chemicals (1988), Appl. Biochem. Biotechnol. Symp. 17,18.
10. Proceedings of the Tenth Symposium on Biotechnology for Fuels and
Chemicals (1989), Appl. Biochem. Biotechnol. Symp. 20,21.
11.Proceedings of the Eleventh Symposium on Biotechnology for Fuels and
Chemicals (1990), Appl. Biochem. Biotechnol. Symp. 24,25.
12. Proceedings of the Twelfth Symposium on Biotechnology for Fuels and
Chemicals (1991), Appl. Biochem. Biotechnol. Symp. 28,29.
13. Proceedings of the Thirteenth Symposium on Biotechnology for Fuels and

Chemicals (1992), Appl. Biochem. Biotechnol. Symp. 34,35.
14. Proceedings of the Fourteenth Symposium on Biotechnology for Fuels and
Chemicals (1993), Appl. Biochem. Biotechnol. Symp. 39,40.
15. Proceedings of the Fifteenth Symposium on Biotechnology for Fuels and
Chemicals (1994), Appl. Biochem. Biotechnol. Symp. 45,46.
16. Proceedings of the Sixteenth Symposium on Biotechnology for Fuels and
Chemicals (1995), Appl. Biochem. Biotechnol. Symp. 51,52.
17. Proceedings of the Seventeenth Symposium on Biotechnology for Fuels
and Chemicals (1996), Appl. Biochem. Biotechnol. Symp. 57,58.
18. Proceedings of the Eighteenth Symposium on Biotechnology for Fuels and
Chemicals (1997), Appl. Biochem. Biotechnol. Symp. 63–65.
19. Proceedings of the Nineteenth Symposium on Biotechnology for Fuels and
Chemicals (1998), Appl. Biochem. Biotechnol. Symp. 70–72.
20. Proceedings of the Twentieth Symposium on Biotechnology for Fuels and
Chemicals (1999), Appl. Biochem. Biotechnol. Symp. 77–79.
21. Proceedings of the Twenty–First Symposium on Biotechnology for Fuels
and Chemicals (2000), Appl. Biochem. Biotechnol. Symp. 84–86.
22. Proceedings of the Twenty–second Symposium on Biotechnology for Fu-
els and Chemicals (2001), Appl. Biochem. Biotechnol. Symp. 91–93.
23. Proceedings of the Twenty–Third Symposium on Biotechnology for Fuels
and Chemicals (2002), Appl. Biochem. Biotechnol. Symp. 98–100.
24. Proceedings of the Twenty–Fourth Symposium on Biotechnology for Fuels
and Chemicals (2003), Appl. Biochem. Biotechnol. Symp. 105–108.
This symposium has been held annually since 1978. We are pleased to
have the Proceedings of the Twenty-Fifth Symposium currently published
in this special issue to continue the tradition of providing a record of the
contributions made.
The Twenty-Sixth Symposium will be held May 7–11, 2004, in
Chattanooga, Tennessee. For more information, visit the following Web-
sites: or />symposium. We encourage comments or discussions relevant to the for-

mat or content of the meetings.
viii
Introduction
ix
Applied Biochemistry and Biotechnology Vols. 113–116, Spring 2004
*For papers with multiple authorship, the asterisk identifies the author to whom corre-
spondence and reprint requests should be addressed.
CONTENTS
Introduction
Brian H. Davison and Mark Finkelstein iii
Volume 113
S
ESSION 1A—FEEDSTOCK SUPPLY, LOGISTICS, PROCESSING, AND COMPOSITION
Introduction to Session 1A
Jim Hettenhaus and David Morris 3
Designing an Effective Federal Biomass Program
David Morris 5
Methodology for Estimating Removable Quantities
of Agricultural Residues for Bioenergy and Bioproduct Use
Richard G. Nelson,* Marie Walsh, John J. Sheehan,
and Robin Graham 13
Pipeline Transport of Biomass
Amit Kumar, Jay B. Cameron, and Peter C. Flynn* 27
Hydrodynamic Separation of Grain
and Stover Components in Corn Silage
Philippe Savoie,* Kevin J. Shinners, and Benjamin N. Binversie 41
A New Class of Plants for a Biofuel Feedstock Energy Crop
James Kamm 55
Fungal Upgrading of Wheat Straw
for Straw-Thermoplastics Production

Tracy P. Houghton, David N. Thompson,* J. Richard Hess,
Jeffrey A. Lacey, Michael P. Wolcott, Anke Schirp,
Karl Englund, David Dostal, and Frank Loge 71
Economic Analysis of Ethanol Production in California
Using Traditional and Innovative Feedstock Supplies
Ellen I. Burnes, John Hagen, Dennis Wichelns,*
and Kristen Callens 95
S
ESSION 1B—ENZYME CATALYSIS AND ENGINEERING
Introduction to Session 1B
Mike Himmel and David Wilson 113
x
Contents
Dynamics of Cellulase Production by Glucose
Grown Cultures of Trichoderma reesei Rut-C30
as a Response to Addition of Cellulose
Nóra Szijártó, Zsolt Szengyel, Gunnar Lidén,
and Kati Réczey* 115
Development and Application of an Integrated System
for Monitoring Ethanol Content of Fuels
Eliana M. Alhadeff, Andrea M. Salgado, Nei Pereira Jr.,
and Belkis Valdman* 125
Model Based Soft-Sensor for On-Line Determination of Substrate
Andréa M. Salgado, Rossana O. M. Folly, Belkis Valdman,*
and Francisco Valero 137
Screening of Dowex
®
Anion-Exchange Resins
for Invertase Immobilization
Ester Junko Tomotani and Michele Vitolo* 145

Effects of Carbon Source on Expression of Alcohol Oxidase
Activity and on Morphologic Pattern of YR-1 Strain,
a Filamentous Fungus Isolated
from Petroleum-Contaminated Soils
Carmen Rodríguez Robelo, Vanesa Zazueta Novoa,
and Roberto Zazueta-Sandoval* 161
Effect of Temperature, Moisture, and Carbon Supplementation
on Lipase Production by Solid-State Fermentation
of Soy Cake by Penicillium simplicissimum
Marco Di Luccio,* Fernando Capra, Najara P. Ribeiro,
Gean D. L. P. Vargas, Denise M. G. Freire,
and Débora de Oliveira 173
The Effect of Temperature, Pressure, Exposure Time,
and Depressurization Rate on Lipase Activity in SCCO
2
Marcelo Lanza, Wagner Luís Priamo,
José Vladimir Oliveira, Cláudio Dariva,
and Débora de Oliveira* 181
Ester Synthesis Catalyzed by Mucor miehei Lipase Immobilized
on Magnetic Polysiloxane–Polyvinyl Alcohol Particles
Laura M. Bruno, José L. de Lima Filho, Eduardo H. de M. Melo,
and Heizir F. de Castro* 189
Effect of pH on Cellulase Production
of Trichoderma reesei RUT C30
Tamás Juhász, Zsolt Szengyel,* Nóra Szijártó, and Kati Réczey 201
Contents
xi
Quantitative Analysis of Cellulose-Reducing Ends
Sasithorn Kongruang, Myung Joo Han, Claudia Isela Gil Breton,
and Michael H. Penner* 213

Properties of a Recombinant β-Glucosidase from Polycentric
Anaerobic Fungus Orpinomyces PC-2 and Its Application
for Cellulose Hydrolysis
Xin-Liang Li,* Lars G. Ljungdahl, Eduardo A. Ximenes,
Huizhong Chen, Carlos R. Felix, Michael A. Cotta,
and Bruce S. Dien 233
Characterization and Performance
of Immobilized Amylase and Cellulase
Bradley A. Saville,* Mikhail Khavkine, Gayathri Seetharam,
Behzad Marandi, and Yong-Li Zuo 251
Immobilized Enzyme Studies in a Microscale Bioreactor
Francis Jones, Scott Forrest, Jim Palmer, Zonghuan Lu,
John Elmore, and Bill B. Elmore* 261
Performance of Chloroperoxidase Stabilization
in Mesoporous Sol-Gel Glass Using In Situ
Glucose Oxidase Peroxide Generation
Abhijeet Borole,* Sheng Dai, Catherine L. Cheng,
Miguel Rodriguez Jr., and Brian H. Davison 273
Integration of Computer Modeling and Initial Studies
of Site-Directed Mutagenesis to Improve Cellulase
Activity on Cel9A from Thermobifida fusca
José M. Escovar-Kousen,* David Wilson, and Diana Irwin 287
Kinetics of Asparaginase II Fermentation
in Saccharomyces cerevisiae ure2dal80 Mutant:
Effect of Nitrogen Nutrition and pH
Maria Antonieta Ferrara,* Josiane M. V. Mattoso,
Elba P. S. Bon, and Nei Pereira Jr. 299
Studies on Immobilized Lipase in Hydrophobic Sol-Gel
Cleide M. F. Soares, Onelia A. dos Santos, Heizir F. de Castro,
Flavio F. de Moraes, and Gisella M. Zanin* 307

Volume 114
S
ESSION 2—INTRODUCTION TO MICROBIAL CATALYSIS AND ENGINEERING
Introduction to Session 2
Thomas W. Jeffries and Lee R. Lynd 323
xii
Contents
Polykaryon Formation Using a Swollen Conidium
of Trichoderma reesei
Hideo Toyama,* Makiko Yano, and Takeshi Hotta 325
Biosynthesis of Poly(3-hydroxybutyrate-co-3-hydroxyalkanoates)
by Metabolically Engineered Escherichia coli Strains
Si Jae Park and Sang Yup Lee* 335
Effect of Corn Stover Concentration on Rheological Characteristics
Natalia V. Pimenova and Thomas R. Hanley* 347
Construction of Recombinant Escherichia coli Strains for Production
of Poly-(3-hydroxybutyrate-co-3-hydroxyvalerate)
Kin-Ho Law, Pui-Ling Chan, Wai-Sum Lau, Yin-Chung Cheng,
Yun-Chung Leung, Wai-Hung Lo, Hugh Lawford,
and Hoi-Fu Yu* 361
Biosynthesis of (R)-3-Hydroxyalkanoic Acids by Metabolically
Engineered Escherichia coli
Si Jae Park, Sang Yup Lee,* and Young Lee 373
Gibberellic Acid Production by Free and Immobilized Cells
in Different Culture Systems
Enrique Durán-Páramo,* Héctor Molina-Jiménez,
Marco A. Brito-Arias, and Fabián Robles-Martínez 381
Screening Genus Penicillium for Producers of Cellulolytic
and Xylanolytic Enzymes
Kristian B. R. Krough, Astrid Mørkeberg, Henning Jørgensen,

Jens C. Frisvad, and Lisbeth Olsson* 389
Production of Ethanol from Cellulosic Biomass Hydrolysates
Using Genetically Engineered Saccharomyces Yeast
Capable of Cofermenting Glucose and Xylose
Miroslav Sedlak and Nancy W. Y. Ho* 403
Secondary Membranes for Flux Optimization
in Membrane Filtration of Biologic Suspensions
Parag R. Nemade and Robert H. Davis* 417
Enzymatic Synthesis of Monolaurin
Carla C. B. Pereira, Mônica A. P. da Silva,
and Marta A. P. Langone* 433
S
ESSION 3—BIOPROCESSING, INCLUDING SEPARATIONS
Introduction to Session 3
Dale A. Monceaux and David R. Short 449
Contents
xiii
Evaluation of Recombinant Green Fluorescent Protein, Under
Various Culture Conditions and Purification with HiTrap
Hydrophobic Interaction Chromatography Resins
Thereza Christina Vessoni Penna,* Marina Ishii,
Adalberto Pessoa Junior, Laura de Oliveira Nascimento,
Luciana Cambricoli de Souza, and Olivia Cholewa 453
Thermal Stability of Recombinant Green Fluorescent
Protein (GFPuv) at Various pH Values
Thereza Christina Vessoni Penna,* Marina Ishii,
Adalberto Pessoa Junior, and Olivia Cholewa 469
Evaluation of Optimization Techniques for an Extractive
Alcoholic Fermentation Process
Aline C. da Costa* and Rubens Maciel Filho 485

Yields from Glucose, Xylose, and Paper Sludge Hydrolysate
During Hydrogen Production by the Extreme
Thermophile Caldicellulosiruptor saccharolyticus
Zsófia Kádár, Truus de Vrije, Giel E. van Noorden,
Miriam A. W. Budde, Zsolt Szengyel, Kati Réczey,
and Pieternel A. M. Claassen* 497
Optimization of Steam Pretreatment of Corn Stover
to Enhance Enzymatic Digestibility
Eniko Varga, Kati Réczey,* and Guido Zacchi 509
Selection of Anion Exchangers for Detoxification
of Dilute-Acid Hydrolysates from Spruce
Ilona Sárvári Horváth, Anders Sjöde, Nils-Olof Nilvebrant,
Andrei Zagorodni, and Leif J. Jönsson* 525
Ethanol Production in Immobilized-Cell Bioreactors
from Mixed Sugar Syrups and Enzymatic
Hydrolysates of Steam-Exploded Biomass
Isabella De Bari,* Daniela Cuna, Francesco Nanna,
and Giacobbe Braccio 539
Silymarin Extraction from Milk Thistle Using Hot Water
Lijun Duan, Danielle Julie Carrier, and Edgar C. Clausen* 559
Extraction of Antioxidant Compounds from Energy Crops
Ching S. Lau, Danielle Julie Carrier, Luke R. Howard,
Jackson O. Lay Jr., Jean A. Archambault,
and Edgar C. Clausen* 569
Cellulase Retention and Sugar Removal by Membrane
Ultrafiltration During Lignocellulosic Biomass Hydrolysis
Jeffrey S. Knutsen and Robert H. Davis* 585
"
xiv
Contents

Controlled Fed-Batch Fermentations of Dilute-Acid Hydrolysate
in Pilot Development Unit Scale
Andreas Rudolf,* Mats Galbe, and Gunnar Lidén 601
Degeneration of β-Glucosidase Activity in a Foam
Fractionation Process
Vorakan Burapatana, Ales Prokop, and Robert D. Tanner* 619
Simultaneous Production of Nisin and Lactic Acid
from Cheese Whey: Optimization of Fermentation Conditions
Through Statistically Based Experimental Designs
Chuanbin Liu, Yan Liu, Wei Liao, Zhiyou Wen,
and Shulin Chen* 627
Effect of Process Parameters on Production
of a Biopolymer by Rhizobium sp.
Flávia Pereira Duta, Francisca Pessôa De França,*
Eliana Flávia Camporese Sérvulo,
Léa Maria De Almeida Lopes,
Antonio Carlos Augusto Da Costa, and Ana Barros 639
Succinic Acid Adsorption from Fermentation Broth
and Regeneration
Brian H. Davison,* Nhuan P. Nghiem,
and Gerald L. Richardson 653
A Hollow-Fiber Membrane Extraction Process for Recovery
and Separation of Lactic Acid from Aqueous Solution
Hanjing Huang, Shang-Tian Yang,* and David E. Ramey 671
Evaluation of Tocopherol Recovery Through Simulation
of Molecular Distillation Process
E. B. Moraes, C. B. Batistella, M. E. Torres Alvarez,
Rubens Maciel Filho, and M. R. Wolf Maciel* 689
High-Productivity Continuous Biofilm Reactor
for Butanol Production: Effect of Acetate, Butyrate,

and Corn Steep Liquor on Bioreactor Performance
Nasib Qureshi,* Patrick Karcher, Michael Cotta,
and Hans P. Blaschek 713
Measurement of Rheology of Distiller’s Grain Slurries
Using a Helical Impeller Viscometer
Tiffany L. Houchin and Thomas R. Hanley* 723
Computation Fluid Dynamics Simulation and Redesign
of a Screw Conveyor Reactor
Yinkun Wan and Thomas R. Hanley* 733
Contents
xv
Production of Biodiesel Fuel by Transesterification of Rapeseed Oil
Gwi-Taek Jeong, Don-Hee Park,* Choon-Hyoung Kang,
Woo-Tai Lee, Chang-Shin Sunwoo, Chung-Han Yoon,
Byung-Chul Choi, Hae-Sung Kim, Si-Wouk Kim,
and Un-Taek Lee 747
Volume 115
S
ESSION 4—BIOTECHNOLOGY FOR FUELS AND CHEMICALS—
P
AST, PRESENT, AND FUTURE
Introduction to Session 4
Charles D. Scott* and Charles E. Wyman 761
Origins of and Changes in the Symposium Series
on Biotechnology for Fuels and Chemicals
Charles D. Scott 765
Optimization of Enzymatic Production of Biodiesel
from Castor Oil in Organic Solvent Medium
Débora de Oliveira, Marco Di Luccio, Carina Faccio,
Clarissa Dalla Rose, João Paulo Bender, Nádia Lipke,

Silvana Menoncin, Cristiana Amroginski,
and José Vladimir de Oliveira* 771
Two-Step Preparation for Catalyst-Free Biodiesel Fuel Production:
Hydrolysis and Methyl Esterification
Dadan Kusdiana and Shiro Saka* 781
Biodiesel Fuel from Vegetable Oil
by Various Supercritical Alcohols
Yuichiro Warabi, Dadan Kusdiana, and Shiro Saka* 793
S
ESSION 5—BIOBASED INDUSTRIAL CHEMICALS
Introduction to Session 5
Douglas C. Cameron and Jeff Lievense 805
Effects of Trace Contaminants on Catalytic Processing
of Biomass-Derived Feedstocks
Douglas C. Elliott,* Keith L. Peterson,
Danielle S. Muzatko, Eric V. Alderson, Todd R. Hart,
and Gary G. Neuenschwander 807
Characterization of Surfactin from Bacillus subtilis
for Application as an Agent for Enhanced Oil Recovery
Kastli D. Schaller,* Sandra L. Fox, Debby F. Bruhn,
Karl S. Noah, and Gregory A. Bala 827
xvi
Contents
Effect of Germ and Fiber Removal on Production
of Ethanol from Corn
Elankovan Ponnampalam,* D. Bernie Steele,
Deborah Burgdorf, and Darold McCalla 837
Production of Fumaric Acid Using Rice Bran and Subsequent
Conversion to Succinic Acid Through a Two-Step Process
Se-Kwon Moon, Young-Jung Wee, Jong-Sun Yun,

and Hwa-Won Ryu* 843
Catalytic Hydrogenation of Glutamic Acid
Johnathan E. Holladay,* Todd A. Werpy,
and Danielle S. Muzatko 857
Opportunities in the Industrial Biobased Products Industry
Tracy M. Carole,* Joan Pellegrino, and Mark D. Paster 871
Continuous Production of Butanol by Clostridium acetobutylicum
Immobilized in a Fibrous Bed Bioreactor
Wei-Cho Huang, David E. Ramey, and Shang-Tian Yang* 887
Lipopeptide Surfactant Production by Bacillus subtilis Grown
on Low-Cost Raw Materials
Fabíula A. S. L. Reis, Eliana Flavia C. Sérvulo,*
and Francisca P. de França 899
Higher-Alcohols Biorefinery:
Improvement of Catalyst for Ethanol Conversion
Edwin S. Olson,* Ramesh K. Sharma, and Ted R. Aulich 913
S
ESSION 6A—BIOMASS PRETREATMENT AND HYDROLYSIS
Introduction to Session 6A
Yong Y. Lee and Bruce E. Dale 935
Fermentation of “Quick Fiber” Produced from a Modified Corn-
Milling Process into Ethanol and Recovery of Corn Fiber Oil
Bruce S. Dien,* Nick Nagle, Kevin B. Hicks, Vijay Singh,
Robert A. Moreau, Melvin P. Tucker, Nancy N. Nichols,
David B. Johnston, Michael A. Cotta, Quang Nguyen,
and Rodney J. Bothast 937
Ammonia Fiber Explosion Treatment of Corn Stover
Farzaneh Teymouri, Lizbeth Laureano-Pérez,
Hasan Alizadeh, and Bruce E. Dale* 951
Initial Evaluation of Simple Mass Transfer Models to Describe

Hemicellulose Hydrolysis in Corn Stover
Michael A. Brennan and Charles E. Wyman* 965
Contents
xvii
Impact of Fluid Velocity on Hot Water Only Pretreatment
of Corn Stover in a Flowthrough Reactor
Chaogang Liu and Charles E. Wyman* 977
Combined Steam Pretreatment and Enzymatic Hydrolysis
of Starch-Free Wheat Fibers
Beatriz Palmarola-Adrados, Mats Galbe, and Guido Zacchi* 989
Application of Xylanase from Thermomyces lanuginosus
IOC-4145 for Enzymatic Hydrolysis of Corncob
and Sugarcane Bagasse
Mônica Caramez Triches Damaso, Aline Machado de Castro,
Raquel Machado Castro, Carolina Maria M. C. Andrade,
and Nei Pereira Jr.* 1003
Predicted Effects of Mineral Neutralization and Bisulfate
Formation on Hydrogen Ion Concentration
for Dilute Sulfuric Acid Pretreatment
Todd A. Lloyd and Charles E. Wyman* 1013
Enhancement of Enzymatic Digestibility
of Recycled Newspaper by Addition of Surfactant
in Ammonia–Hydrogen Peroxide Pretreatment
Sung Bae Kim* and Jin Won Chun 1023
Study on Methane Fermentation and Production
of Vitamin B
12
from Alcohol Waste Slurry
Zhenya Zhang,* Taisheng Quan, Pomin Li, Yansheng Zhang,
Norio Sugiura, and Takaaki Maekawa 1033

Comparison of Two Posthydrolysis Processes
of Brewery’s Spent Grain Autohydrolysis Liquor
to Produce a Pentose-Containing Culture Medium
Luís C. Duate, Florbela Carvalheiro, Sónia Lopes,
Susana Marques, Juan Carlos Parajó,
and Francisco M. Gírio* 1041
Optimization of Brewery’s Spent Grain Dilute-Acid Hydrolysis
for the Production of Pentose-Rich Culture Media
Florbela Carvalheiro, Luís C. Duarte, Raquel Medeiros,
and Francisco M. Gírio* 1059
Comparison of Microbial Inhibition and Enzymatic
Hydrolysis Rates of Liquid and Solid Fractions
Produced from Pretreatment of Biomass
with Carbonic Acid and Liquid Hot Water
Damon M. Yourchisin and G. Peter Van Walsum* 1073
xviii Contents
xviii
Contents
Modeling of Carbonic Acid Pretreatment Process
Using ASPEN-Plus
®
Kemantha Jayawardhana and G. Peter Van Walsum* 1087
Enhanced Enzymatic Hydrolysis of Steam-Exploded
Douglas Fir Wood by Alkali-Oxygen Post-treatment
Xuejun Pan, Xiao Zhang, David J. Gregg, and John N. Saddler* 1103
Effects of Sugar Inhibition on Cellulases and β-Glucosidase
During Enzymatic Hydrolysis of Softwood Substrates
Zhizhuang Xiao, Xiao Zhang, David J. Gregg,
and John N. Saddler* 1115
Kinetics of Glucose Decomposition During Dilute-Acid

Hydrolysis of Lignocellulosic Biomass
Qian Xiang, Yong Y. Lee,* and Robert W. Torget 1127
Conversion of Distiller’s Grain into Fuel Alcohol
and a Higher-Value Animal Feed
by Dilute-Acid Pretreatment
Melvin P. Tucker,* Nicholas J. Nagle, Edward W. Jennings,
Kelly N. Ibsen, Andy Aden, Quang A. Nguyen,
Kyoung H. Kim, and Sally L. Noll 1139
Volume 116
S
ESSION 6B—PLANT BIOTECHNOLOGY AND FEEDSTOCK GENOMICS
Introduction to Session 6B
James S. McLaren and Steven R. Thomas 1163
Expression of UDP–Glucose Dehydrogenase
Reduces Cell-Wall Polysaccharide Concentration
and Increases Xylose Content in Alfalfa Stems
Deborah A. Samac,* Lynn Litterer, Glena Temple,
Hans-Joachim G. Jung, and David A. Somers 1167
Effects of Ammonia Fiber Explosion Treatment
on Activity of Endoglucanase from
Acidothermus cellulolyticus in Transgenic Plant
Farzaneh Teymouri, Hasan Alizadeh,
Lizbeth Laureano-Pérez,
Bruce Dale,* and Mariam Sticklen 1183
Effects of Inoculum Conditions on Growth of Hairy Roots
of Panax ginseng C. A. Meyer
Gwi-Taek Jeong, Don-Hee Park,* Hwa-Won Ryu,
Baik Hwang, and Je-Chang Woo 1193
Contents
xix

S
PECIAL SESSION A—MICROBIAL PENTOSE METABOLISM
Introduction to Special Session A
Bärbel Hahn-Hägerdal and Neville Pamment 1207
S
PECIAL SESSION B—INTERNATIONAL ENERGY AGENCY—BIOENERGY
CURRENT STATE OF FUEL ETHANOL COMMERCIALIZATION
Introduction to Special Session B
Warren E. Mabee, David J. Gregg, and John N. Saddler 1213
Author Index 1215
Subject Index 1219

Volume 113
SESSION 1A
Feedstock Supply, Logistics,
Processing, and Composition

Short Chapter Title
3
Applied Biochemistry and Biotechnology Vol. 113–116, 2004
3
Session 1A
Feedstock Supply, Logistics,
Processing, and Composition
JIM HETTENHAUS
1
AND DAVID MORRIS
2
1
cea Inc, Charlotte, NC; and

2
Institute for Local Self Reliance, Minneapolis, MN
For large, economic, and sustainable harvest of biomass feedstock,
major changes in cropping practice, collection, storage, and transportation
are required. The challenges faced in supplying 0.7–1 million dry short
tons (dt) for a single biorefinery are huge—five times larger than previous
attempts.
Ultimately, the farmer controls biomass sourcing for biorefineries. The
availability of large quantities of residues, stover, and straw is greatly depen-
dent on tillage practice. No tillage results in most of the residue available for
removal, especially when cover crops are employed for erosion control. By
contrast, no excess is available with conventional tillage. Since <20% of crop-
land is no till and >60% is conventional till, a major shift in cropping practice
is needed for sustainable removal of significant quantities.
Present collection costs are 1.5–2 times the delivered cost target—$35/
dt, including $20/acre or more net income for the farmer. Bulk collection is
likely needed, because baling adds cost, $15/dt, and no value. One-pass
harvest can lower the delivered feedstock cost to <$20/dt within a 15- to
20-mi radius. Prototypes for one-pass harvest of straw and stover are under
development, adapting existing equipment. Many variations are possible,
but until a better market definition is available, a new design is probably
limited to paper studies.
One-pass harvest also reduces the risk of corn stover harvest if storage
of wet harvested material is resolved. For the sugar platform, feedstock can
be wet, above 65%, or dry, below 20%. Some are looking at adapting wet,
bagasse-type storage, large 250,000-dt piles built via circulating liquor that
conveys the feedstock from wagons or trailers directly from the field after it
is washed and milled to a particle size that ensures good compaction and
preservation in storage. Less area is required; fire is eliminated when stored
above 65% moisture. The material processes easier because 80% of the

solubles is removed in storage. Water management and other issues remain.
Validation of this method is required for other crops such as stover and straw.
4
Introduction to Section 1A
Applied Biochemistry and Biotechnology Vol. 113–116, 2004
Because of the bulky nature, the cost of transportation is 20–40% of the
cost within a 50-mi radius. Transportation from the field to a storage site
following harvest needs to be kept short if truck requirements are to remain
manageable. Collection within a 50-mi radius for one site requires about
five times the trucks and wagons compared with a 15-mi radius. While bulk
density can be increased, the cost of densification generally offsets any
transport savings. Pipelines require huge initial investment. Short-line rail
delivery from three or more collection sites to supply the plant appears
most advantageous compared to trucking or pipelines.
In conclusion, potential processors want clean liquid, mostly ferment-
able sugars, delivered to the processing plant. Thus, in addition to the
above, using part of the storage time for value-added treatment offers more
potential. Although this processing is probably under different regulatory
requirements than storage, it may be segregated and controlled separately.
Including preprocessing with harvesting, collection, and storage provides
farmers ample opportunity to participate in the value chain, moving away
from simply supplying a commodity.