JPL Publication 88-8 DOE/CS-66001 -11

Distribution Categories

UC-61e&UC-61f

ECUT

ENERGY CONVERSION

AND UTILIZATION

TECHNOLOGIES PROGRAM

Biocatalysis Project

Annual Report

FY 1987

iKaSA-CB-182917) ICUT JEBEEGI COBVEBSICfl 888-24110

AKD UTILIZATION 3ICHNCLOGIIS) FiiCGEAH:.

EICCATflLlSIS;EECJICI fijmual Eepcrt, FY 1987

(JetiPropulsion Lah.) 36 p CSCL 10A Unclas

(G3/44 .01.46446

March 30,1988

Sponsored by

Energy Conversion and Utilization Technologies Division

Office of Energy Systems Research

U.S. Department of Energy

Through an agreement with

National Aeronautics and

Space Administration

by

Jet Propulsion Laboratory

California Institute of Technology

Pasadena, California
TECHNICAL REPORT STANDARD TITLE PAGE

1. Report No. JPL PUB 88-8

4. Title and Subtitle

ECUT — Energy Conversion

Technologies Progi

Blocatalysis Project Annual

2. Government Accession No.

and Utilization

•am

. Report Fiscal Year 1987

7. Author (j)

9. Performing Organization Name or

JET PROPULSION LAB(

California Institul

4800 Oak Grove Drii

Pasadena, Calif orn]

id Address

3RATORY

:e of Technology

/e

La 91109

12. Sponsoring Agency Nome and Address

NATIONAL AERONAUTICS AND SPACE ADMINISTRATION

Washington, D.C. 20546

3. Recipient's Catalog No.

5. Report Date

March 30, 1988

6. Performing Organization Code

8. Performing Organization Report No.

JPL PUB 88-8

10. Work Unit No.

11. Contract or Grant No.

NAS7-918

13. Type of Report and Period Covered

JPL Publication

14. Sponsoring Agency Code

15. Supplementary Notes

16. Abstract

The Annual Report presents the fiscal year (FY) 1987 research activities and

accomplishments for the Biocatalysis Project of the U.S. Department of Energy, Energy

Conversion and Utilization Technologies (ECUT) Division. The ECUT Biocatalysis Project

is managed by the Jet Propulsion Laboratory, California Institute of Technology.

The Biocatalysis Project's technical activities were organized, into three work

elements:

The Molecular Modeling and Applied Genetics work element includes modeling and

simulation studies to verify a dynamic model of the enzyme carboxypeptidase; plasmid

stabilization by chromosomal integration; growth and stability characteristics of

plasmid-containing cells; and determination of optional production parameters for

hyper-production of polyphenol oxidase.

The Bioprocess Engineering work element supports efforts in novel bioreactor

concepts that are likely to lead to substantially higher levels of. reactor productivity,

product yields, and lower separation energetics.

The Bioprocess Design and Assessment work element attempts to develop procedures

(via user-friendly computer software) for assessing the

given biocatalyst process.

economics and energetics of a

17. Key Words (Selected by Author(s)) 18. Distribution Statement

Bioengineering Unlimited/Unclassified

Conversion Techniques

Materials (General)

Chemical Engineering

19. Security Clossif. (of this report)

Unclassified

20. Security Cossif. (of this page) 21. No. of Pages 22. Price

Unclassified '

JPL 0184 R 9(83
JPL Publication 88-8 DOE/CS-66001 -11

Distribution Categories

UC-61e&UC-61f

ECUT

ENERGY CONVERSION

AND UTILIZATION

TECHNOLOGIES PROGRAM

Biocatalysis Project

Annual Report

FY 1987

March 30,1988

Sponsored by

Energy Conversion and Utilization Technologies Division

Office of Energy Systems Research

U.S. Department of Energy

Through an agreement with

National Aeronautics and

Space Administration

by

Jet Propulsion Laboratory

California Institute of Technology

Pasadena, California
The Biocatalysis Project is managed by the Jet Propulsion Laboratory,

California Institute of Technology, for the United States Department of Energy

through an agreement with the National Aeronautics and Space Admini

stration (NASA Task RE-152, Amendment 307; DOE Interagency Agreement

DE-AIO1-81CS66001).

The Biocatalysis Project focuses on resolving the major technical barriers that

impede the potential use of biologically-facilitated continuous chemical production processes.

This report was prepared as an account of work sponsored by an agency of the

United States Government. Neither the United States Government nor any

agency thereof, nor any of their employees, makes any warranty, express or implied, or assumes any legal liability or responsibility for the accuracy, completeness, or usefulness of any information, apparatus, product, or process

disclosed, or represents that its use would not infringe privately owned rights.

Reference herein to any specific commercial product, process, or service by trade

name, trademark, manufacturer, or otherwise, does not necessarily constitute or

imply its endorsement, recommendation, or favoring by the United States

Government or any agency thereof. The views and opinions of authors

expressed herein do not necessarily state or reflect those of the United States

Government or any agency thereof.
ABSTRACT

The Annual Report presents the fiscal year (FY) 1987 research activities

and accomplishments for the Biocatalysis Project of the U.S. Department of

Energy, Energy Conversion and Utilization Technologies (ECUT) Division. The

ECUT Biocatalysis Project is managed by the Jet Propulsion Laboratory,

California Institute of Technology.

The Biocatalysis Project's technical activities were organized into

three work elements:

The Molecular Modeling and Applied Genetics work element includes

modeling and simulation studies to verify a dynamic model of the enzyme

carboxypeptidase; plasmid stabilization by chromosomal integration; growth and

stability characteristics of plasmid-containing cells; and determination of

optional production parameters for hyper-production of polyphenol oxidase.

The Bioprocess Engineering work element supports efforts in novel

bioreactor concepts that are likely to lead to substantially higher levels of

reactor productivity, product yields, and lower separation energetics.

The Bioprocess Design and Assessment work element attempts to develop

procedures (via user-friendly computer software) for assessing the economics

and energetics of a given biocatalyst process.

111
FOREWORD

For further information about the ECUT Biocatalysis Project, contact:

Dr. James J. Eberhardt, Director Dr. Minoo N. Dastoor, Manager

Energy Conversion and Utilization ECUT Biocatalysis Project

Technologies (ECUT), CE-12 M/S 125-112

Forrestal Bldg. 5E-066 Jet Propulsion Laboratory

U.S. Department of Energy California Institute of Technology

1000 Independence Avenue, S.W. 4800 Oak Grove Drive

Washington, D.C. 20585 Pasadena, California 91109

(202) 586-5377 (FTS) 896-5377 (818) 354-7429 (FTS) 792-7429

IV
CONTENTS

I. EXECUTIVE SUMMARY 1-1

II. PROJECT DESCRIPTION 2-1

A. THE ECUT MISSION 2-1

B. HISTORICAL BACKGROUND 2-1

C. RELEVANCE 2-4

D. GOAL AND OBJECTIVE 2-5

E. PROJECT STRUCTURE AND ORGANIZATION 2-5

F. MANAGEMENT SUPPORT FUNCTION 2-6

G. TASK DESCRIPTIONS 2-7

III. FY87 TASK ACCOMPLISHMENTS 3-1

A. MOLECULAR MODELING AND APPLIED GENETICS 3-1

B. BIOPROCESS ENGINEERING 3-3

C. PROCESS DESIGN AND ANALYSIS 3-9

IV. BIBLIOGRAPHY 4-1

FIGURES

2-1. ECUT Role in Energy Conservation Research and Development . . 2-2

2-2. ECUT Program: Projects and Lead Center 2-3
SECTION I

EXECUTIVE SUMMARY

The Biocatalysis Project sponsored by the Energy Conversion and

Utilization Technologies (ECUT) Division of the U.S. Department of Energy

(DOE) is an applied research effort focused on providing the enabling

technology base for new bioprocess applications which have substantial energy

implications. The primary mission of DOE-ECUT is to provide, for the nation,

a bridge between basic research and industrial application for several

technologies (e.g., high temperature materials, tribiology, combustion and

biocatalysis) which have the potential for making positive energy

contributions. Furthermore, the ECUT mission hopes to strengthen the future

U.S. competitive position by conducting research directed towards exploiting

appropriate advances in basic research in which the U.S. has demonstrated

leadership.

As the national lead center, the Jet Propulsion Laboratory of the

California Institute of Technology serves as the technical manager for the

Biocatalysis Project. The primary objective of the Biocatalysis Project is to

resolve the critical technical constraints (e.g., poor productivity, high

separation energetics, presence of aqueous medium, lack of design tools) that

impede the utilization of biocatalysis for the production of chemicals and

materials. To achieve this objective, the Biocatalysis Project supports

generic applied research, as well as development of predictive theoretical

models (that are experimentally verifiable) which address the technological

barriers to the commercial utilization of biochemical catalysis.

The ECUT Biocatalysis Project consists of three major work elements,

each addressing a key technical component of the required enabling technology

base. The selection, as well as the relationship between each of these three

work elements, has been defined by their scale of action. The Molecular

Modeling and Applied Genetics work element focuses on defining optimal

microscale parameters for biocatalysis and pursuing practical applications of

basic molecular biology research findings. Computer graphic models have been

advanced which predict the dynamic behavior and intramolecular conformational

changes of enzymes (biocatalysts), including effects of temperature and

biocatalytic inhibitions. Advances in Applied Genetics have focused on the

issue of biological information (plasmid) stability. Novel high-speed

measurement methods employing flow cytometry have been developed, which now

allow researchers to assay the fraction of plasmid-containing cells in the

recombinant population in approximately three hours, more than one order of

magnitude faster than present day plating methods. Results of such

measurements have been used to develop a new theory of plasmid stability which

recognizes the limited capacity of plasmid-free cells to grow in selective

medium. The Bioprocess Engineering work element emphasizes defining the basic

engineering relationships between cellular scale events and macro-level

parameters. Laboratory tests using an advanced fluidized bed bioreactor

possessing high cell densities (5-15 x 10^-0 cells/ml) have demonstrated

exceptionally high bioreactor productivities: 50 to 100 g/l/h (for ethanol)

as compared to 5 to 10 g/l/h for current conventional processes. Similar

1-1
advances have been noted using a novel multi-membrane bioreactor design in

which the chronic and ubiquitous problem of product feedback inhibition has

been significantly relieved. Substantial progress, in rates and extent of

synthesis, has also been reported in the technology of enzyme reactions in

organic solvents (as opposed to conventional aqueous medium) for promoting

appropriate chemical transformations (e.g., regiospecific oxidation of

aromatics). The Process Design and Analysis work element focuses on

developing user-friendly computer programs which assess the energetics and

economics of biocatalyst chemical production processes. Modular computer

programs have been developed which provide capabilities for rapid assessment

of energy expenditures and costs for unit operations within biocatalyst

processes.

1-2
SECTION II

PROJECT DESCRIPTION

A. THE ECUT MISSION

The Energy Conversion and Utilization Technologies (ECUT) Program seeks

to:

(1) Monitor advances in basic scientific research and evaluate

emerging technologies for applicability to energy

conservation,

(2) Conduct exploratory development and establish feasibility of

innovative or revolutionary conservation concepts,

(3) Effect technology transfer to DOE end-use conservation programs

and/or to private industry, and

(4) Identify energy conservation research needs and feed this

information back to basic research.

In fulfilling these aims, ECUT acts as a transmission in transferring

the power of basic research to the wheels of industry (Figure 2-1). There are

many ways to achieve these aims and since its establishment ECUT has chosen to

support long-term, high-risk generic research and exploratory development for

generation of more energy-efficient materials and processes. The Biocatalysis

Project is one of five projects that make up the ECUT Program (Figure 2-2).

B. HISTORICAL BACKGROUND

The Biocatalysis Project was established in 1980 as the Chemical

Processes Project with the Jet Propulsion Laboratory as the lead center. At

that time the project had two work elements: Catalysis (chemical catalysis and

biocatalysis), and Separation. Within these work elements, several small

contracts were completed that evaluated the energy efficiencies and potential

applications of relatively new separation processes, such as supercritical

extraction, membrane separation, and chromatography. Research was also

started at the California Institute of Technology (Caltech) on chemical

catalytic behavior models and kinetics of expression in recombinant-DNA

microorganisms.

In 1982, the ECUT Program was reorganized and the Chemical Processes

Project was renamed as the Biocatalysis Project. As the name suggests, the

project emphasis was also changed, now stressing biocatalysis as the primary

research focus. The work elements were modified to include: Molecular

Modeling and Applied Genetics, Bioprocess Engineering, and Process Design and

Analysis. Each of these elements addresses a key technical component

necessary for the development of more advanced and efficient bioprocesses.

These three work elements detail a systematic progression of understanding

starting with the basic understanding of biocatalytic mechanisms at the

molecular level, to micro-level effects on process parameters in reactors

2-1
I

exoVQ•O§O0) 0) oiGO

(1)

09cohOV40)CC•HOOSHOOwCM

(1)

2-2
1

1pQo S * CO Z -c CC X Si UJ O -Q > UJ UJ Oz ^ o§ 8 - SS 1 CC N 3- WU SIocc<111COUJ cc_Jz

i

cc12

CD<h- coUJXcc0o

li-es<Q.•

1MP

-.£2

CO CO

w5

>:<

CATAl

BIOCAT

CO<ccUJoCO13mISCIENCES THERMAL

<_j <zzzoOcc<m3z§ « § 3 « Q) §£ -g 1- « LU M ? -o<0 5 2 _1 0 p! 4J, i

 2

Z

 PLI

UJO ^ CC 1-, 3 ° o £gCJu m<-J CM

pu7 ^

ZisO

 <

 Mu

si a i£ s: < _i co -" •m3DCOUJ_l <O<Q•

2-3
(including effects of integrated separation processes), and finally to the

development and assessment of new process concepts for technology transfer to

the industrial sector. With this information the production of large-volume,

low-cost, energy-intensive chemicals from renewable resources may be a

practical alternative in the future.

The selection as well as the relationships between each of the work

elements has been defined by their scale of action. Research activities are

conducted at various scales of action or specific size dimensions. Hence,

Molecular Modeling and Applied Genetics, includes research activities at a

molecular and cellular level, i.e., a scale of 1 urn and smaller. To

successfully exploit the findings at the molecular and cellular level towards

scale-up, Bioprocess Engineering includes research in the area of engineering

kinetics and control, as well as novel concepts in reactor design. The scale

in this work element is generally at the 1-meter dimension or at the reactor

level. Finally, Process Design and Analysis focuses on activities that

operate at the entire process level (0.4 hectares or more). A central tenet

subscribed to by the ECUT Biocatalysis Project is that in the acquisition of

an appropriate enabling technology, it is mandatory to integrate crucial

research findings at the various levels of activity and transfer the findings

to the private sector.

C. RELEVANCE

Three main conclusions can be stated regarding the competitive status

and future potential of the United States petrochemical industry: (1) Many

United States industries are losing their competitive edge as the global

economy is expanding; the U.S. petrochemical industry is an example of just

such an industry. (2) The United States must take advantage of its

competitive strengths if it is to compete effectively in the future. This

includes the application of its world-renowned capabilities in basic science.

Biotechnology is an example of scientific research whose application has

tremendous potential for commercial application. (3) Although biotechnology

is being applied towards the production of specialty chemicals by the chemical

industry, its application to the far larger commodity chemical market is

considered too long term and risky to attract research and development

dollars. This is sensible from the perspective of each individual firm, but

it is unclear whether the specialty chemical markets can sustain all of the

competitors who are restructuring their operations towards specialty chemical

production. A key government role in the future competitiveness of the

industry may be the support of generic research which can be applied to the

development of economical commercial processes.

The application of biocatalytic processes to the production of largescale chemicals would capitalize on the three major areas in which the United

States currently exerts leadership and whose successful exploitation would

contribute to its global competitive position in the future. First, the

United States is considered a world leader in basic research advances; our

lead in molecular biology and biochemistry is unquestioned. Commercial

utilization of that lead could increase the competitive advantage of our

domestic chemical industries. Second, we can exert our leadership further by

2-4
using the tools of macromolecular modeling and energy optimization to design

entirely novel biocatalysts and to test such catalysts by design concepts

through computer simulation. Such a design strategy would be useless without

a means for inexpensive synthesis; however, the newly developed techniques of

site-directed mutagenesis provide the methodology to synthesize and test

experimentally these design principles. The use of this design strategy would

enhance our productivity in the future by significantly and intelligently

reducing the time spent on experimental and empirical procedures in the

laboratory. Third, the United States is one of the most efficient and

productive producers of agricultural products in the world. Because biomass

is often the feedstock for the biocatalyst production of organic chemicals, we

have a clear competitive advantage in biomass production within our domestic

agricultural sector.

D. GOAL AND OBJECTIVE

(1) Goal. The goal of the ECUT/Biocatalysis Project is to exploit the

United States' competitive advantage in biotechnology by

facilitating the production of chemicals efficiently via

biocatalytic processes. The Biocatalysis Project supports

high-risk applied generic research and development aimed at

advancing an enabling technology base that will allow the rational

development and scale-up of large biocatalyst chemical production

processes. The Biocatalysis Project does this by conducting

exploratory development on, and by establishing the feasibility

of, novel, innovative, or revolutionary basic research advances in

biotechnology.

(2) Objective. To meet this goal, the Biocatalysis Project has

focused on developing predictive models and supporting novel

bioprocessing concepts which can be utilized by commercial

producers for large-scale chemical production. The strategy of

the Biocatalysis Project is to reduce production costs by

increasing product yields, by increasing reactor productivity, and

by decreasing energy requirements for production. These problems

are generic in a very large number of biocatalyst processes.

E. PROJECT STRUCTURE AND ORGANIZATION

The activities of the Biocatalysis Project have been incorporated as

three major work elements and a Management Support function. The three major

work elements and their supporting tasks are:

(1) The Molecular Modeling and Applied Genetics work element focuses

on defining optimal microscale parameters for biocatalysts and

developing practical applications of basic molecular biology

research findings. The primary roles of this work element are to

provide a database for defining kinetic models of biocatalyst

reactivity, and to develop genetically engineered solutions to the

generic technical barriers that preclude widespread applications

of biocatalysis.

2-5
(a) Biocatalysis by Design.

(b) Chromosomal Amplification of Foreign DNA.

(c) Kinetics of Recombinant Cells.

(d) Hyperproduction and Secretion of Polyphenol Oxidase.

(2) The Bioprocess Engineering work element emphasizes defining the

basic engineering relationships between molecular scale events and

macro-level parameters required for designing scaled-up

biocatalyst chemical production processes. Additionally, this

work element has a responsibility for establishing the technical

feasibility of critical bioprocess monitoring and control

subsystems.

(a) Metabolic Engineering.

(b) Immobilized Cell System for Continuous Efficient Biocatalyst

Processes.

(c) Enzyme Catalysis in Nonaqueous Solutions.

(d) Applications of Molecular Hydrogen in Chemical Fermentations.

(e) Multimembrane Bioreactor for Chemical Production.

(f) Multiphase Fluidized Bed Bioreactor.

(g) Biocatalyst Hydroxylation in Organic Solvents,

(h) Separation by Reversible Chemical Association.

(i) Bioseparation of Phosphate.

\

(j) Protein Engine~el:ihg for Nonaqueous Solvents.' ~ ~ '~

(3) The Process Design and Analysis work element provides overall

assessments, via energetic and economic analysis of biocatalyst

chemical production processes, and initiation of technology

transfer for advanced bioprocesses.

(a) Bioprocess Synthesis, Integration, and Analysis.

(b) Assessment: Biotechnology and Chemical Production.

F. MANAGEMENT SUPPORT FUNCTION

The Management Support function has three areas of responsibility: Task

Management and Planning; The Guidance and Evaluation Panel; and Industry

Technology Transfer.

2-6
(1) The Task Management and Planning area administers and coordinates

the task elements of the Biocatalysis Project. These

responsibilities include: developing statements of work and

evaluation criteria; defining the sequence of accomplishments that

will achieve the outlined objectives; monitoring and evaluating

in-house and contract research progress; preparing and

implementing the Annual Operating Plan (AOP); technical multi-year

plans and budgets; assigning personnel; identifying new areas of

research and issues to be addressed; and timely publishing of

research results and developments and their dissemination to DOE

headquarters, industry, and other researchers.

(2) The Guidance and Evaluation Panel includes industrial and academic

representatives who are leading authorities in the science and

technology of biocatalysis. The panel is responsible for

reviewing ongoing activities and future project plans, as well as

evaluating the Biocatalysis Project's performance.

(3) The Industry Technology Transfer area's task is to ensure strong,

interactive relationships between the Biocatalysis Project and

industry that will assist the transfer of the enabling technology

required by the private sector for the production of chemicals via

biocatalysis.

G. TASK DESCRIPTIONS

(1) Molecular Modeling and Applied Genetics

The Molecular Modeling and Applied Genetics work element is

comprised of four research tasks: Biocatalysis by Design,

Chromosomal Amplification of Foreign DNA, Kinetics of Recombinant

Cells, and Hyperproduction and Secretion of Polyphenol Oxidase.

The focus is on defining optimal microscale parameters for

biocatalysts and developing practical applications of basic

molecular biology research findings.

(a) Biocatalysis by Design (W.A. Goddard, III, Caltech). A

critical problem in the design of new. biological systems

(biopolymers, biocatalyst) is the prediction of the

structure and properties of the new system. Thus, given a

specific primary sequence of amino acids, prediction of the

secondary and tertiary structures (folding) of the

functional protein complex is desirable. The problem is

that the conformation is determined by weak forces (van der

Waals, hydrogen bonding, electrostatic), with the result

that an enormous number of conformations give similar

energies. A further complication is that the time scale of

a system refolding from one conformation to another is slow

(maybe seconds). Thus, in a molecular dynamics simulation,

the system may oscillate about one conformational minimum

for 10" to 10" time steps before it begins to unfold

and refold to a better conformation. As a result, it is not

2-7
yet possible to predict the conformation (tertiary

structure) of even small proteins (say, 50 amino acids or

500 atoms). However, to design new biochemical systems, it

is essential that we be able to predict the structure so

that the concomitant properties can be properly predicted.

Because of difficulties in predicting structure, the focus

of this task was on systems where the conformation was

known. The various amino acids were then modified and the

molecular dynamics techniques of annealing and quenching

were used to equilibrate the system. However, in designing

new biosystems, it is critical to develop the ability to

predict the globally minimum conformations so that more

drastic changes can be utilized in designing new systems.

Three overall approaches are being considered, all of which

combine random fluctuations (to allow sampling of new

regions of conformational space) with molecular dynamics

(allowing the forces to guide optimization of local

structure): unconstrained optimization using Monte Carlo

simulated annealing and simulated growth techniques combined

with molecular dynamics; tube-constrained conformation

searches where the centerline of the electron density from

low-resolution X-ray studies is used as a conformation

constraint for molecular dynamics; and pairwise-constrained

conformation searches where pairwise distances from

two-dimensional NMR are used.

(b) Chromosomal Amplification of Foreign DNA (G. Bertani, JPL).

Chromosomal amplification (and productivity of recombinant

microorganisms, discussed below) represent new advances in

the area of biological information stability. Information

is a dominant scientific issue in the case of genetically

engineered organisms. Since the "new" information is

exogenously added to the biological system (microorganism),

it is a perturbation from the natural state and, hence, the

stability characteristics of the engineered information with

respect to time are central for success. The objective of

this research is to determine the possibility of solving the

plasmid stability problem by inserting the recombinant DNA

plasmid directly into the bacterial chromosome. Because the

plasmid carrying the desired genetic trait would then be

part of the chromosome, it could not be easily lost as the

bacterium multiplies.

(c) Kinetics of Recombinant Cells (J.E. Bailey, Caltech). This

research employs experimental methods in concert with

mathematical models at the molecular and population level

for increasing the productivity of bioreactors using

genetically engineered recombinant microorganisms. There

are two main research areas that are necessary for

successful development of this method: kinetics of DNA

replication, which determines the rates of growth and

2-8
stability of desired recombinant cells; and control and

monitoring of plasmids, which are the DNA elements that

contain the genetic instructions needed to make desired

products more efficiently and selectively.

Quantitative descriptions (kinetic expressions) of the

growth and stability (plasmid retention) of recombinant

cells are needed to optimize the combination of natural

cellular products and components introduced to maximize the

efficiency of the overall system. Although cells can be

genetically engineered to increase efficiency, specificity,

and product yield, the presence of recombinant DNA can have

deleterious effects on the activity of cells with the

desired characteristics.

(d) Hyperproduction and Secretion of Polyphenol Oxidase (W.V.

Dashek and A.L. Williams, Atlanta). The objectives of this

research are to: purify intracellular and extracellular

polyphenol oxidases from Coriolus versicolor maintained in

liquid culture; and determine if intracellular polyphenol

oxidase is either synthesized de novo or activated. The

approach is to: establish the time courses for the

appearance of intracellular and extracellular polyphenol

oxidase and subsequently quantify oxidase specific activity;

and determine optimum growth conditions of C. versicolor for

maximum production of polyphenol oxidase, including

determination of cofactor requirements.

(2) Bioprocess Engineering

This work element consists of ten tasks: Metabolic Engineering,

Immobilized Cell System for Continuous Efficient Biocatalyst

Processes, Enzyme Catalysis in Nonaqueous Solutions, Applications

of Molecular Hydrogen in Chemical Fermentations, Multimembrane

Bioreactor for Chemical Production, Multiphase Fluidized Bed

Bioreactor, Biocatalyst Hydroxylation in Organic Solvents,

Separation by Reversible Chemical Association, Bioseparation of

Phosphate, and Protein Engineering for Nonaqueous Solvents. They

are primarily concerned with the definition of basic engineering

relationships between molecular and microscale events and

macro-level parameters required to design and scale-up

bioprocesses.

(a) Metabolic Engineering (J.E. Bailey, Caltech). The objective

is to formulate different types of mathematical structures

for analysis and synthesis of directed metabolic changes

accomplished by recombinant DNA genetic engineering

methods. One approach is the definition of a highly

structured, single-cell model of E. coli which includes many

of the mechanistic details of nutrient assimilation, cell

growth, and protein synthesis.

2-9
(b) Immobilized Cell System for Continuous Efficient Biocatalyst

Processing (C.E. Scott, ORNL). The task objectives are to

enhance productivity and operability of a fluidized bed

reactor system containing immobilized microorganisms, and to

investigate bioreactor dynamics, including the formulation

and investigation of kinetic properties of biocatalyst

particles. This will lead to a better understanding of

reactor behavior and control predictability. The reactor

consists of a column containing immobilized cells; the

substrate (glucose) and nutrients are passed into the bottom

of the column and the product ethanol (in water) is

continuously withdrawn from the top. The primary advantage

of this type of bioreactor is increased rates of

fermentation resulting from the high concentration of cells

in the reactor.

(c) Enzyme Catalysis in Nonaqueous Solutions (H.W. Blanch, U.C.

Berkeley). Techniques for increasing the solubility and

transport rate of organics at the site of enzyme action must

be found before enzymes will be widely used for production

of large-scale chemicals. The approach proposed here is

based on the use of a second organic liquid, in which the

substrate or product is soluble to a much greater extent

than in an aqueous system. A second aspect of the use of

enzymes in nonaqueous environments is the potential to run

many reactions "backwards." In cases where Water is a

reaction substrate, its high activity in aqueous solution

generally results in a shift of equilibrium to favor the

normal products of the reaction. In a nonaqueous system

such reactions may be forced in the reverse direction, as

water will be present at low concentrations. Examples of

this type of reaction are esterification and synthesis of

tryptophan, an important amino acid.

(d) Applications of Molecular Hydrogen in Chemical Fermentations

(P.O. Maxwell, Celgene). Glucose, derived from renewable

biomass, currently has two serious limitations as a

fermentation raw material: selectivity to form specific end

products is often low and the variety of end products that

can be made is limited. It has recently been found that if

fermentation is conducted in the presence of hydrogen, the

formation of oxidized coproducts is decreased so that

selectivity is increased. In addition, the types of primary

products formed may contain less oxygen. Two target

fermentations have been selected to demonstrate these

concepts: fermentation of glucose to succinic acid which

will demonstrate a desirable shift in product mix

composition, and fermentation of glucose to 1,3-propanediol

to demonstrate the ability to extend the range of end

products.

2-10
(e) Multimembrane Bioreactor for Chemical Production (M.L.

Shuler, Cornell). A four-layer, multimembrane reactor

concept is being developed: one for cell entrapment, one for

substrate flow, one for gas flow, and one for the flow of an

extractant which selectively removes the product. Task

efforts will include: proof-of-concept experiments;

development of a mathematical model relating the

micro-environment to reactor performance; development of a

process simulator from the mathematical model; and

experiments to validate the model and to monitor the

interrelationship of cell physiology to immobilization.

(f) Multiphase Fluidized Bed Bioreactor (B. Allen, Battelle

Columbus). This task is directed towards the development of

a novel, fluidized-bed bioreactor for the continuous

production of low-value, high-volume chemicals. The concept

incorporates the features required to minimize the effects

of end-product inhibition, a fundamental biochemical/

microbiological limitation that adversely affects the

economics of current bioprocess technologies. End-product

inhibition decreases biocatalyst activity which leads to low

productivity, high-volume reactors, and low-product

concentration, which requires capital/energy-intensive

aqueous separation systems.

The bioreactor concept in this task combines the use of an

immobilized biocatalyst with in-situ, nonaqueous sorbent

separation to minimize end-product inhibition. This is

achieved by the use of multi-phase fluidized-bed (MPFB)

bioreactor process. The biocatalyst is immobilized as

relatively large or dense particles (dense phase) which are

fluidized in a conventional manner using the fermentation

media. Either a solid or an insoluble, non-aqueous solvent

(entrained phase) is then circulated through the dense bed

to remove secreted products. After circulating through a

separation/desorption unit to recover products, the

entrained phase is regenerated.

(g) Biocatalyst Hydroxylation in Organic Solvents (A. Klibanov,

MIT). As discussed earlier, one of the major obstacles in

utilization of enzymes in organic chemistry is the necessity

to carry out enzymatic reactions in water, which is a poor

medium for most industrial chemical processes. For example,

the enzyme polyphenol oxidase has a great potential for

selective hydroxylation of aromatic compounds, an often

desirable but very difficult problem in organic chemistry.

However, polyphenol oxidase-catalyzed oxidations cannot be

conducted in water because: most phenolic substrates are

only slightly soluble in water; quinones (formed as the

product of the enzymatic oxidation) spontaneously polymerize

in water; and during that polymerization the enzyme is

chemically modified and consequently inactivated.

2-11
It has recently been discovered that several different

enzymes can function in nearly anhydrous organic solvents.

Under such conditions, polyphenol oxidase can quantitatively

convert phenols to the corresponding o-quinones which then

can be chemically reduced to catechols. The overall process

affords preparative regioselective hydroxylation of a

phenol. This effort includes determination of the

mechanism, implications and preparative use of the above

phenomenon.

(h) Separation by Reversible Chemical Association (C.J. King,

U.C. Berkeley). The objective of this task is to examine

and evaluate reversible chemical association, or

complexation, using organic agents as a method for

separating polar organic substances from dilute aqueous

solutions (e.g., bioprocess product or waste streams). The

goal is to obtain sufficient understanding of underlying

chemical, equilibrium, and transport behavior to enable

rational selection of separating agents, and methods of

implementation, as well as rational conceptual design and

economic evaluation. In the production of carboxylic acids

and other products, processes such as fermentation yield low

concentrations of products in water. The subsequent

separation and purification of the desired products is often

difficult and energy intensive.

(i) Bioseparation of Phosphate (Rogers, INEL). The phosphate

industry utilizes about 0.3 quads/yr for the separation of

phosphate from apatite ore. Therefore, this task is

directed toward development of bioprocessing for

solubilization and separation of phosphate from ore. The

specific objectives of the proposed research are to: define

a microbiological system which will extract phosphate from

its ore; develop a basic understanding of the biochemical

mechanisms involved; and, through the use of modern

biotechnology, develop a bioprocessing system for transfer

to the phosphate industry. If a more efficient recovery

process can be developed, it could also be applied to

phosphate mine waste because ore containing <26% phosphorus

is currently considered waste and is used as mine backfill.

The approach will be to gain a thorough understanding of the

biochemical interactions which cause the microbial release

of phosphate from its ore. This will be accomplished by

screening microorganisms obtained from areas of high

phosphate content (i.e., phosphate mines, process waste

streams, fertilized agricultural lands, etc.) for their

ability to solubilize phosphate. Those organisms which are

positive for the desired trait will be selected for further

biochemical studies. When a better understanding of the

mechanism of solubilization has been established, the work

will be focused on development of methods to enhance

production. Enhancement could be through physical/chemical

stimulation, genetic manipulations, or both.

2-12
(j) Protein Engineering for Nonaqueous Solvents (F.H. Arnold,

Caltech). The industrial applications of biocatalysts have

been severely limited by constraints on the solvent

environment of proteins, which normally require an aqueous

medium for effective operation. With the advent of

convenient methods for altering the amino acid composition

and for synthesis of entirely new proteins, it is worthwhile

to consider engineering proteins that would be effective in

nonaqueous solvents. However, the success of a rational

design procedure for constructing proteins to use in organic

solvents depends on understanding relationships among

various factors: amino acid sequence, secondary and tertiary

protein structure, and activity and stability in nonaqueous

solvents. The goal of this research is to begin to define

these relationships, by implementing an integral and

iterative protein engineering approach based on the model

hydrophobic protein crambin.

(3) Process Design and Analysis

The Process Design and Analysis work element includes development

of design and process technology for scaling up bioprocesses to

the pilot-plant level and overall assessments (i.e., systems

analyses) of biocatalyst chemical production processes. This work

element is comprised of two research tasks: Bioprocess Synthesis,

Integration, and Analysis; and Assessment: Biotechnology and

Chemical Production.

(a) Bioprocess Synthesis, Integration, and Analysis (J.D.

Ingham, JPL). The purpose of this task is to assemble a

series of candidate bioprocesses and systematically conduct

relevant energy-economic analyses and comparisons of their

projected potential for commercial development within the

next decade. Bioprocesses to be assessed will include

projected research advances (e.g., genetically engineered

microorganisms, bioreactor modeling and verification,

membrane development, and biocatalyst immobilization) for

energy-economic comparative assessments. The four

parameters that determine bioprocess economics and total

energy requirements are cell density, productivity, product

recovery energy and yield.

(b) Assessment; Biotechnology and Chemical Production (Chem

Systems Inc.). The objective of this work is to carry out

techno-economic assessments of specific bioprocesses where

most of the new technology has already been developed,

primarily in support of technology transfer activities.

2-13
SECTION III

FY87 TASK ACCOMPLISHMENTS

A. MOLECULAR MODELING AND APPLIED GENETICS

(1) Kinetics of Recombinant Cells (J. Bailey, Caltech). This research

focuses on control and monitoring of plasmids and kinetics of DNA

expression. Novel high-speed measurement methods employing flow

cytometry are used to characterize the cell population in a

reactor in terms of cell size and cellular plasmid concentration.

These experimental methods are used in concert with mathematical

models at the molecular and population level to improve

understanding of plasmid stability and increasing productivity of

bioreactors using genetically engineered recombinant

microorganisms.

The single-cell measurement method based upon labelling now

permits detection of single-plasmid copies in yeast. This is

accomplished through the use of flow cytometry in which light

scattering and fluorescence measurements can be made on a single

cell in a flowing cell suspension. Using this experimental

method, the fraction of plasmid-containing cells in the

recombinant population can be determined in approximately three

hours.

Results of these measurements have been used to develop a plasmid

stability model for recombinant populations propagating unstable

multicopy plasmids. The model incorporates plasmid replication

and partition functions, as well as the effect of plasmid presence

on host growth rate. It has been found that the amount of a

cloned enzyme produced by a genetically engineered cell depends

upon the differences between the rate of enzyme or messenger RNA

(mRNA) synthesis and the rate of enzyme or mRNA degradation. The

rates of mRNA and protein turnover have been found to be

significantly influenced by changes in the number of plasmids per

cell, the longevity of the mRNA, the physiological condition of

the cell, and the frequency of plasmid segregation of the plasmid

between sister cells at cellular division. For example, as the

number of plasmids within the cell increases, the quantity of

DNA-directed mRNA increases, which in turn produces higher

quantities of gene product (protein). This increase in gene

product is not gratis: costs include the additional energy to

manufacture the gene, its mRNA, and its specific protein. This

additional energy cost places these cells at a competitive

disadvantage in the bioreactor when compared to non-plasmidcontaining cells, leading to product production failure. Because

plasmids are transferred between cells, there is also a chance for

their loss in division. Normally the genetic content of the

plasmid is adjusted such that the cell is dependent upon its

presence for its survival. It is then assumed that if the plasmid

is lost, the necessary material (enzyme) for survival is lost and

3-1
the cell will be lost; Yet it has now been shown that the

availability of the enzyme depends on the half life of the raRNA.

The longer the half life of the mRNA, the longer the cell can

continue to grow without a plasmid. The cell that lacks a plasmid

is not then penalized and can grow for some time without

contributing to the reactor performance. This same phenomenon of

continued growth without a plasmid can also occur if the quantity

of mRNA is high. Thus, the overall productivity of the reactor

depends on a number of variables. This model now allows workers

in the field to estimate the impact of plasmid instability on

bioreactor performance.

(2) Chromosomal Amplification of Foreign DNA (G. Bertani, JPL).

Another approach to obviate the intrinsic instability of

plasmids, and therefore of any foreign genetic material that is

built into them, is to insert a copy of the plasmid, with whatever

genes are carried by it, into the chromosome of the bacterium.

This approach has now been successfully demonstrated. To increase

the number of copies of the genes thus introduced on the

chromosome, and make the structure equivalent to that of a typical

bacterium loaded with plasmids, an indirect method of selection

for chromosome amplification was applied, also successfully. Such

chromosomally amplified structures can be undone by normal genetic

recombination, however. During the year, attempts at reducing the

effects of normal genetic recombination have been made, by

introducing mutations that reduce recombination in the bacterium.

This work is still in progress. Another problem that is being

investigated is the unexpectedly low growth rate of those strains

where plasmid amplification has been produced within the

chromosome.

(3) Hyperproduction and Secretion of Polyphenol Oxidase (W.V. Dashek

and A.L. Williams, Atlanta University.). Two Ph.D students have

prepared task plans that define each of their activities that will

be implemented to accomplish the objectives of this research.

Experimental work has been started to determine kinetics of

production of extracellular oxidases from C. versicolor, establish

optimum growth conditions, and compare specific activities for the

enzyme utilizing different phenolic substrates.

Experiments have been completed to unequivocally establish time

courses for the formation of intra- and extracellular polyphenol

oxidases (PPO). Cycloheximide addition (to C. versicolor

intracellular total protein) inhibited total protein levels by

57.4% and enhanced PPO activity by a factor of 2.3, to suggest

that PPO is activated, rather than synthesized de novo. Protocols

for the purification of extracellular PPO are being developed.

Presentations of this and related work were made in St. Louis, MO

and at the University of Cambridge, England.

3-2
B. BIOPROCESS ENGINEERING

(1) Protein Engineering for Nonaqueous Solvents (F.H. Arnold,

Caltech). The goal of this research is to begin to define the

relationship between the amino acid sequence and protein

structure, based on the model hydrophobic protein crambin. The

unusual stability and solubility of crambin in a wide range of

nonaqueous solvents will be investigated. The structures,

stabilities, and solubilities of crambin mutants will be measured

and correlated with specific alterations in the protein amino acid

sequence. This research includes studies of the properties of

crambins, design of new mutant crambins by computer/molecular

graphics, production of new mutants by site-directed mutagenesis,

and detailed NMR studies of crambin stability and response to

nonaqueous solvents. The results of this work will be used to

formulate a set of criteria and a method for rational design of

proteins to be used in the presence of nonaqueous solvents.

The synthetic gene for crambin has been expressed in E. coli. The

gene was inserted into pKK223-3, containing the tac promoter.

Crambin is expressed intracellularly at reasonably high levels.

The protein produced in the bacteria is reduced and unfolded.

Attempts to refold the recombinant crambin in a mixture of

oxidized and reduced thiols have been successful. The resulting

protein has an NMR proton spectrum very similar to the native

material. The genes for five variants of crambin have been

produced, including one with an additional disulfide bridge

between residues 12 and 30. A preliminary set of design criteria

for engineering proteins to be stable in nonaqueous solvents has

been proposed and presented at the IX Enzyme Engineering

Conference in. Santa Barbara.

(2) Enzyme Catalysis in Nonaqueous Solutions (H.W. Blanch, U.C.

Berkeley). This research pursues conversion of cholesterol to

cholestenone by using an enzyme (with and without enzyme-water

encapsulation) dispersed in an organic solvent. Kinetics of

enzymatic conversion in a two-phase system (toluene-water, without

encapsulant) have been determined as a basis for comparison with

the encapsulated system and determination of the effects of

surfactant concentration and stirring rate. Oxidase activity at

25°C decreased after one day, probably because of denaturation

at the solvent interface, followed by no further decrease before

termination after 5 days. Although the surfactant (Span 85, >0.5

vol%, used to disperse the solvent in the aqueous phase) increased

the initial rate by a factor of about five, the effect of

surfactant on interfacial enzymatic activity is not well

understood. Therefore, the effect of a biological surfactant,

phosphatidylcholine (PC), on the rate was determined, and the

relative effects of the two surfactants were rationalized on the

basis of differences in their chemical structures. A model was

developed to account for various effects on rates of reaction in a

two-phase system, and oxidase activity assay and control of pH in

the two-phase system were investigated.

3-3
A method has been developed to avoid capsule membrane swelling

when the capsules are transferred from the encapsulant formation

solvent (cyclohexane) to the reaction solvent (toluene or

benzene), and to control pH within the microcapsules, by using

cyclohexane-toluene (1:1) as a common solvent: the mixed solvent

does not cause swelling and allows a higher acid concentration in

the solvent. In the oxidase-catalyzed conversion of cholesterol

to cholestenone within microcapsules, control of pH is required to

maximize enzymatic activity. To provide for transfer of acid

across the membrane and into the capsules, they are exposed to HC1

with Aliquat 336 in the organic phase. A non-invasive technique

has been developed to monitor acidity within the capsules by using

NMR (31p) spectrometry to determine encapsulated mono- and

dihydrogen phosphate. The relative amounts of each phosphate type

are then used to calculate the pH inside the capsules. The pH for

maximum oxidase activity was determined to be from 6.0 to 6.5.

Additional research concerns tryptophan synthesis and extractive

catalysis. Although it was originally planned to study urea

synthesis, attention has been redirected to tryptophan formation

as a model to minimize experimental difficulties (e.g., definition

of an appropriate solvent system for urea, and development of a

fundamental understanding of extractive catalysis and the effect

of product water on reaction equilibria). In this conversion,

ammonium pyruvate or serine plus indole react to form tryptophan

and water in the presence of tryptophanase. The normal reaction

is hydrolysis, because of the high effective concentration of

water in most physiological environments. A liquid membrane

(cyclohexane) or micellar solution is used to isolate the enzyme

in an aqueous environment at pH 9 from the aqueous outer phase at

pH 7, into which the product is extracted, as a result of the pH

gradient across the liquid organic membrane. (Micelles consist of

microscopic drops of water within an organic phase, which is

dispersed as an emulsion in water.) Because of limited ammonia

transport across the organic phase, it was found that serine,

rather than ammonium pyruvate, must be used in a micellar '

environment.

A model has been developed and experimentally verified to describe

mass transfer kinetics for substrate and products of this system.

Another significant accomplishment is a detailed description and

clarification of the effects of solvents on the equilibria of

enzymatic reactions in two-phase systems, which will be extremely

useful in the definition of future biocatalyst conversion

processes based on extractive catalysis (i.e., catalyzed reactions

in two-phase systems). In this work, equilibrium equations have

been derived in terms of partition coefficients and activities of

the reactants; these equations can be used to predict product

yields in two-phase systems. Further work on tryptophan synthesis

includes initiation of studies using EPR spectrometry and spin

labels to investigate water and enzyme interactions (e.g.,

conformational mobility of enzymes within micelles).

3-4
(3) Biocatalyst Hydroxylation in Organic Solvents (A.M., Klibanov,

MIT). The purpose of this research is to develop new methods for

enzymatic oxidative. catalysis in organic .solvents, e.g., for

selective oxidation of aromatic compounds to produce quinones and

hydroxylated aromatic chemicals, for peroxidase-catalyzed

depolymerization of lignin, and for potential polymerization of

aromatic compounds.

Conditions for maximum enzymatic depolymerization of polyconiferyl

alcohol as a model for lignin have been determined. These include

utilization of a solvent consisting of 95% dioxane in water at a

pH of 5.0 with 0.5 mg/ml of the enzyme (horseradish peroxidase)

and 300 ug/ml of polyconiferyl alcohol. Alternative solvents were

also investigated. While peroxidase-catalyzed depolymerization of

synthetic lignin did not take place in aqueous solutions,

significant depolymerization was accomplished in organic solvents

using either horseradish or lactoperoxidases. A mechanistic

evaluation of the ability of horseradish peroxidase to catalyze

the cleavage of specific lignin interunit linkages using low

molecular weight model compounds indicated a preference for

benzylic ether cleavage over aryl-alkyl cleavage if both were

present. Molecular oxygen was shown to stimulate

depolymerization, and this was interpreted to be the result of

formation of free radical lignin species produced by the

peroxidase-initiated depolymerization reactions. The ability of

horseradish peroxidase to depolymerize lignin in organic solvents

may enable useful low molecular weight chemicals to be produced

economically from lignin or lignin waste products.

The depolymerization of lignin has been extended to show that

extracted lignins from milled wood or kraft pine can be

successfully depolymerized using the same methods as for

polyconiferyl alcohol. Also, milled wheat straw has been

delignified with a modified, dioxane-soluble peroxidase. No

depolymerization took place in aqueous solutions, to show the

advantage of enzymatic reaction in organic solvents. Studies have

also been initiated on horseradish peroxidase-catalyzed reactions

to polymerize phenols in organic solvents as an alternative to

phenol-formaldehyde resins. Two inventions developed in this work

have been licensed for commercial applications: Enzymatic

Reactions in Liquid and Solid Paraffins: Application for

Temperature Abuse Sensors; and Method and Apparatus for Detecting

Compounds Utilizing Enzyme-Catalyzed Reactions in the Gaseous

Phase.

Separation by Reversible Chemical Association (C.J. King, LBL,

U.C. Berkeley). In the production of carboxylic acids, processes

such as fermentation yield low concentrations of carboxylic acids

in an aqueous multicomponent solution. The subsequent separation,

purification, and concentration of the carboxylic acids is

difficult and energy intensive.

3-5
A novel processing approach for recovery of low-volatility

carboxylic acids has been developed. This method involves use of

two diluents that are substantially more volatile than the amine

and the acid. One (e.g., chloroform) is an "active" diluent,

which solvates the complex and greatly increases the equilibrium

distribution coefficient. The other (e.g., n-heptane) is an

"inert" diluent, which does not interact with the complex and

gives lower distribution coefficients. The active diluent is

present during extraction of the acid from water. It is then

removed by distillation and replaced by the inert diluent, which

is present during back-extraction of the acid into water. The

inert diluent is then removed by distillation and replaced with

the active diluent for reuse of the solvent mixture. Equilibrium

measurements for extraction of succinic acid with amine present in

a diluent containing different proportions of chloroform and

heptane show that this approach is promising. Extraction of

lactic acid by Alamine 336 in chloroform and m-cresol diluents

shows very high distribution coefficients, which indicates that

this system can be applied to energy-efficient recovery of lactic

acid.

Fixed-bed adsorption runs have been made with Porapak Q (Waters

Assoc.) to determine the effect of particle size on adsorption.

Succinic acid, acetic acid, and 1,3-butanediol have been adsorbed

from aqueous solutions, using the bed in both wetted and

non-wetted conditions. In the latter case, separation follows the

order of volatilities. This enables a separation among solutes

that is not possible in the wetted state. The shapes of

breakthrough curves for the non-wetted state display a number of

transfer units about a factor of 10 less than expected.

Preliminary indications are that this is the result of

channelling, caused by preferential wetting of the wall of the

glass column. The possibility of using a polycarbonate column is

being investigated. Sorption properties of a large number of

basic resins have also been examined experimentally.

Preliminary infrared spectroscopic measurements have been made for

succinic acid dissolved in dry amine with chloroform diluent to

try to confirm the presence of specific complexes. Equilibrium

data have been obtained for extraction of ethanol by various

phenolic compounds in different diluents to attempt to identify

structural factors that lead to favorable equilibria. Many

phenolics are not amenable as alcohol extractants because they are

solids at practical temperatures that cannot be dissolved in

diluents at high concentrations. Alkyl groups on the phenol

decrease solubility loss in water, but distribution coefficients

are unfavorably decreased. It was also determined that there is

no significant advantage in using pentachlorophenol or

dihydroxybenzenes. Sorption properties of various basic resins

(e.g., polyethyleneimine, epoxy polyamines, and polyvinylpyridine)

have been examined experimentally to .identify attractive

regenerable sorbents for carboxylic acids.

3-6
(5) Metabolic Engineering (J. Bailey, Caltech). The objective of this

study is to formulate mathematical models [i.e., the Metabolic

Pathway Synthesis Program (MPS)] for analysis of directed

metabolic changes accomplished by recombinant DNA methods. The

MPS program can be used to predict, in a qualitative way, the

effect of adding or deleting enzymatic activities to or from the

cellular environment, to classify pathways with respect to

cellular objectives, arid to extract information about metabolic

regulation. MPS can be used for the identification of appropriate

genotypes or genetic modifications that will redirect metabolism

towards amplified production of desirable bioproducts. These

capabilities have been illustrated by synthesizing the classical

conversion of glucose 6-phosphate to pyruvate (Embden-MeyerhofParnas, pentose phosphate, and Entner-Doudoroff pathways), and

biosynthesizing L-alanine that does not necessitate the use of

alanine aminotransferase from pyruvate.

(6) Multiphase Fluidized Bed Bioreactor (B. Allen, Battelle Memorial

Institute, Columbus). A Multiphase Fluidized Bed Reactor (MPFB)

was built by Battelle Memorial Institute and its design and

operation was tested. This effort was successful at elucidating

many operational characteristics of fluidized beds used as

bioreactors for the bacterium Clostridium acetobutylicum.

The bacterium C. acetobutylicum was successfully encapsulated in

alginate beads with bead sizes ranging from 0.5 mm to 3 mm (and

larger). The inclusion of aluminum oxide or iron was also

successfully achieved to create beads with greater density, which

operate more effectively as the dense phase in the fluidized bed.

The bacterium was active and was viable for over 300 hours of

continuous operation with active production of butanol for over

240 hours. Operationally, it was found that the small diameter

beads were required for successful fluidization. The specific

organism was chosen because of its acute sensitivity to product

inhibition. Thus, the relief of such inhibition by the sorbent

extraction in this system should be better observed.

The operational mode for the extractive or sorbent phase was

examined. It was determined that a liquid sorbent which operates

in a co-current mode would be best to test the concept. Several

candidate sorbents were chosen based on published distribution

coefficients for butanol in a biphasic liquid system and tested

for toxicity towards cells and for actual distribution coefficient

values. Some sorbents were found that possessed appealing

distribution coefficient values. However, most were found to be

toxic to the cells and the final choice was made to use oleyl

alcohol. Although not the best choice for processing, it serves

well to test the feasibility of this concept.

An actual working prototype of the MPFB system was built and used

for cold flow model testing in order to obtain operational data.

It was 3" in diameter by 6' in height with a working volume of

about 7 to 8 liters. Bed expansion volumes, velocity

3-7
measurements, pumping and circulation requirements, and sorbent

recirculation parameters were measured. A novel measurement tool

was employed to measure bed and solvent velocities: a Fiber Optic

Laser Doppler Anemometer. This instrument permitted a

non-invasive measurement of fluid flows, although it had a

restricted range. A major result of this work was to show that

the fluidized bed operates as a reactor that is intermediate

between plug flow and backmixed operation. These data will be

helpful in designing larger units for scale-up.

The prototype of the MPFB was reconfigured to be sterilizable,

thus permitting operation with bacterial cells. This unit will

provide a unique facility for testing authentic microbial cultures

with the MPFB concept. This unit has undergone initial testing

and operation to eliminate "bugs" prior to the actual feasibility

tests. Tests to demonstrate sorbent extraction of butanol in the

fluidized bed were conducted. The results indicated successful

partitioning of butanol into the oleyl alcohol from beads

containing butanol (no microbial encapsulation in this case).

An independent technoeconomic analysis of the MPFB concept was

conducted by the engineering firm, Bio En-Gene-Er Assoc., Inc.

Their analysis was quite favorable and concluded that this

fluidized bed concept could increase the effective concentration

of the product ten-fold while maintaining actual concentrations

near the organism below the threshold of inhibition. As currently

configured, the MPFB would reduce the cost of butanol by about 34%

(to yield a 30% pretax return on investment). Further

optimization could reduce this cost even further. Guidelines for

further research were proposed, including a need for better

product stoichiometry from the microbe and an improvement in the

sorbent.

(7) Multimembrane Bioreactor for Chemical Production (M. Shuler,

Cornell). The Multimembrane Bioreactor with extraction was shown

- to operate and exhibit improved performance over-conventional

batch and immobilized fermenters. In the MMB system with

extraction, glucose concentration nearly approached zero, lowering

production costs. In addition, ethanol productivity was

increased by a factor of 5 to 7. Operational problems with this

system were encountered which were overcome by defining nutrient

requirements. One new concept utilizing the MMB system was

attempted by operating the system with pressure cycling in the gas

phase. It was anticipated that such operation would enhance mass

transfer and permit increased cell densities. Experiments were

conducted to test this hypothesis.

It was subsequently demonstrated that a five-fold improvement in

average fermentation rate for a 200 g/1 glucose feed for a

pressure cycle reactor could be achieved over the normal

diffusion-controlled system (8.7 g glucose consumed/hr versus

1.8 g glucose consumed/hr). The pressure cycle removes ethanol

more efficiently from the cell layer (2.8 g/l/hr versus 1.1

3-8
g/l/hr). Additional characteristics of this reactor system were

examined, such as the effect of high cell densities on operation

and operation at high temperatures (above 40°C).

The effect of high cell densities on the reactor system were

studied. The analysis evaluated growth rate as a function of cell

density. The apparent growth rate at 41 g/1 was roughly half of

that at 9 g/1 (0.16 versus 0.29/hr). The reduction appeared to be

due to cell contact blocking uptake sites at high cell

concentrations. The operation of the pressure cycle reactor at

high temperatures was also evaluated. Previous attempts to

operate such fermentations at higher temperatures in the diffusion

mode showed non-performance at 40° and 45°C. In the pressure

cycle mode, operation at 40°C was very effective (i.e., rapid

glucose consumption), but at 45°C cell viability was lost and

the fermentation became "stuck" (or inhibited). Work with the

pressure cycled MMB system is now being directed towards

continuous operation and/or increasing the size of the system by

operating two or more units in series.

Additional areas of research involved an analysis of the

separations and aeration requirements for this extractive system,

and modeling of the ethanol yeast fermentation system.

Preliminary experiments to evaluate separations were carried out.

In a simple two-step distillation with "spent" TBP and ca. 30 g/1

of ethanol and ca. 60 g/1 of water in the TBP, a top product with

80 wt% ethanol and virtually no TBP was recovered. If the spent

TBP was dehydrated with MgSO^ then the top product is nearly

pure ethanol. These results indicated that purification is

achievable. The effects of aeration on the specific yeast strain

employed were studied. This strain operated poorly under strictly

anaerobic conditions, but required a level of oxygen at 2 to 3% of

100% saturation.

An initial formulation of a mathematical model for yeast growth

and metabolism was completed. After considerable work and

improvement, the model is considered to be one of the most

complete and powerful simulators of yeast behavior available. It

can accurately predict transient behavior in batch culture, a

predictive behavior not found in models currently available. The

model will continue to be improved until the end of this effort to

better fit empirical data obtained in the laboratory.

C. PROCESS DESIGN AND ANALYSIS

(1) Bioprocess Synthesis, Integration, and Analysis (J. D. Ingham,

JPL). The purpose of this task is to derive or synthesize a

series of candidate bioprocesses and systematically conduct

relevant energy-economic analyses and comparisons for added-value

commodity chemicals to determine potential for process technology

transfer and commercial development. These bioprocesses will be

3-9
modified to include projected research advances (e.g., genetically

engineered microorganisms, bioreactor modeling and verification,

membrane development, and biocatalyst immobilization) for

energy-economic comparative assessments. The four parameters that

determine bioprocess economics and total energy requirements are

cell density, productivity, product recovery energy, and yield.

Extensive utilization of bioprocesses for the production of

commodity chemicals could significantly decrease U.S. dependence

on the supply of foreign and domestic non-renewable petroleum

resources, but because of relatively high feedstock costs and some

specific limitations of biocatalyst processes, most of them are

not yet competitive with current petrochemical routes with respect

to either process energy consumption or economics. Since these

limitations often result in either relatively low rates of

reaction and production, low product concentration, or both,

assessments of a series of processes for ethanol (as a model of a

high volume industrial chemical) have ,been completed to establish

a more quantitative basis for estimation of the relative effects

of production rates and product concentration in the bioreactor on

process energy and economics. The results of model bioprocess

simulations for ethanol show that energy consumption is sensitive

to product concentration, is essentially independent of rate of

production, and that the effect of production rate on economics is

at least an order of magnitude less than the effect of product

concentration. It is also suggested that concentration is likely

to be even more important for other potential industrial chemical

biocatalyst processes (e.g., for n-butanol or acetic acid) where

product recovery is more energy intensive than for ethanol. It

can be concluded that future research advances to improve the

energy, economics, and effective synthesis of new, competitive

biocatalyst processes for industrial chemicals should be directed

toward significantly increasing product concentration, rather than

by attempting to independently maximize only rates of production.

A computer model has been-formulated-where fundamental-kinetic

equations and intrinsic parameters (e.g., maximum specific rates,

saturation constants, and yield factors) result in fixed

relationships with respect to conversion, product yield, product

concentration, and productivity. Calculations show that there are

maximum limits for these parameters for specific bioprocesses: the

highest productivity normally will occur at a relatively low yield

and concentration. Therefore, investigations have been initiated

to establish reactor configurations and conditions that result in

minimum energy requirements and production costs. Preliminary

calculations for simulated processes of rate, yield, and

concentration combinations agree with previous experimental

observations. This bioreactor kinetic study will be completed and

used to attempt to, confirm that earlier assessments are realistic

and consistently applicable to potential advanced bioprocesses, to

ensure that assumed reactor conditions are reasonable, and that

projected improvements could be realized.

3-10
SECTION IV

BIBLIOGRAPHY

Allison, J. M., and Goddard, W. A., "Oxidative Dehydrogenation of Methanol to

Formaldehyde," J. Catal.. Vol. 92, pp. 127-135, 1985.

Allison, J. M., and Goddard, W. A., "Active Sites on Molybdate Surfaces,

Mechanistic Considerations for Selective Oxidation, and Ammoxidation of

Propene," ACS Symposium Series No. 279, pp. 23-36, 1985.

Bertani, L. E., and Bertani, G., "Chromosomal Integration and Excision

of a Multicopy Plasmid Joined to Phage Determinants for Site Specific

Recombination," in Plasmids in Bacteria, D. R. Helinski, ed., Plenum

Press, NY, p. 917, 1985.

Bertani, L. E., and Six, E. W., "The P2-like Phages and Their Parasite, P4,"

to appear in The Viruses, editors; Wagner, R. R., and FraenkelConrat, H., Plenum Publishing Corp, 1988.

Biocatalysis Research Activity Annual Report; FY 1983, Jet Propulsion

Laboratory, JPL Publication 84-27, DOE/CS-66001-4, April 15, 1984.

Biocatalysis Project Annual Report; FY 1984, Jet Propulsion Laboratory,

JPL Publication 85-31, DOE/CS-66001-8, April 15, 1985.

Biocatalysis Project Annual Report; FY 1985, Jet Propulsion Laboratory, JPL

Publication 86-32, DOE/CS-66001-9, July 30, 1986.

Biocatalysis Project Annual Report; FY 1986, Jet Propulsion Laboratory,

JPL Publication 87-11, DOE/CS-66001-10, April 30, 1987.

Celniker, S., Sweder, K., Srienc, F., Bailey, J. E., and Campbell, J. L.,

"Deletion Mutations Affecting the Yeast Autonomously Replicating

Sequence, ARSI," Mol. Cell. Biology, Vol. 4, p. 2455, 1985.

Chem Systems, Inc., "Technical and Economic Assessment of Processes for the

Production of Butanol and Acetone - Phase Two: Analysis of Research

Advances," JPL 9950-928, DOE/CS-66001-5, Prepared for Jet Propulsion

Laboratory by Chem Systems, Inc., Tarrytown, NY, August 1984.

Chem Systems Inc., "Techno-Economic Assessment of Microbial Ammonia

Production," prepared for Jet Propulsion Laboratory by Chem Systems

Inc., Tarrytown, NY, February 1986.

Chem Systems Inc., "Assessment of ORNL Immobilized Cell Ethanol Fermentation

Process," prepared for Jet Propulsion Laboratory by Chem Systems Inc.,

Tarrytown, NY, March 1986.

Chem Systems Inc., "Implications of ORNL Immobilization Technology on Citric

Acid Production," prepared for Jet Propulsion Laboratory by Chem Systems

Inc., Tarrytown, NY, June 1986.

4-1
Cho, T., and Shuler, M. L., "Multi-Membrane Bioreactor for Extractive

Fermentation," Biotech. Prog., Vol. 2, pp. 53-60, 1986.

Davison, B. H., and Scott, C. D., "Ethanol Production on an Industrial

Feedstock by Zymomonas Mobilis in a Fluidized Bed Bioreactor," Eighth

Symposium on Biotechnology for Fuels and Chemicals, Gatlinburg, TE,

May 13-16, 1986.

Davison, B. H., and Scott, C. D., "Fermentation Kinetics of Zymomonas mobilis

under Mass Transfer Limitations," 192nd ACS National Meeting, Anaheim,

CA, September 7-12, 1986.

Eggers, D. K., Lira, D. J., and Blanch, H. W., "Enzymatic Production of

1-Tryptophan in Liquid Membrane Systems," submitted to Bioprocess Eng.

Eggers, D. K. , Blanch, H. W., and Prausnitz, J. M., "Extractive Catalysis:

Solvent Effects on Equilibria of Enzymatic Reactions in Two-Phase

Systems," submitted to Bioprocess Eng.

England, C., "Isothermal Separation Processes Update," JPL 9950-927,

DOE/CS-66001-6, prepared for Jet Propulsion Laboratory by Engineering

Research West, Santa Monica, CA, August 1984.

Goddard III, W. A., Low, J. J., Olafson, B. D., Redondo, A., Zeiri, Y.,

Steigerwald, M. L., Carter, E. A., Allison, J. N., and Chang, R., "The

Role of Oxygen and Other Chemisorbed Species on Surface Processes for

Metals and Semiconductors; Approaches to Dynamical Studies of Surface

Processes," Proceedings of the Symposium on the Chemistry and Physics of

Electrocatalysis, Vol. 84-12, The Electrochemical Society, Ind.,

Pennington, NJ, pp. 63-95, 1984.

Goddard III, W. A., "Theoretical Chemistry Comes Alive: Full Partner with

Experiment," Science. Vol. 227, pp. 917-923, 1985.

Hatcher, H. J., "Biocatalysis Program Bioseparations Task," Summary Report,

E. G. and G. Idaho, Inc., Idaho National Engineering Laboratory, Idaho

Falls, ID, February 1986.

Ingham, J. D., Biocatalyst Processes for Production of Commodity Chemicals;

Assessment of Future Research Advanced for N-Butanol Production, Jet

Propulsion Laboratory, JPL Publication 84-60, July 1, 1984.

Kertes, A. S., and King, C. J., "Extraction Chemistry of Fermentation Product

Carboxylic Acids," Biotechnology and Bioengineering, Vol. 28,

pp. 269-282, 1986.

King, C. J., chapter on Chemical Association in Handbook of Separation

Process Technology, R. W. Rousseau, ed., John Wiley and Sons, 1987.

King, C. J., et al., "Non-wetting Polymeric Adsorbents for Selective Uptake of

Carboxylic Acids and Glycols," presented at the Symposium, Fundamentals

of Adsorption, Meeting of the American Chemical Society, Denver,

Colorado, April 1987,.and Foundation Conference on Advances in

Separation Technology, West Germany, April 1987.

4-2
Klibanov, A. M., "Polyphenol Oxidase - Catalyzed Hydroxylation of Aromatic

Compounds in Organic Solvents," Quarterly Report No. 1, Massachusetts

Institute of Technology, Cambridge, MA, March 1986.

Kuhn, R. H., and Linden, J. C., "Effects of Temperature and Membrane Fatty

Acid Composition on Butanol Tolerance of Clostridium acetobutylicum,"

Eighth Symposium on Biotechnology for Fuels and Chemicals, Gatlinburg,

TE, May 13, 1986.

Lee, S. B., Seressiotis, A., and Bailey, J. E., "A Kinetic Model for Product

Formation in Unstable Recombinant Populations," Biotechnology and Bioengineering, Vol. 27, pp. 1699-1709, 1985.

Lee, S. B., and Bailey, J. E., "A Mathematical Model for v Plasmid Replication:

Analysis of Wild-Type Plasmid," Plasmid. Vol. 11, No. 151, 1984.

Lee, S. B., and Bailey, J. E., "A Mathematical Model for v Plamid Replication:

Analysis of Copy Number Mutants," Plasmid, Vol. 11, No. 151, 1984.

Lee, S. B., and Bailey, J. E., "Analysis of Growth Rate Effects on Productivity

of Recombinant Escherichia coli Populations Using Molecular Mechanism

Models," Biotechnol. Bioeng.. Vol. 26, No. 66, 1984.

Lee, S. B., and Bailey, J. E., "Genetically Structured Model for Lac PromoterOperator Function in the Chromosome and in Multicopy Plasmids: Lac

Promoter Function," Biotechnol. Bioeng., to be published.

Linden, J. C., and Kuhn, R. H., "Biochemistry of Alcohol Effects on

Clostridia," in Ethanol Toxicity at the Cellular Level, Van Uden, N.,

ed., CRC Press, Cleveland, OH, in press.

Miskowski, V. M., "The Electronic Spectra of u-Peroxodicobalt (III)

Complexes," Comments Inorg. Chem., in press.

Peretti, S. W., and Bailey, J. E., "A Mechanistically Detailed Model of

Cellular Metabolism for Glucose-Limited Growth of Escherichia coli

B/r-A," Biotechnol. Bioeng.. Vol. 28, p. 1672, 1986.

Peretti, S. W., and Bailey, J. E. , "Simulations of Host-Plasmid Interactions

in Escherichia coli; Copy Number, Promoter Strength, and Ribosome

Binding Site Strength Effects on Metabolic Activity and Plasmid Gene

Expression," Biotechnol. Bioeng., Vol. 29, p. 316, 1987.

Scott, C. D., "Techniques for Producing Mono-dispersed Biocatalyst Beads for

Use in Columnar Bioreactors," Proceedings of the Seventh Symposium on

Biotechnology for Fuels and Chemicals, Gatlinburg, TE, May 16, 1985.

Scott, C. D., "Innovative Techniques for Gel Immobilization of Microorganisms

and Enzymes," International Conference on Enzyme Engineering, Helsingor,

Denmark, September 1985, to be published in The Annals of the New York

Academy of Sciences.

4-3
Scott, C. D., and Thompson, J. E., "Mass Transfer Properties of Biocatalyst

Beads," Eighth Symposium on Biotechnology for Fuels and Chemicals, .

Gatlinburg, TE, May 13-16, 1986.

Seo, J. H., and Bailey, J. E., "Effects of Recombinant Plasmid Content on

Growth Properties and Cloned Gene Product Formation in Escherichia

coli," Biotechnology and Bioengineering, Vol. 27, pp. 1668-1674, 1985.

Seressiotis, A., and Bailey, J. E., "Optimal Gene Expression and Amplification

Strategies for Batch and Continuous Recombinant Cultures," Biotechnol.

Bioeng., Vol. 29, p. 392, 1987.

Seressiotis, A., and Bailey, J. E., "MPS: An Algorithm and Data Base for

Metabolic Pathway Synthesis," Biotechnol. Left.. Vol. 8, p. 837, 1986.

Srienc, F. , Campbell, J. L., and Bailey, J. E., Biotechnol. Letters, Vol 5,

p. 43, 1985. .

Srienc, F., Bailey, J. E., and Campbell, J. L., "Effect of ARSI Mutations on

Chromosome Stability in Saccharomyces Cerevisiae," Mol. Cell. Biology,

Vol. 5, No. 7, pp. 1676-1684, 1985.

Srienc, F., Campbell, J., and Bailey, J. E., "Flow Cytometry Characterization

of Plasmid Stability in Saccharomyces cerevisiae," Third European

Congress on Biotechnology, Verlag Chemie, Weinheim, Federal Republic of

Germany, Vol. Ill, pp. 111-187, 1984.

Srienc, F., Campbell, J. L., and Bailey, J. E., "Analysis of Unstable

Recombinant Saccharomyces cerevisiae Population Growth in Selective

Medium," 1984, submitted for publication.

"System for Biotechnology Assessment," Final Report, Phase I; August 1984 -

July 31, 1985, Washington University, St. Louis, MO, August 1985.

Tamada, T. A., Kertes, A. S., and King, C. J., "Solvent Extraction of Succinic

Acid from Aqueous Solutions," International Conference on Solvent •

Extraction, Munich, Germany, September 1986.

The ECUT Biocatalysis Project. Jet Propulsion Laboratory, ECUT Program

Bulletin, DOE, ECU-86/1, PB86-900401, Jan.-Feb., 1986.

The ECUT Biocatalysis Project, Jet Propulsion Laboratory, submitted for

Publication in the ECUT Program Bulletin, May 1987.

Warren, L. F., "Electrocatalysis Research," Final Report for the Period

August 25. 1982 through December 30, 1983, JPL 9950-926, DOE/CS-66001-7,

prepared for Jet Propulsion Laboratory by Rockwell International,

Science Center, Thousand Oaks, CA, August 1984.

Wilcox, R. E., Industry, University, and Research Institute Interest in the

U.S. Department of Energy ECUT Biocatalysis Research Activity, Jet

Propulsion Laboratory, JPL Publication 83-90, DOE/CS-66001-3,

November 1, 1983.

4-4