SERI/TR-231-2093

UC Category: 61a

QE84013015

Ethanol Production by

Enzymatic Hydrolysis

Parametric Analysis of a

Base-Case Process

Steven H. Isaacs

May 1984

Prepared under Task No. 1372.35

Solar Energy Research Institute

A Divisio n of Midwest Research Institute

1617 Cole Boulevard

Golden, Colorado 80401

Prepared for the

U.S. Department of Energy

Contract No . DE-ACQl2-83CH10093
Printed in the United States of America

Available from:

National Technical Information Service

U.S. Department of Commerce

5285- Port Royal Road

Springfield, VA 22161

Price:

Microfiche A01

Printed Copy AD5

NOTICE

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

Government. Neither the United States nor the United States Department of Energy,

nor any of their employees, nor any of their contractors, subcontractors, or their

employees, makes any warranty, express or implied, or assumes any leg'al 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.
S=~IIi*;1 TR-2093 ----------------------------------=:::..::..--=-.:.--=--=. -~ .

PREFACE

The Solar Fuels Research Division of the Solar Energy Research Institute is

responsible for managing a program of research and development for the U. S.

Department of Energy Office of Alcohol Fuels. One emphasis of the program is

the hydrolysis of lignocellulosic feedstock to sugars and the subsequent fermentation of these sugars to fuel ethanol. One of the more promising means to

accomplish this is with an enzymatic process. The specificity and relatively

mild conditions of the enzymatic reaction provide the potential for complete

utilization of cellulose and inexpensive materials of construction for process

equipment. This report presents an analysis of one enzymatic hydrolysis process in which the unit operations of enzyme production, hydrolysis of cellulose to glucose, and fermentation of the glucose to ethanol are performed

separately.

This report contains a process flow sheet and an economic analysis of a basecase design. Background information on the technology of enzyme hydrolysis

and on the bases for the base-case design assumptions is included. A

parametric analysis of the base-case design is provided to identify key

research issues and determine the ultimate potential of this process.

This study was carried out by Steven Isaacs of the Alcohol Fuels Program

Office with assistance and guidance from Larry Douglas, Bill Hoagland, John

Wright, and Karel Grohmann.

Steven H. Isaacs

~.\~ L. J .\DougIS

Approved for

SOLAR ENERGY RESEARCH INSTITUTE

Stanl

Solar

iii
S=~II.I ---------------------------------=.=-..::::....:.~ TR-2093

SUMMARY

Objective

To present a base-case flowsheet for an enzymatic hydrolysis process, to

perform a parametric sensitivity analysis to identify key research issues, and

to assess the potential of this technology. Background information concerning

the enzyme hydrolysis technology is included.

Discussion

The plant discussed in the report is a large-scale facility, producing

50 million gallons of ethanol per year. The plant design is based on the

process originally conceived by the U.S. National Army Command and consists of

these process steps: pretreatment; enzyme production; enzyme hydrolysis;

fermentation; and distillation. The base-case design parameters are based on

recent laboratory data from Lawrence Berkeley Laboratories and the University

of California at Berkeley. The selling price of ethanol is used to compare

variations in the base-case operating parameters, which include hydrolysis

efficiencies, capital costs, enzyme production efficiencies, and enzyme

recycle.

Conclusion

The economic evaluation of the base-case process design indicates a selling

price of $2.13 /gal. Major contributions to this cost include the aspen wood

feedstock cost (46.3i/gal), power costs (14.3i/gal), and capital-related

costs. The capital costs are dominated by the steam explosion and enzyme

production sections (35.4% and 42.6% of the total ansite purchased capital).

The most significant improvement to the base-case process economies is caused

by the inclusion of enzyme recycle. Sixty percent recycle via adsorption onto

freshly pretreated cellulosics decreases the selling price to $1.67/gal.

Ability to ferment xylose, a five-carbon sugar derived from the hydrolysis of

the hemicellulose functions of biomass, reduces the selling price to $1.70/gal

with no enzyme recycle and $1.38 with 60% enzyme recycle.

Other process parameters that have significant impact on ethanol selling price

include hydrolysis conversion, enzyme loading per gram of cellulose, and

reduction in the capital cost associated with the equipment for enzyme

production and steam explosion pretreatment. Combinations of improvements in

these parameters cause a reduction in the base-case selling price to

99.0i/gal.

Results of the parametric analyses indicate potential for this particular

enzymatic hydrolysis process to become competitive with the current market

price for ethanol and for ethanol produced from biomass via an acid hydrolysis

process. Uncertainties in the scale up of laboratory results, uncertainties

in actual industrial scale agitation, aeration and sterility requirements, and

lack of detail in the computer simulation model make the selling prices quoted

tv
S=~II.I ------------------------=:.=--=..:::~ TR-2093

tn this report only approximate. Additional research at both the laboratory

sca l,e and the larger scale is required for a more accurate design and cost

estimation.

v
TR-2093

TABLE OF CONTENTS

1.0 Introduction •••••••••••••••••••••••••••••••••••••••••••••••••••••••• 1

2.0 Objective and Scope ••••••••••••••••••••••••••••••••••••••••••••••••• 2

3.0 Enzymatic Hydrolysis•••••••••••••••••••••••••••••••••••••••••••••••• 3

3.1

3.2

3.3

3.4

3.5

Feedstock Characteristics••••••••••••••••••••••••••••••••••••••

Cellulase Enzymes ••••••••••••••••••••••••••••••••••••••••••••••

Limits on Cellulose Hydrolysis•••••••••••••••••••••••••••••••••

Pretreatment •••••••••••••••••••••••••••••••••••••••••••••••••••

Enzymatic Activity Measurement•••••••••••••••••••••••••••••••••

3

5

7

8

9

4.0 Enzymatic Hydrolysis Process Design ••••••••••••••••••••••••••••••••• 11

19

22

11

11

11

16

17

and Offsites••••••••••••••••••••••••••••••

Carbon Dioxide Recovery,

Enzyme Recovery •••••••••••••••••••••••••••••••••••••••••

Enzyme Hydrolysis •••••••••••••••••••••••••••••••••••••••

Enzyme Production •••••••••••••••••••••••••••••••••••••••

Fermentation,

Purification,

4.2.5

Overall Process Design •••••••••••••••••••••••••••••••••••••••••

Detailed Process Design ••••••••••••••••••••••••••••••••••••••••

4.2.1 Pretreatment••••••••••••••••••••••••••••••••••••••••••••

4.2.2

4.2.3

4.2.4

4.1

4.2

5.0 Simulation Model •••••••••••••••••••••••••••••••••••••••••••••••••••• 24

6.0 Process Simulation Studies•••••••••••••••••••••••••••••••••••••••••• 28

6.1

6.2

Base-Case Design •••••••••••••••••••••••••••••••••••••••••••••••

Parametric Analysis••••••••••••••••••••••••••••••••••••••••••••

6.2.1 Qualitative Analysis of Process Improvements••••••••••••

6.2.2 Variations in Single and Coupled Parameters•••••••••••••

6.2.3 Summary of Individual Parameter Variations••••••••••••••

6.2.4 Selling Price versus Substrate Cost •••••••••••••••••••••

28

29

33

34

40

46

7.0 Discussion •••••••••••••••••••••••••••••••••••••••••••••••••••••••••• 48

7.1

7.2

Base-Case Process Verification •••••••••••••••••••••••••••••••••

Basic Laboratory Research ••••••••••••••••••••••••••••••••••••••

48

49

8.0 Conclusions••••••••••••••••••••••••••••••••••••••••••••••••••••••••• 52

9.0 References•••••••••••••••••••••••••••••••••••••••••••••••••••••••••• 54

Appendix A: Alternative Enzymatic Hydrolysis Processes•••••••••••••••••• 56

Appendix B: Enzymatic Hydrolysis Examples••••••••••••••••••••••••••••••• 59

vi
S=~I TR-2093

LIST OF FIGURES

3-1a

3-1b

Cross Section of a Wood Fiber Cell•••••••••••••••••••••••••••••

Model of a Conifer Tracheid ••••••••••••••••••••••••••••••••••••

4

4

3-2 Chemical Structure of a Cellulose Chain, a-D-glucose, and

~-D-glucose••••••••••••••••••••••••••••••••••••••••-.. • • • • • • • • • • 5

3-3

3-4

4-1

4-2

4-3

4-4

4-5

4-6

4-7

4-8

5-1

6-1

6-2

6-3

6-4

6-5

Genealogy of Mutants of Trichoderma reesei (formerly

Trichoderma viride) that Produce High Yields of Cellulase ••••••

Separate Activities of the Cellulase Complex •••••••••••••••••••

Enzyme Hydrolysis Process Block Diagram••••••••••••••••••••••••

Process Block Diagram Including Enzyme Recovery ••••••••••••••••

Steam Explosion Block Diagram••••••••••••••••••••••••••••••••••

Hydrolysis Section (three feed stages) •••••••••••••••••••••••••

Enzyme Production Section ••••••••••••••••••••••••••••••••••••••

Fermentation Section•••••••••••••••••••••••••••••••••••••••••••

Purification Section•••••••••••••••••••••••••••••••••••••••••••

Two-Stage Countercurrent Enzyme Recovery Section•••••••••••••••

Simulation Model Block Diagram•••••••••••••••••••••••••••••••••

Variation of Selling Price, Depreciation, and Costs Due

to Electricity with Percentage Enzyme Recovery and Reuse •••••••

Ethanol Selling Price versus Capital Cost of Enzyme Production

Equipment for Various Percentages of Enzyme Recovery •••••••••••

Ethanol Selling Price versus Enzyme Production Batch Time

at Two Levels of Enzyme Recovery •••••••••••••••••••••••••••••••

Ethanol Selling Price versus Enzyme Loading (per gram of

cellulose) at Various Levels of Enzyme Recovery ••••••••••••••••

Ethanol Selling Price versus Hydrolysis Residence Time

at Various Levels of Cellulose Conversion••••••••••••••••••••••

vii

6

8

12

13

14

16

18

20

21

23

25

34

36

36

37

38
S=~II.I -------------------------==---=~ TR-2093

LIST OF FIGURES (Continued)

6-6 Selling Price of Ethanol versus Hydrolylsis Solids

Load at Various Levels of Cellulose Conversion ••••••••••••••••• 38

6-7 Ethanol Selling Price v~rsus Cellulose Conversion at

Various Levels of Enzyme Loading (FPU/g Cellulose) ••••••••••••• 38

6-8 Ethanol Selling Price versus Various Levels of Base-Case

Steam, Explosion Steam, and Capital Costs•••••••••••••••••••••• 39

6-9 Effect of Base-Case Process Improvements on Selling Price

of Ethanol..................................................... 42

6-10 Ethanol Selling Price versus Two Scenarios of Process

Improvement Implementations•••••••••••••••••••••••••••••••••••• 44

6-11 Ethanol Selling Price versus Process Improvement

Implementation................................................. 45

6-12 Cost Components for Cases of Curve 1, Figure 6-10 •••••••••••••• 46

6-13 Ethanol Selling Price versus Aspen Wood Cost (Wet Basis) ••••••• 47

A-I Gulf/University of Arkansas Process•••••••••••••••••••••••••••• 58

A-2 Battelle Design of MIT Process••••••••••••••••••••••••••••••••• 58

B-1 Example of Enzyme Hydrolysis at High Enzyme Loading ••• e •••••••• 59

B-2 Hydrolysis of Steam-Exploded Corn Stover at Low Specific

Cellulase Activity............................................. 60

B-3 Glucose Conversion versus Specific Activity •••••••••••••••••••• 60

B-4 Example of Interaction of Substrate Loading (FPU/mL),

Reaction Time, and Enzyme Loading on Hydrolysis of AcidTreated Corn Stover•••••••••••••••••••••••••••••••••••••••••••• 61

B-5 Effect of Pretreatment on Hydrolysis of Corn Stover•••••••••••• 61

B-6 Adsorption of Cellulase Enzyme Activities onto 5 wt %

Steam-Exploded Corn Stover at 150 C and Enzyme Load of

25 FPU/g Solids•••••••••••••••••••••••••••••••••••••••••••••••• 63

B-7 Adsorption and Desorption of Cellulase Enzymes during

Hydrolysis of Steam-Exploded Corn Stover••••••••••••••••••••••• 63

viii
S=~II.I TR-2093 -----------------------------------=-=---:..:.....::....:...-=-

LIST OF FIGURES (Concluded)

B-8 Cellobiose Concentration Observed after 48 Hours of

Hydrolysis versus Specific Cellobiase (~-glucosidase)

Activity ...•...••••••..•.••..•.••••••.••••.•....•...•.••••• e... 64

B-9 Glucose Production versus Specific Cellulase Activity for

Different Ratios of Cellobiase (~-glucosidase) to

Cellulase. . . . . . . • • • • • • . . . . . . . . . • • • • . • • • . . • . . . . . . . . . . . . . • • • . . . . . 64

ix
TR-2093

3-1

4-1

4-2

LIST OF TABLES

Feedstock Composition •••••••••••••••••••••••••••••••••••••••••••••••

Steam Explosion Reaction Equations Developed by Chern Systems Inc ••••

Cellulase Production Medium•••••••••••••••••••••••••••••••••••••••••

3

15

19

6-1 Cost of Production Estimate for Ethanol Process-Enzyme

Hydrolysis•••••••••••••••••••••••••••••••••••••••••••••••••••••••••• 30

6-2

6-3

6-4

6-5

Base-Case Capital Cost Summary••••••••••••••••••••••••••••••••••••••

Base-Case Utilities Summary•••••••••••••••••••••••••••••••••••••••••

Cost Components (i/gal)•••••••••••••••••••••••••••••••••••••••••••••

Contribution to Depreciation of Capital Cost Components (i/gal) •••••

x

31

32

41

43
S=~I r_1 TR-2093 ---------------------.---.....:=-.:::..::....=...= -~ .

SECTION 1.0

INTRODUCTION

Current research at SERI includes the development of processes that produce

ethanol from cellulose for use as a fuel. Ethanol may be used as an octane

enhancer t as a fuel extender when blended with unleaded gasoline t or as a neat

fuel. Cellulose is attractive as a raw material because it is abundant and

renewable; an estimated 609 billion metric tons of biomass (wood t crop

residues t and municipal waste) are produced each year in the United States

alone (SRI International 1981).

Currently most ethanol intended for industrial use is produced from ethylene,

a petroleum product. Beverage ethanol is produced by fermentation of starchand sugar-rich feedstacks. Use of cellulose will provide a renewable feedstock for industrial ethanol t yet will not compete with the supply of food.

Cellulose may be converted to ethanol in two discrete steps. The cellulose is

hydrolyzed to glucose, which is fermentated to ethanol. Fermentation

technology has been in development for years by the beverage industry.

Therefore, more recent research efforts have concentrated on the development

of economical means of carrying out the hydrolysis step.

Cellulose may be hydrolyzed to glucose chemically with acid or enzymatically.

Acid hydrolysis processes have the disadvantage that they require severe conditions of high temperature and low pH. Such conditions make it necessary to

employ expensive corrosive-resistant equipment. Conversion efficiency is

limited due to glucose degradation. Enzymatic processes have been severely

hampered by the high costs associated with enzyme produc t Lon , the limited

accessibility of the substrate to the enzyme, and slow reaction rates. However, enzymatic processes are performed at less severe reaction conditions and

have the potential, due to the high specificity of enzymatic reactions, for

total conversion of cellulose to glucose.

Many flowsheets for the enzymatic cellulose-to-ethanol process have been proposed and all fit into one of three categories: (a) separate hydrolysis,

fermentation, and enzyme production steps; (b) simultaneous saccharification

and fermentation (SSF), which combines the hydrolysis and fermentation reactions into one step; and (c) direct microbial conversion, which combines all

three steps of enzyme production, hydrolysis, and fermentation. The objective

of this work is to evaluate an enzymatic hydrolysis process that fits into the

first category because this type of process is furthest along in development

toward operation at a scale larger than bench scale. A base-case flowsheet is

presented based on the model developed by Chern Systems Tnc , , under a SERI

subcontract, and on recent laboratory results obtained at Lawrence Berkeley

Laboratories. Using this simulation model, a parametric analysis is performed

to assess the economic feasibility of such a process and to determine key

research areas for future process improvement.

1
S=~II.I--__ TR-2093 ----------_._--_..:.=.:..-.=

SECTION 2.0

OBJECTIVE AND SCOPE

The objective of this report is to present the economics of a base-case enzymatic hydrolysis cellulose-to-ethanol plant, and to describe the effect of

variations in process parameters on the selling price of ethanol.

The base-case process consists of steam-exploded pretreatment, enzyme production by fed-batch fermentation of the RUT C30 strain of the fungus Trichoderma viride, hydrolysis of cellulose to glucose followed by glucose fermentation in a separate process step, and vapor reuse distillation. A detailed

process description is presented in Section 4.0.

In the parametric analysis, changes are considered in operating conditions

(e.g., enzyme to cellulose ratio); anticipated operational results (e.g.,

cellulose conversion); technology in the form of breakthroughs (e.g., xylose

fermentation by yeast); and anticipated equipment costs (e.g., for the enzyme

production and steam explosion sections). The only major change in process

configuration is the addition of an enzyme recycle section.

The purpose of the parametric analysis in this report is to assess the economic feasibility of this particular enzymatic process at various levels of

implementation of process improvements, as well as to determine the areas of

fundamental and process research that would produce significant cost reduction. This report does not provide a comparison of the various enzymatLc

hydrolysis processes, and only a brief discussion of alternative processes

(e.g., simultaneous saccharification and fermentation [SSF] and direct conversion) is included in Appendix A.

It is also beyond the scope of this report to produce a detailed process

design and an exact cost for ethanol production. The simulation program

models the back end of the process (fermentation, distillation, waste

treatment, and heat generation) with little detail. Emphasis is placed on the

enzymatic portion of the plant (pretreatment, enzyme production, and

hydrolysis). A comprehensive kinetic model to describe cellulose conversion

for various pretreatments, feedstocks, and hydrolysis conditions is not

currently available, and the parametric analysis does not account for these

interactions. Finally, industrial-scale equipment costs and operating

conditions for some of the processes are speculative, particularly those of

steam explosion and enzyme production.

2
TR-2093 ----------------------=-=-=-----=..::::...::...::::. -~ .

SECTION 3.0

ENZYMATIC HYDROLYSIS

This section presents a brief overview of the fundamentals of enzymatic

hydrolysis of biomass to provide background information for the process design

and parametric analysis of this report.

3.1 FEEDSTOCK CHARACTERISTICS

Cellulose does not occur in nature in a pure form. It is always found associated with other materials necessary for biomass structure and growth.

Typical compositions of corn stover and aspen wood are shown in Table 3-1.

Aspen wood has a higher water content than field-dried corn stover (50 wt %

and 30.7 wt %t respectively). On a dry basis, aspen wood has a higher cellulose content than corn stover. For the example of Table 3-1, the cellulose

contents are 38 wt % for corn stover and 49 wt % for aspen wood. The drybasis lignin content of the aspen wood (17 wt %) is higher than that of the

corn stover (10 wt %), but both exhibit similar dry-basis hemicellulose

contents. The ash content of corn stover is significantly higher than that of

aspen wood.

Table 3-1. Feedstock Composition

Wet Basis Dry Basis

Component Field Dry Aspen Wood Field Dry Aspen Wood Corn Stover (wt %) Corn Stover (wt %) (wt %) (wt %)

Water 30.7 50.0 0.0 0.0

Crystalline cellulose 22.5 20.7 32.5 41.4

Amorphous cellulose 3.9 3.7 5.6 7.4

Hemicellulose

Pentosan 16.1 10.9 23.2 21.8

Hexosan 6.5 4.4 9.4 8.8

Carbohydrates 5.3 0.0 7.6 0.0

Insoluble lignin 7.0 8.3 10.1 16.6

Insoluble protein 3.0 0.0 4.3 0.0

Ash 3.9 0.1 5.6 0.2

Extractives 1.1 1.9 1.6 3.8

Total 100.0 100.0 100.0 100.0

3
S=~II']] TR-2093 ---------------------------------=.:::..:....-=...::....:..-=-

Figure 3-1 shows a schematic representation of wood. Lignins and hemicelluloses are located primarily in the middle lamella, forming a protective adhesive shell for the cells. Cellulose is located in the primary and secondary

cell walls. Many texts provide a more detailed description of wood chemistry,

one of which is Wenzl (1970).

Cellulose is a linear polYmer of n-glucose molecules (a six-carbon sugar) held

together by ~-1,4-glycosidic bonds (Figure 3-2). The average chain length is

highly dependent on the type of biomass and pretreatment, but analyses reveal

typical degrees of polymerization between 700 and 2000 units (Wenzl 1970).

The structure of cellulose in plant tissue has been shown -to exist in

crystalline and amorphous forms. Cellulose chains aggregate in parallel

fashion, bound together by hydrogen bonding between hydroxyl groups, to form

crystalline regions of high mechanical strength and high chemical and

enzymatic stability. Primary chains may be up to 30 times the length of the

region of crystallinity and may emerge from either end in a more tangled

fashion, forming less ordered, less stable amorphous regions (Burr 1947).

Hemicellulose in crop residues and hardwoods is a complex polysaccharide composed mainly of n-xylose (a five-carbon sugar), and D-glucose, L-arabinose,

and organic acids. Hemicellulose is less crystalline than cellulose and

therefore can be readily hydrolyzed in dilute alkali and warm dilute acid

solution.

P

SI

.....;..J..j---S2

~~::;::::::~""?1tr-S3

-..." r-

\ \

Figure 3-1a. Cross Section of a

Wood Fiber Cell

Figure 3-1b. Model of a Conifer

Tracheid. M =

middle lamella;

P = primary wall;

51 = outer layer

of the secondary

wall; 52 = secondary wall; 53 =

inner layer of the

secondary wall

(also called the

tertiary wall)

4
---~-~----~--------------- TR-2093 -~ ~~.

"-

LD

LD

o

o

0-

H

Cellulose Chain

H

H

a-D- Glucose /3-0- Glucose

Figure 3-2. Chemical Structure of a Cellulose Chain, a-n-glucose, and

p-n-glucose

Carbohydrate (starch) is a polymer of glucose held together by a-glycosidic

bonds. Starch consists of both linear polymers (amylose) with a degree of

polymerization of 60-300 and branched polymers (amylopectin) with a degree of

polymerization of 300-6000. The tertiary structures of both forms, similar to

that of hemicellulose, do not allow for close packing to form a stable crystalline form, and therefore the starch is amorphous and readily hydrolyzed.

Lignin is a heterogeneous, amorphous, branched polymer based primarily on the

phenylpropane unit. It is intermixed with the hemicellulose in a layer surrounding cellulose. The exact structure of lignin is not known.

In addition to cellulose, hemicellulose, carbohydrates, and lignins, plant

matter is composed of a variety of other materials, including ash and extractives. Due to the high variability of these materials with species and seasons, only a limited amount of information is available on them.

3.2 CELLULASE ENZYMES

Enzymatic hydrolysis of cellulose to ethanol is performed by a class of

enzymes termed cellulases. Each component of the cellulase multienzyme system

plays a particular catalytic role in the depolymerization of cellulose to

glucose monomer units.

Sources

aerobic

enzymes

of cellulase enzymes include anaerobic protozoa, aerobic fungi, and

and anaerobic bacteria. The most extensively studied cellulase

have been produced from aerobic, mesophilic fungi, and of these,

5
S=~II.I ---------------------------------.:..---.:..~ TR-2093

current research is focused primarily on strains of Trichoderma reesei. These

fungi are of particular interest because they produce, extracellularly, all

the enzyme components necessary for cellulose hydrolysis; the cellulase

produced is resistant to chemical inhibitors and stable at temperatures up to

SOoC for up to 48 hours (Mandels 1980).

Figure 3-3 presents the genealogy of mutational research of T. reesei strains

(Wilke 1981). The wild type strain, QM6A, was isolated by Reese in 1950

(Reese 1950). Many hydrolysis studies have been performed with the QM9414

mutant isolated at Rutgers, but the extracellular cellulase from this strain

is deficient in the enzyme necessary for the cleavage of cellobio~e to glucose

(~-glucosidase). The RUT C30 strain, also isolated at Rutgers

(Montenecourt 1977), overcomes this obstacle since it displays high

~-glucosidase activity. The RUT C30 strain is also of interest because it is

resistant to catabolite repression, is hyper-cellulase producing, and exhibits

high xylanase activity.

Hyper-cellulase production signifies that the strain produces the cellulase in

abundance, perhaps producing greater amounts than required for culture growth.

Catabolite repression, a form of metabolic control, occurs where the

production of an enzyme is stopped due to the presence of a breakdown product

of the reaction catalyzed by that enzyme. Resistance to catabolite repression

implies that the RUT C30 strain will continue to produce enzymes even after

Trichoderma reesei (viride)

QM9123

QM9414

1

OM6A (wild type)

~V

High energy l

electrons RUT M-7

/1Nitros09uanidine

( RUT NG-14

RUT L-5 (Hyper producing)

luv

RUT C-30

(Catabolite repression

resistant)

Figure 3-3. Genealogy of Mutants of Trichoderma reesei (formerly Trichoderma

viride) That Produce High Yields of Cellulase

6
TR-2093

-~ .

;*; -----------------------~

significant amounts of cellulose, present for cell growth and enzyme

induction, have been degraded. Xylanase activity, which is a term for the

enzyme activity for the hydrolysis of xylans to xylose or short-chain polymers

of xylose, would be beneficial to a biomass-to-ethanol process both in the

utilization of the five-carbon sugars, either through fermentation to ethanol

or the production of other salable by-products, and for removal of the

protective coating that the xylans may provide against the enzymatic

degradation of cellulose.

Several theories have been proposed for the exact mode of action of ceLl.ulases. Originally, Reese and co-workers proposed the Cl-Cx concept (Reese

1950). This was a mechanism in which two types of enzymes, C1 and Cx' acted

sequentially to depolymerize the cellulase chain. It was thought that the C1

component converted the cellulose to a reactive state that was subsequently

depolymerized by the Cx component.

More recent fractionation studies suggest another mechanism of cellulase

action. The cellulase complex has been shown to consist of three main types

of enzymes: ~-1,4-glucan glucanohydrolase, an endo-enzyme; ~-1,4-g1ucan cellobiohydrolase, an exo-enzyme; and ~-glucosidase.

The first enzyme, ~-1,4-g1ucan glucanohydrolase, randomly acts on the interi.or

of the cellulose polymer to generate new chain ends. The second enzyme,

~-1 ,4-glucan cellobiohydrolase, catalyzes the cleavage of a cellobiose unit

from the nonreducing end of the cellulose chain. It appears to be end product

inhibited and required for the hydrolysis of highly ordered substrates.

The last major component of cellulase, ~-glucosidase, acts to split cellobiose

and other short-chain polymers of glucose to the monomeric glucose unit.. This

component has been found to be end product inhibited, acting by a noncompetitive mechanism.

Figure 3-4 illustrates the separate activities of the cellulase complex (from

Wilke 1981). The Cx enzyme activity from the original postulate of cellulase

mechanism is considered equivalent to the random-acting endoglucanase from the

current theory. Likewise, the old C1 activity is considered equivalent to the

current exoglucanase activity. Both enzymes have been found to act

synergistically; that is, fractionation studies have indicated that the

combined enzymes have a greater activity against cellulose than the sum of

each component.

3.3 LIMITS ON CELLULOSE HYDROLYSIS

In theory, due to the specificity of the enzymatic reaction, cellulose can be

converted to glucose with 100% efficiency. However, in application, yields of

glucose will be bounded by factors that influence the rate and extent of cellulose hydrolysis.

The hydrolysis rate will depend on cellulose accessibility and crystallinity. Surface area available for enzyme-substrate interaction will be

influenced by pore size and shielding effects by lignins. The crystalline

structure excludes water molecules as well as any larger molecules and thus

7
S=~II.: --~-------~------_--=.:~~ TR-2093

Endoglucanases: Random action on

amorphous cellulose

Cx

G-G-G-G 1:- G-G-G-G-G-G 8

Exoglucanases: Endwise action on

crystalline and

amorphous cellulose

C1

t G-G- G-G-G-G-G-G-G-G

f3-GIucosidases: Hydrolysis of

cellobiose to glucose

Figure 3-4. Separate Activities of the Cellulase co.plex

reduces available surface area. Crystalline regions will be hydrolyzed at a

much slower rate than amorphous cellulose due to the greater stability

imparted by interchain hydrogen bonding.

In the case of a high-cost biomass substrate, a more important factor than

rate of reaction may be the extent of reaction. Steric effects imparted by

crystalline structure may effectively halt reaction. More importantly,

shielding effects due to lignins may place a limit on cellulose available for

reaction.

Product inhibition may limit reaction rate and extent. Glucose has been shown

to inhibit ~-glucosidase, and cellobiose has been shown to inhibit the cellulose-hydrolyzing enzymes.

3.4 PRETREATMENT

The primary aim of pretreatment is to enhance the enzymatic susceptibility of

native biomass, through chemical or physical means. Physical pretreatments

include various forms of milling, shredding, and mulching. All act to

decrease the particle size and increase surface area as well as decrease the

degree of polymerization and crystallinity. Ball and roll milling are the two

major techniques. An improvement over ball milling appears to be simultaneous

wet milling and enzymatic hydrolysis (Kelsey 1980). The high energy requirements make the economic feasibility of industrial-scale operation of these

physical pretreatments questionable.

8
;;;: -- II II

~.".~

TR-2093

Chemical pretreatments enhance enzymatic susceptibility by removing the

shielding effects of lignin, reducing crystallinity, and increasing cellulose

solubility by addition of chemical subst itutes or hydration to swell cellulosis fibers. A SERI report (Chum 1983) discusses the relative costs of some

of these pretreatments.

Feedstock costs, which are predicted to be $30/dry ton, will playa major role

in the production cost of ethanol. For example, at 100% cellulose conversion

to glucose and 90% efficiency in fermentation of glucose to ethanol, and

assuming 50% cellulose content on a dry basis, costs associated with the

feedstock are about 37 rilgal. At only 50% cellulose conversion, these costs

become about 74ilgal. Due to the importance of enhanced biomass utilization,

pretreatment effectiveness must be considered as well as pretreatment cost.

Recent laboratory results indicate that three methods of chemical pretreatment

may prove to yield high enzymatic conversions and be cost effective.

Organosolv methods currently suffer from the high costs of solvent recovery,

but separat ions research may produce an economical process. The Dartmouth

acid pretreatment exhibits high conversions, but currently suffers from the

acid costs and the need for a milled substrate. Steam explosion may not

require milling, but has the disadvantage of high steam requirements, costly

equipment, and is a batch, rather than continuous, process.

Lignin removal prior to cellulose hydrolysis may prove beneficial due to

(a) the possibility of a toxic effect of lignins on yeasts; (b) the

possibility that some cellulase ~omponents may bind to lignins, reducing

efficiency and limiting enzyme recovery and reuse; (c) reduction of inert

solids in the hydrolysis section; and (d) separation of lignins for sale as a

chemical by-product. Pretreatments such as organosolv or alkali wash will

separate lignins from cellulose.

3.5 ENZYMATIC ACTIVITY MEASUREMENT

Cellulose activity is reported in terms of filter paper units (FPU) in this

study. A brief discussion of cellulase activity measurements is included

here.

The activity of an enzyme is usually defined in terms of the amount of product

formation per unit of time. An exact determination of cellulase activity presents a difficult task. The functions of the various cellulase components are

not completely understood, and different cellulase preparations may not be of

the same component mixture. Also, the cellulase enzymes act synergistically

and cellulase preparations may exhibit a difference in relative activities for

different substrates. It is therefore important that the cellulase assay be

based on a standard assay procedure with a model substrate that closely

approximates the actual hydrolysis conditions.

A widely used test to measure the composite cellulase activity is the filter

paper test. A 1 em x 3 em strip of Whatman 1f1 filter paper is the substrate. The filter paper is neither too susceptible nor too resistant to

enzymatic attack and is universally available. This assay yields a measurement of the combined cellulase activities in terms of FPU. One FPU is the

9
S=~II.I ---------------------------.:=.::.=....-..=~ TR-2093

amount of enzyme required to produce 1.0 umo.l of reducing sugar per minute.

(In some of the literature, however, the cellulase activity as defined here is

termed an international unit [IU], and an FPU is defined as milligrams of

sugar produced per hour.)

Three other substrates are widely used to measure the activity of subsets of

the celluase complex. Cotton, a highly crystalline substrate, is used to

determine the C1 activity, or rather, the activity against crystalline cellulose. Carboxymethyl cellulose (CMC), a soluble derivative of cellulose, is

used to measure the Cx activity, which is the activity against amorphous cellulose. Cellobiose, a soluble dimer of glucose, is used to measure the

~-glucosidase activity.

Except perhaps for the ~-glucosidase assay, none of the above-mentioned assays

should be considered to determine the amount of a particular cellulase component. Due to the complex interactions present, these assays determine the

combined activities of several enzymes.

10
TR-2093

SECTION 4.0

ENZYMATIC HYDROLYSIS PROCESS DESIGN

The enzymatic hydrolysis cellulose-to-ethanol process presented in this study

is described in this sec t t on , The process flowsheet is the same as that

described by Chern Systems Inc. (Chern Systems Ince 1982a) with modifications to

the enzyme production and enzyme hydrolysis sections. These modifications

were made in order to implement recent results in enzyme production, enzyme

hydrolysis, and enzyme recycle obtained from H. Blanch and co-workers at the

Lawrence Berkeley Laboratories.

4.1 OVERALL PROCESS DESIGN

A major block diagram of the base-case process is presented in Figure 4-1. In

this design, enzyme production, cellulose hydrolysis, and glucose fermentation

are performed in separate vessels. Incoming biomass feedstock is sent through

a steam explosion pretreatment. After steam explosion, a portion of the feedstock is sent to enzyme production and the remainder is sent to the hydrolysis

section. Cellulase enzyme is produced via fed-batch fermentation in the

enzyme production section and is sent to the hydrolysis section. After hydrolysis, the sugar-rich liquid stream is sent to fermentation. The ethanol-rich

stream from fermentation is purified and dehydrated in the purification

sect ion. Residual cellulosics and lignins from the hydrolysis vessels are

sent to a lignin boiler to provide process steam. Yeast and cell mass from

the fermentation and enzyme production sections are sent to the by-product

recovery section for sale as single-cell protein (SCp). Stillage from the

distillation section is sent to waste disposal.

Figure 4-2 is a block diagram for a process that includes enzyme recycle.

Recycle is accomplished by adsorption of the soluble cellulase enzyme in the

hydrolysate onto fresh biomass feedstock. This process differs from the one

depicted in Figure 4-1 only in that the hydrolyzate liquors are directed

through a two-stage contacting reactor prior to being sent to concentration

and fermentation. A portion of the fresh hydrolysis biomass feed is directed

through this contacting reactor after steam explosion.

4.2 DETAILED PROCESS DESIGN

4.2.1 Pretreatment

The base-case process design incorporates steam explosion as a pretreatmente A

block flow of this section is presented in Figure 4-3.

The biomass feedstock is assumed to be supplied in a form suitable for direct

feeding into the steam explosion guns. In the case of a hardwood feedstock

this assumption implies that the wood has been chipped prior to delivery and

that the chipped wood requires no further size reduction, such as milling.

11


- I( '"' l\II

- ~=~

TR-2093

600 psi steam

I ,

I /

Flash vapors

Steam -, explosion / Cyclone

guns

I ,

,

o >

Aspen wood chips

Exploded biomass

to enzyme hydrolysis

or enzyme production

Figure 4-3. Steam Explosion Block Diagram

The steam explosion process developed by Iotech is a batch process. An

explosion gun is charged with biomass and steam is injected until the desired

cook temperature is attained. After the required cook time, the reactor pressure is quickly reduced, explosively discharging the contents into a cyclone.

Some water and degradation products are flashed in the cyclone, and the

resultant product is sent on to enzyme production or hydrolysis.

Chern Systems Inc. analyzed the Iotech experimental results and determined the

kinetics of hemicellulose conversion for the following operating conditions:

Steam: 400 lb/ton of feedstock, 560 psig, 247 0C

Cook time: 5 seconds.

Recent information obtained from researchers involved with the Iotech process

indicates that the steam requirements per ton of wet feedstock will be much

greater than the 400 lb/ton used in this report. Heat reuse to preheat the

incoming feed will reduce these estimates, but 400 lb/ton may indeed be overly

optimistic.

The equations for hemicellulose conversion and product formation are listed in

Table 4-1 (Chern Systems Inc. 1982b). In determining these equations, Chern

Systems made the following assumptions:

• Iotech experimental data (Iotech Corporation 1980) form the basis for the

xylose and degradation product conversions from hemicellulose and

cellulose.

14
Table 4-1. Steam Explosion Reaction Equations Developed by

Chem Systems Inc.

Xyloses produced from pentosans:

0.0018 x hemicellulose (lb) x 0.711 = xyloses produced (lb)

0.88 x xyloses produced (lb) pentosan consumed (lb)

0.12 x xyloses produced (lb) = H20 consumed (lb)

H20 consumed (lb)

degradation products formed (lb)

Degradation products produced from

0.013 x hemicellulose (lb) x 0.711

products (lb)

0.136 x pentosan consumed (lb)

1.136 x pentosan consumed (lb)

pentosans:

pentosan consumed to degradation

Furfural produced from pentosans:

y x 0.64 x degradation products formed (lb) = furfural formed (lb)

0.36 x degradation products formed (lb) = H20 formed (lb)

Pseudolignin produced from pentosans:

Z x 0.813 x furfural formed (lb) = pseudolignin formed (lb)

0.187 x furfural formed (lb) = H20 formed (lb)

H20 consumed (lb)

degradation products produced (lb)

Degradation products produced from

0.013 x hemicellulose (lb) x 0.289

products (lb)

0.11 x hexosan consumed (lb)

1.11 x hexosan consumed (lb)

hexosans:

= hexosan consumed to degradation

liMF produced from hexosans:

0.7 x degradation products produced (lb)

0.3 x degradation products produced (lb)

HMF produced (lb)

H20 produced (lb)

Pseudolignin produced from hexosans:

0.86 x HMY formed (lb) pseudolignin formed (lb)

0.14 x HMF formed (lb) = H20 produced (lb)

sugars and degradation prod-

- H2 0 . pseudolignin + Lf.gntn

H20

l' .~ pseudolignin • 19n1n

Hemicellulose and cellulose are converted to

ucts according to the following reactions:

+ H20 - 3 H20

pentosan xylose furfural

+ H20 - 3 H20

hexosan glucose HMF

•

• No net glucose is formed within the operating range studied (5 to 32

seconds cook time) and the decomposition of cellulose does not occur

prior to 60 seconds cooking time.

15
TR-2093 S=~IIr*;1 ------------------------ -~ .

• The weight percent of hemicellulosic hexosans converted to degradation

products is equal to pentosans converted to xylose and degradation

products.

• The xylose formation from hardwoods is the same as from corn stover, and

the equations listed in Table 4-1 apply to either substrate.

4.2.2 Enzyme Hydrolysis

Figure 4-4 presents a diagram of the enzyme hydrolysis section. The hydrolysis reactors are constructed of carbon steel and are rated for atmospheric

use. To facilitate high biomass loading, the hydrolysis feed is split and fed

in up to four feed stages. Due to the decrease in slurry viscosity during the

initial hours of hydrolysis, this allows a greater loading of biomass than if

one feed stage was used. The following staging sequence is used:

Substrate Loading (wt %)

less than 10%

10% to 15%

15% to 20%

20% to 25%

Number of Feed Stages

1

2

3

4

Pretreated biomass

Wash water

-r

0J

.,.

CJ

CJ

Solids to

heat generation

.---.,._ Rotary filter

Pump Pump Pump Pump

Enzymes

from

enzyme prOd£» =

Pump Agitated

vessel

Figure 4-4. Hydrolysis Section (three feed stages)

16
S=~II.~I ~------------------------------_....=..::.~:::..::::...:::...:::.... TR-2093

For the base-case design, the hydrolysis section is run with a solids loading

of 20 wt %, with 3 feed stages, at 4SoC and pH 5.0, and with a total residence

time of 48 hours.

Enzymes produced in the enzyme production section are continuously fed along

with process water to the first hydrolysis feed stage. After hydrolysis, the

slurry is washed in a rotary wash filter. Residual solids are sent to the

lignin boiler to provide process steam. The glucose-rich hydrolysate is sent

either to fermentation or concentration.

At present, a well-defined kinetic model of the hydrolysis reaction allowing a

determination of cellulose conversion based on the parameters of feedstock,

pretreatment, residence time, and enzyme loading is not available. From

recent data it has been assumed that 80% cellulose conversion to glucose can

be achieved at the base-case design parameters described above with an enzYme

loading of 25- FPU/g of cellulose.

4.2.3. Enzyme Production

The enzyme production section is based on the design reported by Perez

(1981). Modifications have been made to the operating conditions as reported

by Perez to incorporate recent results on fed-batch enzyme production (Hendy

1981, Blanch 1983).

A schematic of the enzyme production section is shown in Figure 4-5. The

enzyme is produced batchwise from the RUT C30 strain of Trichoderma reesei in

fermenters constructed of 304 stainless steel, rated to 2.5 atm to enable

steam sterilization. Batch cycle time is 13.5 days, which includes 13 days

fermentation and 0.5 day for charging, discharging, and sterilizations.

Inoculum for each batch is produced in two stages of inoculation fermenters.

The second stage has a working volume of 10% of the daily production and the

first stage has a working volume of 10% of the second stage. The medium

described in Table 4-2 is mixed and sterilized continuously prior to entering

the fermenter.

Steam-exploded btomass "feedstock is washed on a rotary wash filter to remove

soluble lignins. The washed biomass is then added in increments of 50 g/L to

the batch fermenters until a final loading of 150 g/L is attained.

After fermentation the enzyme broth is washed on a rotary wash filter. The

filtrate is stored in a surge tank to facilitate continuous introduction to

the hydrolysis section. The solids, consisting of approximately 18 g/L of

mycelia, 7.5 g/L of cellulose, and the biomass noncellulosic material, are

either sent to by-product recovery or burned in the lignin boiler to provide

process steam.

Laboratory tests on the fed-batch enzyme production process have produced

enzyme activity as high as 32 FPU/mL with the RUT C30 1.. reesei strain, using

Solka Flok as a carbon source. Recent results indicate that this titre may be

achieved also by using the less expensive biomass feedstock as carbon source

(Blanch 1983). The base-case process described in this report assumes that an

enzyme strength of 30 FPU/mL can indeed be attained using the biomass as

carbon source.

17

S=~II~'!! TR-Z093

Table 4-2. Cellulase Production

Medium

Component

(NH4) Z S04

UH ZP04

MgS04

CaC1Z • 2HZO

Corn Steep Liquor

Tween-80 (ml)

FeS04 • 7 H20

MuS04 • H20

ZnS04 • 7 HZO

COC1 Z

Concentration

(gIL)

34.8

11.4

0.9

Z.4

8.7

0.2

5.0

1.6

1.4

2.0

Medium is a variation on that presented in Perez 1981. The concentrations of the first 5 components

above have been increased by a

factor of 3 over that described by

Perez for 50-gIL cellulose, due to

the increase in cellulose concentration to 150 gIL (Blanch 1983).

4.2.4 Fermentation, Carbon Dioxide Recovery, Purification, and Offsites

The remaining process sections of fermentation, carbon dioxide recovery, and

purification as well as the offsites are identical to the process reported by

Wright (1982), and the following descriptions are exerpts from that report.

The fermentation block (Figure 4-6) consists of three parts:

fermentation, and carbon dioxide (COZ) recovery. The ethanol

in the continuous cascade fermenters is sent to purification.

is recovered, liquefied, and sold as a by-product. A yeast

sold as single-cell protein animal feed.

detoxification,

stream produced

Carbon dioxide

purge stream is

The neutralized sugar solution is passed through columns of activated carbon

to remove HMF, furfural, and any other trace hydrolysis degradation products

that may be toxic to the yeast.

19
TR-2093

CO2

Recovery "'" CO2

Sugar Fermentation Yeast Ethanol to

Solution Separation Purification

, Yeast Purge

Make-Up Yeast Split to

Yeast - Preparation By-Product

Recovery (15%)

Recycle

Figure 4-6. Fermentation Section

Fermentat ion is carried out at 300C (8SoF) in a cont inuous cascade scheme.

The fermentation time is 24 hours. Ninety-five percent of the glucose is converted by the yeast to ethanol and carbon dioxide by the following reaction:

.. 2C2HSOH + 2C02

(46) (44)

In this reaction, 51 wt % of the glucose is converted to ethanol and 49% to

CO2• Three percent of the glucose is converted to glycerol, and 2% is converted to yeast. The yeast grows 10% during the cycle. Eighty-five percent

of the yeast is recycled to the reactor, while the remainder is sold as a single-cell protein by-product. The concentration of yeast in the fermenter is

augmented with fresh yeast to 9% of the total glucose input. The xylose

passes through the fermenter unchanged.

The fermenters used are closed vessels. The CO 2 produced is collected, liquefied, and sold. The collected CO2 is scrubbed with water to remove soluble

impurities and then compressed to 2.02 MPa (300 psia). The compressed gas is

passed through beds of activated carbon to remove any remaining impurities.

The gas is then chilled, dried on a desiccant bed, and liquefied.

The ethanol-water stream from the fermenter is concentrated to the 94 wt %

ethanol/6% water azeotrope in the beer still and then dehydrated to produce

anhydrous ethanol by. a ternary benzene distillation (Figure 4-7). The aqueous

stillage (mainly water and xylose) is sent to the waste ponds. Steam usage is

minimized by heat integration.

20
S=~II.}I ---------------------------------.-::::..;~=....:::..~ TR-2093

Make-Up

Benzene

Water to

Benzene

Stripper

Column

Waste

Aqueous Disposal

Component

Ethanol Product

Azeotrope

Dehydration

Column

Beer

Still

Aqueous

Stillage

to

Waste Ponds

Ethanol

from

Fermentation

Figure 4-7. Purification Section

The beer feed from fermentation enters the rectification column (beer

still). Heat to run the beer still is provided by 1.3 MPa (200 psia) steam.

The upper part of the column" utilizes sieve trays while the bottom uses disk

and doughnut trays to handle the water, xylose, and suspended solids. The

water stream (stillage) is sent to waste disposal. The column overhead stream

contains the binary water-ethanol azeotrope.

The overheads from the beer still enter the dehydration column where benzene

ternary distillation is used to break the azeotrope. Pure ethanol is removed

from the bottom of the dehydration column. The dehydration column overhead

stream is a tertiary azeotrope of ethanol, water, and benzene. The water is

separated from the benzene and ethanol in a stripper column. The heat

required for the dehydration and stripper columns is cascaded from the beer

still, and the azeotropic distillation therefore does not require additional

energy.

The offsites section includes heat generation, waste disposal, storage for raw

materials and products, and the various utilities.

In the heat generation block the unreacted solids from the hydrolysis reactor

(mainly lignin and unreacted crystalline cellulose) are neutralized, dried to

50% solids, and burned in the lignin boiler to produce 4.04 MPa (600 psia)

steam. The lignin boiler is sized to dispose of the entire hydrolysis purge

stream. The high pressure steam provides heat for the hydrolysis reactor.

Excess steam is throttled to 1.35 MPa and used to heat the prehydrolysis reactor and distillation section, and to dry the boiler feed. If excess steam is

available after the plant load is met, it is sold as a by-product at a price

competitive with steam generated by a coal boiler, or at a lower price based

21
5=~11.' --------------------------=.;:..:...-::~ TR-2093

on the value of the unreacted solid feed. If the lignin boiler is not large

enough to supply the plant demand, a coal boiler with flue gas desulfurization

is added to make up the difference. The boilers constitute the single largest

capital investment in the plant.

The aqueous stillage and various condensed flash vapors are collected and sent

to a waste treatment pond. Electrical and cooling water systems are sized to

meet the plant load. Storage for ethanol, calcium hydroxide, sulfuric acid,

feedRtock, and by-products is sized to accommodate two weeks of these

materials.

4.2.5 Enzyme Recovery

Enzyme recovery via countercurrent adsorption has been included as a process

option. A schematic of the equipment for this section is shown in Figure 4-8.

Enzyme recovery is accomplished by adsorption of the soluble cellulase enzymes

contained in the hydrolysate stream onto pretreated biomass feed. Two contacting stages are used, each with a residence time of 0.5 hour. Each stage

consists of an agitated carbon steel tank, conveyor, rotary filter, and pumps.

Laboratory results have indicated that up to 80% of the hydrolysis section

filter paper activity may be released back into solution at high levels of

cellulase conversion (Orinchowskyj 1982). Detailed computer modeling of the

two-stage enzyme recovery section, using experimentally determined Langmuir

isotherm constants for pretreated biomass to describe the enzyme adsorption,

indicates that as much as 98% of this filter paper activity may be adsorbed

onto the freshly pretreated biomass. Sixty percent enzyme reuse was therefore

considered in the parametric analyses of Section 6.0.

22

S=~II.I -----------------------~~~ TR-2093

SECTION 5.0

SIMULATION K>DEL

In order to determine the effect of operating conditions, process configuration, and assumed kinetics, it is desirable to model the operation of the

entire biomass-to-ethanol plant. Earlier efforts have concentrated on the

enzymatic hydrolysis portion of the process and modeled only on the basis of

selling price of glucose (Perez 1981; Wald 1981). However, this method does

not allow an integration of offsites (i.e., utilities and waste disposal)

between the glucose production portion (front end) and the subsequent

concentration, fermentation, and purification portion of the plant (back end).

The calculations presented in this report were executed by a simulation model

developed by Chern Systems Inc., under subcontract to SERle This model simulates the entire plant and determines a selling price for ethanol at the

desired return on investment. Some modifications were made to the Chern Systems model as described in the previous section and will be discussed below,

along with a brief description of the Chem Systems version. A more detailed

documentation of the unmodified Chem Systems model can be found elsewhere (Chern Systems Inc. 1982).

The simulation model was developed for implementation on an IBM 5120 computer,

using "A Programming Language" (APL). Minor modification allowed the model to

be also implemented on a Control Data Corporation 720 mainframe computer,

allowing for quicker execution time and direct data plotting.

Figure 5-1 shows the flow path of the simulated model. The first block,

called EDITENZ, simply loads in operational data for the current run. ENZ1,

the second block, calculates all stream flows and material balances. Once

these flows have been established, they are scaled up in ENZ2 such that the

desired production rate is achieved. ENZ3 calculates the capital cost of

equipment based on major stream flows. The last block, ENZ4, determines the

plant economics and the selling price of ethanol. '

Capital cost for each major piece of equipment is determined based on the

equation

Cost = base cost ( Size )a x base size •

The base cost and base size were determined by Chem Systems Inc. for a

50 million gal/yr plant using the Icarus Cost Program developed by the Icarus

Corp. of Rockville, MD. The exponent a was determined for each equipment type

also using the Icarus Cost Program by perturbation of the base-case size and

plotting of the resultant costs. Costs of equipment for the enzyme

hydrolysis, enzyme production, and enzyme recovery sections were determined

from costing estimates as described by Perez (1981).

The discounted cash flow (DCF) analysis used in ENZ4 to calculate the selling

cost contains the following assumptions (from Chern Systems Inc. 1982):

24

TR-2093 S=~II.I ------------------------=-::...:.......=...:....:....=..

• Plant basis is first quarter 1982 at a U.S. Gulf Coast location.

• Time of construction is two years with uniform expenditure of capital

throughout this period.

• Working capital is the sum of

Feedstock inventory--0.5 month of raw materials at delivered price

Finished product inventory--0.5 month of products and by-products

Accounts receivable--one month gross cost of production

Cash--one month expenses (gross cost of production less depreciation)

Warehouse/space parts inventory--3% of ISBL capital cost

Less:

Accounts payable--one month of raw materials at delivered prices.

• Total federal and local taxes is 50%.

• Depreciation is straight line, over a 5-year period for onsite investments and a 10-year period for offsite investments, with no salvage value

for the plant.

• Cost of sales is 6% of the product selling price.

• A gradual sales buildup of 60% of capacity in the first year, 80% in the

second, and 100% return from the third year on.

• Return on investment of ]5%.

Modifications to the Chern Systems model were made to the subroutines performing the enzyme hydrolysis and enzyme production materials balances and costs.

Additionally, subroutines were added to implement enzyme recycle via countercurrent adsorption. Material balance calculations for these sections are

based upon the process descriptions presented in Section 4.0. Capital costs

of equipment for these sections are determined as described by Perez (1981)

and are based upon cost estimates found in the literature.

This simulation model is limited in its ability to determine an exact selling

price of ethanol due to (a) lack of detail in the model and (b) lack of a full

understanding of kinetics and processing constraints.

Regarding the first reason, the purpose of the Chern Systems model was to provide a method of comparison of process configurations and assumed kinetics, to

aid in understanding major cost considerations, and to provide a direction for

further research. The model was designed to provide not a detailed but rather

a realistic estimate of the cost to produce ethanol. Particularly, major

attention in the model is focused on the front end of the plant, the biomass

hydrolysis, and lesser detail has been provided for the back end, ethanol

production from the hydrolysis sugars.

Regarding the second reason, kinetics and materials handling processes are not

well understood. Currently, there are no kinetics models to predict glucose

conversion and ethanol fermentation that encompass all the possible variations

in pretreatment conditions, feedstock, enzyme source, enzyme loading,

hydrolysis conditions, and the presence of toxins. Also, the factors

involving materials handling and exact costs and operating conditions for

novel processes (e.g., steam explosion) are not completely known at this time.

26
TR-2093 S=~II.I -------------------------------------

The simulation model used for this report requires as input values for glucan

conversion in the enzymatic hydrolysis section as well as for all operating

conditions such as residence time and enzyme loading. It is up to the user to

realize that the model does not contain a kinetic equation for enzymatic

hydrolysis and that in order to obtain a realistic ethanol cost estimate, the

user must input values based on realistic laboratory and pilot-plant results.

27
S=~II.I -----------------------------=~=~ TR-2093

SECTION 6.0

PROCESS SIMULATION STUDIES

6.1 BASE-CASE DESIGN

Plant capacity was set at 50 x 106 gal of ethanol per year. Economies of

scale usually dictate that the ethanol cost would decrease with increased

plant capacity. However, with a biomass feedstock, increased plant capacity

results in increased feedstock cost, due to increased collection radius. As

discussed in an earlier report (Wright 1982), 50 x 106 gal/yr may be the

largest capacity plant that can be reasonably built.

Aspen wood was chosen as the feedstock. A hardwood was chosen over an agricultural residue, such as corn stover, because of the inclusion of steam

explosion as pretreatment. Currently available data on the steam explosion

process do not indicate a change in operating conditions to effect a similar

enzymatic conversion for different feedstocks. Due to the high cost of steam

explosion, hardwoods may be more economical because they have a higher glucan

content than agricultural residue (50% cellulose as compared with 35%-40% for

corn stover on a dry basis). The cost for aspen wood was assumed to be

$30/dry ton, or $0.75/wet pound, assuming 50% moisture content. The wood was

assumed delivered to the plant site in a chipped form suitable for steam

explosion without additional size reduction.

Since a comprehensive kinetic model for cellulose hydrolysis encompassing all

the various parameters such as pretreatment, nature of substrate, and hydrolysis conditions is not available, an estimation must be made of the percentage

of cellulose-to-glucose conversion for a given set of ope rat ing conditions.

Recent laboratory results have indicated conversions in excess of 80%

resulting in sugar solutions in excess of 12 wt% using steam-exploded biomass

(Blanch 1983). For the base-case process, 80% cellu10se-to-glucose conversion

was assumed, with a residence time of 40 hours, solids loading of 20 wt %, and

an enzyme loading of 25 FPU/g of cellulose.

This combination of 80% conversion and 20 wt % solids loading produced a glucose stream of approximately 10 wt %, which eliminated the need for a concentration step prior to fermentation. This was shown by simulation of the basecase process while incorporating the concentration step. The selling price of

ethanol is $2.39 /ga1 with concentration to 20 wt % glucose solution prior to

fermentation, and $2.13/ga1 with no concentration. The savings in purification and fermentation capital and utilities when concentration is incorporated

are less than the increased costs associated with the multieffect evaporator. However, a more detailed design of the waste treatment section than is

currently available in the model may alter these results.

Recent laboratory results have indicated that as high as 32 FPU/mL enzyme

titre can be produced from RUT C30 using corn-steep liquor as a nitrogen

source and Solka Flok at a level of 150 gIL as a carbon source, in fed-batch

fermentations (Blanch 1983). Batch residence time was approximately 13

days. Recent results also indicate that similar high enzyme titres can be

achieved using water-washed, steam-exploded biomass as the carbon

28
S=~III.i ------------------------.:::...::..::....-::~ TR-2093

source (Blanch 1983). The base-case design of the enzyme production section

assumes an enzyme strength of 30 FPU/mL produced in a 13. 5-day cycle time,

utilizing the washed, steam-exploded aspen wood as the carbon source.

The base-case design does not include enzyme recovery and reuse, and sends the

solubilized xylose to the waste treatment ponds.

The production cost summary for the base-case design is shown in Table 6-1.

For the 15% return on investment (ROI), a selling price of $2.13/gal is

indicated.

The largest contributor to the net cost of production is depreciation, at

66.2ri/gal. Table 6-2 shows the summary of capital cost due to offsites and

onsites. Column 2 shows the percentage contributions to onsite purchased

equipment cost for each of the major processing steps. The most capitalintensive processing steps are steam explosion and enzyme production, accounting for 35% and 43% of the onsite purchased capital, respectively. Due to

depreciation, these processing steps contribute l8i and 22i/gal, respectively,

to the net cost of production. It must be noted that with the economic analysis of this simulation model, overhead, operating costs, and offsite equipment

costs are based in part on the onsite capital equipment cost, and therefore

these two processing steps contribute heavily to the production costs in these

areas as well.

The second largest contribution to ethanol cost is due to raw materials. Aspen

wood costs dominate the raw material costs, contributing 46ri/gal.

Utilities account for 2Ii/gal. Table 6-3 presents a summary of the utilities

for each processing step. Power requirements are highest for enzyme production, accounting for 7.4i/gal. Hydrolysis and fermentation also consume large

amounts of power, contributing 3.3ri/gal each. Steam costs are not a major

factor in the base-case costs, due to the onsite production of most of the

process steam in the lignin boilers. The boiler costs contribute Sri/gal to

the depreciation costs. When utilities are considered together, however,

steam consumption becomes an important factor, because any excess Btu value

from residential biomass can conceivably be converted onsite to electrical

energy. Electricity alone contributes lSri/gal to ethanol costs. Steam costs

also will become more' of a factor if residual lignins are converted into a

salable by-product, and are not available for process steam production.

Operating costs account for 19.6i/gal. Of this cost, 84% or 16.4i/gal are due

to maintenance, which is calculated as a percentage of onsite capital cost,

which, as shown above, is due mainly to the steam explosion and enzyme production steps.

6.2 PARAMETRIC ANALYSIS

This section presents the findings of a parametric analysis of the base-case

process. First, a qualitative discussion provides the rationale for the

chosen process variations. Next the results of single-parameter variations,

along with some coupled-parameter variations are presented to illustrate their

effects on the selling price of ethanol. The process component costs of

29
TR-2093 S=~II.I ------------------------=.::..::..---..:...-

Table 6-1. Cost of Production Estimate for Ethanol Process-Enzyme Hydrolysis

Basis Capital Cost Summary ($M)

u.S. Gulf Coast Location

First Quarter 1982

50.0 x 106 gal/yr capacity

149,335 metric tons/yr

8000 h/yr str. time

Battery limits 137.2

Offsites 56.5

Total fixed inventory 193.6

Working capital 15.3

Production Cost Summary

Component (units)

Units

per gal

of Ethanol

Price

(t/unit)

Annual

Cost ($M)

Cents

per gal

of Ethanol

$/

Metric

Ton

Raw materials

Aspen wood (lb)

Sulfuric acid (lb)

Nutrients

Catalyst and chemicals

Total raw materials

61. 7198

0.0000

0.6191

0.8

4.3

6.5

23,145

o

2,012

5,681

30,838

46.29

0.00

4.02

11.36

61.68 206.50

Utilities

Power (kWh)

Cooling water (106 gal)

Process water (10 6 gal)

Boiler feedwater (106 gal)

Steam, 200 psia (10 6 lb)

Steam, 55 psia (106 lb)

Total utilities

3.11395

0.25803

0.01634

0.00195

0.00130

0.00520

4.6

7.3

65.0

113.0

480."0

470.0

7,162

942

531

110

313

1,221

10,279

14.32

1.88

1.06

0.22

0.63

2.44

20.56 68.83

6.55120

0.40484

Operating costs

Labor, 46 men at $26,000

Foremen, 9 men at $29,600

Supervision, 3 man at $35,600

Maintenance, material, and labor, 6% of ISBL

Total operating cost

Overhead expenses

Direct overhead, 45% labor and supervision

General plant overhead, 65% operating costs

Insurance and property tax, at 1.5% total

fixed inventory

Total overhead expenses

By-product credit

Carbon dioxide (lb)

Single-cell protein (lb)

Total by-product credit

Cash cost of production

Depreciation, 20% ISBL + 10% OSBL

Net cost of production

2.8

15.0

1,196

266

107

8,230

9,799

706

6,369

2,905

9,980

-9,172

-3,036

-12,208

48,688

33,080

81,768

19.60

19.96

-24.42

97.38

163.54

65.62

66.83

-81.75

326.03

547.54

Sales price at 15% DCF 212.6 711.8

30
TR-2093 S=~II.I ----~--~-----------~--------=~ -~ .

Table 6-2. Base-Case Capital Cost Summary

Cost ($) % of

Purchased Equipment

Purchased Equipment Costs

Raw material handling

Enzyme production

Enzyme hydrolysis

Fermentation

Purification

Heat generation

Total Purchased Equipment Cost

16,700,602

20,093,196

3,869,203

2,648,985

2,323,179

1,515,542

47,150,707

35.42

42.61

8.21

5.62

4.93

3.21

100.00

Total Installed Equipment Cost 97,149,624

Engineering and construction overhead

Engineering fee and contingency

16,879,953

15,371,130

CO 2 recovery system package

Total plant ISBL cost

7,758,127

137,158,834

Offsites Capital Cost

Ethanol storage, 14 days

Sulfuric acid storage, 14 days

By-product storage, ·14 days

Yeast storage, 14 days

Nutrient storage, 14 days

Lignin steam boiler, 600 psia

Steam boiler, 600 psia

Cooling water system

Electrical

Buildings, 3% ISBL

General utilities, 5% ISBL

Site development, 3% ISBL

Piping, 3% ISBL

Pollution control, 2% ISBL

Total offsites cost

826,946

968

241,804

65,765

723,278

19,690,764

5,510,085

2,416,167

5,060,175

4,114,760

6,857,942

4,114,765

4,114,765

2,743,177

56,481,367

Total plant capital cost 193,640,201

select process variations are then summarized, both with respect to their

individual contributions and to various combinations. Finally, the sensitivity of the selling price of ethanol to feedstock cost is presented for a few

alternatives to the base-case design.

31
Table 6-3. Base-ease Utilities Summary

Processing Steam (1000 lb/h) Power Cooling Water Process Water

Step 15 psia 55 psia 200 psia 600 psia (hp) (gal/min) (gal/min)

Steam explosion -73.4 0.0 0.0 101.9 385.7 0.0 0.0

Enzyme production 15.0 0.0 0.0 0.0 13,529.3 1,137.6 933.1

w Enzyme hydrolysis 26.7 0.0 0.0 0.0 7,200.8 0.0 769.3 N Fermentation 0.0 0.0 0.0 0.0 6,023.3 5,863.0 0.0

Purification 0.0 0.0 192.1 0.0 124.7 6,997.5 0.0

Heat generation 0.0 64.2 -184.0 -101.9 94.4 7,191.7 0.0

Waste treatment 0.0 0.0 0.0 0.0 2.0 5,688.4 0.0 -- 31.7 64.2 8.1 0.0 27,360.2 26,872.2 1,702.3

UI

III

N

-

I

N

o

\0

W
S=~II.;i ---------------------:::~~ TR-2093

-~ .

6.2.1 Qualitative Analysis of Process Improvements

One of the major contributors to the ethanol cost was shown to be feedstock

costs. For a feedstock cost greater than zero, cost reduction can be achieved

with an increase in feedstock utilization--either an increase in cellulose

conversion or the utilization of xylose to produce additional ethanol.

Approximately 7% of the pretreated feedstock is diverted to the enzyme production section, which accounts for 3.2¢ of the 46.3i/gal feedstock cost. Substitution of this portion with a less expensive carbon source can perhaps be

accomplished using whey, or in the case of development of a constitutive

enzyme producer, with a soluble carbon source. This would increase feedstock

utilization to produce ethanol as well as reduce the capacity of the steam

explosion section.

Cost of production due to the steam explosion pretreatment was shown to be a

major factor in the selling price of ethanol. Enhanced biomass utilization

would decrease pretreatment capacity and thus lower costs. Substitution of

the steam explosion process with a less costly pretreatment or a reduction in

the estimated capital cost and steam requirements would reduce costs due to

the pretreatment, but this reduction may be offset by any increases necessitated by lesser enzymatic conversions caused by less effective pretreatment.

A reduction in enzyme production costs can be achieved by decreasing the

enzyme production capacity. This would be accomplished if it were possible to

reduce enzyme loading per gram of cellulose, incorporate enzyme recovery,

increase enzyme titre per batch, or reduce the batch cycle time. Laboratory

results discussed in Appendix B indicate that up to 65% of the filter paper

activity of the cellulase enzymes may be recycled and reused by adsorption

onto the pretreated hydrolysis solids feed. Hydrolysis data shown in Appendix B indicate that enzyme loading may be decreased below 25 FPU/g perhaps at

the expense of an increase in hydrolysis residence time. The base-case values

for enzyme titre and batch time are at the best reported levels, and improvements here may be more speculative with the RUT C30 organism.

Capital cost for the enzyme production section is very high due to stainless

steel construction and use of fermentors designed to withstand 2.5 atm and

equipped with cooling coils. The base-case enzyme product ion sect ion is

designed as described by Perez (1981), and may be conservatively expensive for

this section. Few data are available on large-scale studies of cellulase production, and this design is included as perhaps a worst case. Some pilotplant data suggest that the enzyme product ion can be carried out in less

expensive equipment (University of Arkansas 1981). A reduction of equipment

cost for enzyme production would have a large effect on the ethanol price.

Additional salable by-products from lignins or xylose will also effectively

increase the feedstock utilization and decrease the ethanol selling price.

However, in the case of lignins, the by-product credit would be offset by the

loss of fuel value to produce process steam. Fermentat ion of xylose to

ethanol would allow a decrease in plant size for a given yearly ethanol production rate.

A decrease in hydrolysis residence time would decrease the costs due to hydrolysis capital and ope rat ing costs. An increase in hydrolysis solids loading

33
TR-2093 S=~II.I -----------------------------------

would decrease fermentation, distillation, and waste treatment costs due to

the resulting higher concentration of glucose in the processing streams. However, it is expected that correlations exist between hydrolysis residence

time, solids loading, and conversion, and that parametric analyses of one of

these parameters with the others held constant may not represent a possible

set of conditions.

6.2.2 Variations in Single and Coupled Parameters

Figure 6-1 shows the selling price of ethanol for levels of enzymatic recycle

from 0% to 75%. At 75% enzyme recycle, the selling price was reduced 27% to

$I.sS/gal. Addition of the enzyme recycle section adds approximately

$0 •.5 million in capital cost and increased power consumption. However, at 75%

recycle, the capital cost of the enzyme production section decreases from

$20 million to $S.4 million. Figure 6-1 also indicates that over this range,

the enzyme production depreciation contribution decreases from 23.4i/gal to

6.3r/./gal and the power requirements drop from 7.4;' to 2r/./gal. Even at the

more realistic value of 60% enzyme reuse, the data indicate a reduction in

selling price of 21.2% to $1.67/gal.

25

-

20

200 (ij

Q>

OJ 15 c::

0

Q) .~

0 '0 '§' Q)

OJ Ci

§ Q)

10 "0

Q) (; (/) iii

0

0

5

20 60

150 "'"'-----'-----..I.----L------l------l-__-L__--.....JL-...Jo

o

Figure 6-1. Variation of Selling Price, Depreciation, and Costs Due to

Electricity with Percentage Enzyme Recovery and Reuse

34
TR-2093 S=~II.I ----------------------------~-------

To assess the effects of less costly enzyme production equipment, simulations

were performed in which the equipment was taken as 25% to 100% of the basecase costs. Capital costs for enzyme production may be reduced if the fermentation can be performed in less complex equipment. Chemical rather than

steam sterilization would negate the need for 2.5-atm rated vessels. External

heat jackets would be less costly than internal cooling coils. Carbon steel

construction, if feasible, would also reduce costs. Figure 6-2 shows the

resulting selling price for a range of levels of enzyme recovery. For the

case of no enzyme recovery, the selling price is reduced 19% to $1.73/gal at

25% of the base-case enzyme production cost. For this case, the enzyme production capital cost has been reduced from 43% to 15.7% of the total onsites

capital.

Figure 6-2 shows that the effect of the reduction of enzyme production capital

cost is reduced as enzyme production capacity is reduced, in this case by the

use of enzyme recycle. At 75% enzyme recycle, the selling price is reduced to

$1.45/gal at 25% enzyme production capital costs, but this is only a 7% reduction over the case of 75% enzyme reuse and base-case capital costs.

An increase in enzyme titre or a decrease in enzyme production batch time will

similarly reduce enzyme production costs. Figure 6-3 shows the selling price

of ethanol as a function of enzyme batch time. A 9.5-day cycle time reduces

the price to $1.95/gal for a decrease of 8.1%, and a 5.5-day cycle time

reduces the price to $1.78/gal for a decrease of 16.5i/gal. Capital cost for

both these cases is reduced from $20 million to $14.7 million and $9.1 million, respectively. Like Figure 6-2, Figure 6-3 shows that as enzyme reuse is

incorporated, the relative effect of a decrease in batch cycle time is

reduced.

Simulation runs were performed in which the enzyme loading was decreased from

25 FPU/g to 5 FPU/g of cellulose for various levels of enzyme recycle; the

result s are presented in Figure 6-4. In all these runs, the percentage of

glucan conversion was held at the base-case value of 80%. It is unlikely that

this level of cellulose conversion could be achieved at lower enzyme loadings

without other process modifications such as a more efficient pretreatment or a

large increase in hydrolysis residence time. These simulations merely illustrate the effect on costs produced by a reduction in enzyme loading alone.

When the enzyme load is reduced to 5 FPU/ g, the selling price is reduced by

29.4% to $1.50/gal at 0% enzyme reuse and by 35.8% to $1.36/gal at 75%

enzyme reuse. The major contributor to this reduction in price is the reduced

enzyme production capital and operating costs due to reduced enzyme production

capacity. A minor contribution is due to the reduction in feedstock cost and

pretreatment costs. At 25 FPU/g and no enzyme recycle, approximately 7% of

the feed is directed to enzyme production; but at 5 FPU/g and no enzyme

recycle, this is reduced to 1.5%.

The base-case design assumes 80% conversion of cellulose at 40-hours residence

time and base-case hydrolysis conditions. Variations in process parameters,

such as pretreatment, feedstock, substrate loading, and enzyme loading, may

alter the residence time required to make a particular conversion. The cost

effect of the hydrolysis residence time was determined at various cellulose

conversions.

35

TR-2093

At constant conversion, the selling

price is not greatly affected by

small reductions in residence

time. This is to be expected,

because changes in residence time

alone affect only hydrolysis capital

and operating costs, which are not

major contributors to the cost of

ethanol. Figure 6-5 shows that at

80% conversion, there is a reduction

of only 4.7% to $-2.03/gal at

12 hours. As the residence time for

80% conversion is increased by

roughly a factor of 3 to 125 hours,

the increase in price is 14% to

$2.43/gal. This is due to an

increase in hydrolysis capital cost

to 20.7% of the onsites, for a total

of $11.2 million purchased price.

It must be noted that- the effect of

process improvements that would be

intended to reduce production costs

(e.g., enhanced cellulose conversion, lowered enzyme loading, less

expensive pretreatments, etc , ) may

be somewhat offset if kinetics

necessitated a significant increase

in hydrolysis residence time.

15 30

Enzyme loading (FPU/g)

130L--__L.-__l--__L.-_----''--_---'

5

200

Cii

rn

Q)

u

.~ 150

en

c

Q)

(J)

Figure 6-4. Ethanol Selling Price

versus Enzyme Loading

(per gram of cellulose) at Various

Levels of Enzyme

Recovery

Figure 6-6 illustrates the effect on selling price of the solids loading in

the hydrolysis section at various cellulose conversions. At constant conversion, an increase in substrate loading results in a decrease in selling price.

This is due to the smaller capacity of the hydrolysis, fermentation, and purification sections, which results from the processing of streams of greater

concentration. An increase in the base-case loading from 20 to 25 wt %

decreases the selling price by only 3.5% to $2.05/gal. At 2 wt % solids

loading and 80% cellulose conversions, the resulting sugar product stream concentration is high. Lower solids loading and conversions would have a more

pronounced effect on the selling price of ethanol due to fermentation and distillation costs of the more dilute streams.

Some interactions between conversion and solids loading are illustrated in

Figure 6-6. For example, in order to achieve the base-case cost of

$2.13/ gal, the solids loading could be as low as 13 wt % if at the same time

(perhaps due to removal of product inhibitions) the conversion is increased to

90%.

Figure 6-7 shows results of simulations in which the cellulose conversion was

varied from 60% to 100% at a variety of enzyme loads. For 25 FPU/g, the selling price varies from $2.58/gal at 60% conversion, to $1.84/gal at 100% conversion. Total plant capital costs decrease from $240 million to $165.7 million over this range of conversions, because of the change in capacity

37
S=~II.I TR-2093 ---------------------------------=:.:........;=''''-=-

Residence time (h)

10 20

Wt % solids load

\

\\

\~'e \ , <, \ ......... . \ ."....... 60% conversion .... . ............_._---e • ...... __e1Q% conversion

... ' ......_.. 80% conversion -.

----.-----.. 90% conversion

150 '--__L...-__L- L...- L...-_---J'--_~

o

200

400

450,..------------------__

:::J

300

'§.

C>

.s

Q5

C/)

24 48 60 72 96 120

175 L...-.1-J::::::..---I__---!._...1----L__--L._-----'-_---'

o

200

.~225

Q)

CI)

275

ai

0'

-250

Q)

o

.~

Figure 6-5. Ethanol Selling Price

versus Hydrolysis Residence Time at Various Levels of Cellulose Conversion

Figure 6-6. Selling Price of

Ethanol versus

Hydrolysis Solids Load

at Various Levels of

Cellulose Conversion

Figure 6-8 shows the results of simulations in which the steam explosion capital cost and steam

requirements were varied between 0%

and 100% of the base-case values.

As the costs associated with pretreatment decrease, so does the selling price. At 80% conversion, the

selling price reaches a, minimum of

$1.74/gal at zero steam explosion

required to produce 50 x 106 gal/yr

of ethanol. Utilities costs increase from 16.5i to 23.0i/gal, due

to less residual biomass available

for plant steam production. Aspen

costs decrease from 59.5i to

37.8i/gal. An increase in the basecase conversion from 80% to 90% produces only a 15.8i/gal decrease in

selling price. The aspen costs decrease 10.2% from 46.3i to

41.6<!/gal.

80 90 100

% conversion

Ethanol Selling Price

versus Cellulose Conversion at Various

Levels of Enzyme

Loading (FPU/g

cellulose)

----""'G...

---

Figure 6-7.

130L...-_...1-_--L._----J'--_...L.-_..........._----J'--_""""--_-»'

60 70

150

ai

Ol

Q)

.g 200 COl

:§

Q5

CI)

38
S=~I i;.' --------------~-·---------- _____"'~~LL:L TR-2093

ro

OJ

0)

o

OJ

C

Q)

(J)

200

..... E 1Il~

o 0) 0 ..... O(J)

C§. 0 OC§.

00

r- L{)

E

- 0) Ill ..... DC/)

U D

DC§. C§.o

00

L{) rE - CO III D_0)

OC/)

E

CO

0)

~U5

O-g.

-R 0 D 0 0...-

E

CO -0) Ill ..... O(J)

o 0

DC§. C§.o

OU')

E -CO III 0) 0 ..... O(fJ

170 '-----'------------L--------.L.- ____l._ _L__---.J

3 5

Run number

7 9

Figure 6-8. Ethanol Selling Price versus Various Levels of Base-Case Steam,

Explosion Steam, and Capital Costs

costs. At only 50% reduction in both capital and steam usage, the price is

reduced by 9.2% to $1.93!gal.

The base-case cost of $2.13!gal can be achieved where the costs due to pretreatment are practically zero (case 8, 0% capital and 50% steam usage) if 60%

conversion can be attained. Reported conversions for milled, unpretreated

biomass are below 60%, which illustrates the need for some form of pretreatment to enhance enzymatic susceptibility.

Substitution of steam explosion by a less costly pretreatment, or a redefinition of the steam explosion equipment and steam requirements, may decrease the

ethanol selling price. However, as seen from Figure 6-8, enzymatic susceptibility is also a major factor to be considered in the pretreatment design.

When 90% of the available soluble xylose is "magically" converted to 5/6 its

weight of glucose prior to fermentation, its selling price is reduced 17.2% to

$1.76/gal. This simulation approximates a system in which an organism is

available that can ferment both xylose and glucose with kinetics similar to

that of glucose. Utilization of xylose enhances the feedstock utilization and

decreases overall plant size. This results in the following reductions over

the base case:

•

•

•

•

From 61.7 to 48.7 lb aspen wood/gal ethanol

From 46.3~ to 36.5¢:/gal aspen cost

From 66.2r/. to 55.6r/./gal depreciation

From $193.6 million to $164 million total plant capital cost.

39
S=~II.I ------------------~~ TR-2093

6.2.3 Summary of Individual Parameter Variations

To evaluate the relative merits of individual process improvements, results of

select simulations are tabulated, representing 11 process designs. The basecase design is included as the first design for comparison. The next seven

designs represent the following individual changes from the base case:

• Inclusion of 60% enzyme reuse

• Fermentation of xylose

• Enzyme load decrease from 25 to 15 FPU/g

• Cellulose conversion increase from 80% to 90%

• Increase in hydrolysis solids loading from 20 to 25 wt %

• Reduction in pretreatment capital and utilities by 50%

• Reduction of enzyme production capital cost by 50%.

The final three designs represent combinations of these cases; the ninth

design includes the first five improvements listed above, the tenth also

includes 50% reduction in pretreatment costs, and the eleventh includes all

seven of the improvements.

Selection of these seven improvements was based on the potential feasibility

of implementation. Recent studies have indicated greater than 60% enzyme

reuse may be possible with countercurrent adsorption (Orinchowskyj 1982).

Currently much research is being undertaken to provide organisms that can ferment glucose and xylose. Many hydrolysis experiments have been performed with

enzyme loadings at less than 25 FPU/g of cellulose (see Appendix B). Recent

results have indicated that conversions of greater than 90% may be achieved

with steam-exploded biomass (Blanch 1983). The cost of steam explosion is

speculative and further pretreatment process research may reduce costs. Some

pilot-plant data have indicated that enzyme production may be carried out in

simpler, less expensive equipment. Finally, at high cellulose conversions,

hydrolysis solids loading greater than 20 wt % may be possible with a staged

method of feed introduction.

Reduct ions in hydrolysis residence time were not considered here. The residence time will be dependent on other, more cost-controlling parameters, such

as enzyme load, pretreatment, desired conversion, and feedstock. As the

change in selling price due to change in residence time is significant only at

high residence times (as shown in Figure 6-5), residence time was considered

of secondary importance to these other parameters.

Table 6-4 summarizes the cost components for each of the eleven cases. Columns 1 through 5 add up to the values in coLumn 6, the cash cost of production. Column 8, the net cost of production, is the sum of depreciation, in

column 7, and column 6. The selling price at 15% ROI in column 9 is calculated from column 8 by an algorithm in the Chern Systems simulation model.

Column 10 shows the percentage decrease in selling price from the base-case

price. Increasing the hydrolysis solids loading to 20 wt % only reduces the

price 3.5% to $2.05/gal. The greatest single contribution to price reduction

is seen to be addition of 60% enzyme recycle, which contributes a 21.2% reduction. Xylose utilization contributes 17.2% reduction.

40
Table 6-4. Cost Components (I./gal)

Reduction

Raw Operating By-Product Cash Cost of Net Cost of Selling Price in Selling

Utilities Overhead Depreciation from Materials Cost Credit Production Production at15% ROI Base-Case

Cost (%)

1. Base case 61.7 20.6 19.6 20.0 -24.5 97.4 66.2 163.5 212.6

2. 60% enzyme recycle 54.4 15.5 15.3 15.9 -22.5 78.6 50.2 128.8 167.4 21.2

3. Xylose utilization 50.1 19.4 16.8 17.3 -23.9 79.7 55.6 135.4 176.0 17.2

4• 15 FPU/g enzyme ...... loading 56.8 17.1 16.7 17.2 -23.2 84.6 55.3 139.8 181. 8 14.5

5. 90% cellulose

conversion 56.1 21.9 18.2 18.6 -24.2 90.6 ·60.8 151.4 196.8 7.4

6. 25 wt % solids load 62.1 16.9 19.3 19.6 -24.5 93.4 64.3 157.8 205.1 3.5

7. 50% reduction of

steam explosion 61.7 17.7 17.6 17.9 -24.4 90.5 58.1 148.6 193.1 9.2

cost

8. 50% reduction of

enzyme production

cost 61.7 20.6 15.9 16.5 -24.4 90.2 52.8 143.0 185.9 12.6

9. Combined cases 2-6 40.1 13.2 11.5 12.1 -21.9 55.0 35.5 90.5 117.6 44.7

10. Combined cases 2-7 40.1 11.2 10.2 10.8 -22.0 50.3 30.0 80.2 104.3 50.9

11. Combined cases 2-8 40.1 11.2 9.4 10.1 -22.0 48.8 27.3 76.1 99.0 53.4

::cl

I

N

o

'-D

LA.>
TR-2093 S=~II~.;; ~~~---------------

The three combined-parameter cases show a decrease in selling price of 44.7%,

50.9%, and 53.4%, for selling prices of $1.18, $1.04 and $0.99/gal, respectively. All three cases represent significant cost reductions over the base

case. The selling prices of column 9 are shown in bar chart form in

Figure 6-9.

Table 6-5 provides a breakdown of the cents-per-gallon contribution to depreciation of the capital cost components for these 11 cases. Recycle of enzymes

significantly reduces enzyme production capital (from 22.1/ to 8.9//ga1),

slightly reduces the steam explosion capital, and produces the greatest

200

co

Ql

Ql

.g 150 c,

Ol

,§

(j)

en

100

2 3 4 5 6 7

Run number

8 2-6 2.-7 2-8

Key

1. Base case

2. 60% enzyme recycle

3. Xylose utilization

4. 15 FPU/g enzyme load

5. 90% cellulose conversion

6. 25 wt % solids

7. 50% reduction in steam

explosion costs

8. 50% reduction in enzyme production costs

9. Combined cases 2· 6

10. Combined cases 2 - 7

11. Combined cases 2 - 8

Figure 6-9. Effect of Base-ease Process Improvements on Selling Price of

Ethanol

42
Table 6-5. Contribution to Depreciation of Capital Cost Components (l./ga1)

Steam Enzyme Heat CO2 Total Total Total Capital

Explosion Production Hydrolysis Fermentation Purification Generation Recovery Onsites Offsites Depreciation

1. Base case 18.3 22.1 4.2 2.9 2.6 1.7 3.1 54.9 11.3 66.2

2. 60% enzyme recycle 17.1 8.9 4.1 2.8 2.5 1.6 3.1 40.7 9.6 50.2

3. Xylose utilization 14.6 17.7 3.6 2.7 2.5 1.4 3.1 45.5 10.1 55.6

4. 15 FPU/g enzyme loading 17.6 13.3 4.2 2.8 2.5 1.6 3.1 45.2 10.1 55.3

+:--

VJ 5. 90% cellulose conversion 16.6 19.9 3.9 2.8 2.5 1.3 3.1 50.1 10.7 60.8

6. 25 wt % solids load 18.5 22.1 3.4 2.7 2.5 1.7 3.1 54.0 10.4 64.4

7. 50% reduction of steam

explosion cost 9.7 23.4 4.5 3.1 2.7 1.8 3.1 48.2 9.9 58.1

8. 50% reduction of

enzyme production cost 17.7 10.7 4.1 2.8 2.5 1.6 3.1 42.5 10.3 52.8

9. Combined cases 2-6 above 12.1 4.2 2.5 2.3 2.3 1.1 3.1 28.0 7.5 35.5

10. Combined cases 2-7 6.5 4.5 2.7 2.5 2.5 1.2 3.1 23.4 6.6 30.0

11. Combined caSes 2-8 6.4 2.2 2.6 2.5 2.4 1.1 3.1 20.9 6.4 27.3

III

III

N

-
S=~I TR-2093 (;*'i ----~-----.----------...=.=.......:=..:::...=..::.

-~ '.

reduction in depreciation for the 7 individual cases (from

50. 2ilgal, a 24% reduction). The combined cases reduce the

components 46.4%, 54.7%, and 58.8%, respectively, for a cost of

and 27.3ilgal--once again, a very significant reduction.

66.2tlgal to

capital cost

35.5i, 30.0i,

This summary shows that process enhancements that would most reduce ethanol

price include enzyme reuse, xylose fermentation, and reduction of enzyme loading. The other parameters reduce the price by 3.5% to 12.6%. No single

parameter has been shown to reduce the price to the $l/gal range, but as shown

by the three combined cases, this price range can be achieved with a combination of the process improvements.

Figure 6-10 shows the implementation of the seven process improvements in a

stepwise fashion. The two curves differ only in the order in which the

improvements are implemented. Curve 1 includes xylose fermentation immediately after enzyme recycle, but curve 2 includes xylose fermentation as the

last improvement. In both cases, the same final selling price of 99i/gal is

reached.

The cents-per-gallon reduction due to a specific improvement depends on the

state of the process prior to its implementation, as seen with the case of

xylose fermentation. In curve 1, xylose fermentation causes a 29.0ilgal

reduction ($1.67 to $1.38/gal) but in curve 2, only 14ilgal ($1.14/gal to

99ilgal).

KEY

Run no. Curve 1 Curve 2 -

200 1. Base case Base case

2. Plus enzyme Plus enzyme

recovery (60%) recovery (60%)

3. Plus xylose Plus reduction in

(ij utilization enzyme load (15 FPUI

Ol gram)

Ql 4. Plus reduction in Plus increased 0 enzyme load cellulose con- '§'

Ol 150 (15 FPU/gram) version (90%)

§ 5. Plus increased eel- Plus increased hydrol- Q)

(/) lulose conversion ysis load (25 wt. %)

(90%)

6. Plus increased hydrol- Plus 50% reducysis load (25 wt. %) tion in steam explosion costs

7. Plus 50% reduc- Plus 50% reduc100 tion in steam explo- tion in enzyme prosion costs duction capital

90 8. Plus 50% reduc- Plus xylose utili2 3 4 5 6 7 8 tion in enzyme pro- zation

Run number duction

Figure 6-10. Ethanol Selling Price versus Two Scenarios of Process

Improvement Implementations

44
TR-2093

Figure 6-11 has been included to show that in addition to the major cost

reductions provided by enzyme reuse, xylose fermentation, and decreased enzyme

load, many smaller reductions can additively cause a significant reduction in

price. The simulation shown in this figure also includes reduction of the

hydrolysis residence time and reduction of enzyme load to 10 FPU/g, to provide

an ethanol price of 96~/gal.

Figure 6-12 shows the cost contributions, except for by-product credit, for

the simulations of curve 1 of Figure 6-10. Costs due to depreciation decrease

by the greatest amount. Raw materials costs change significantly with the

200

C'l

Q)

o

'§' 150

C'l .'=

CD

en

100

90

2 3 4 5 6 7 8 9

Run number

10 11 12 13 14

Key

Run No.

1.

2.

3.

4.

5.

6.

7.

Improvement

Base-case

60% enzyme reuse

Enzyme load reduction to 15 FPU/g

75% of base-case steam explosion capital cost

50% of base-case steam explosion capital cost

75% of base-case enzyme production capital

cost

50% of base-case enzyme production capital

cost

Run No.

8.

9.

10.

11.

12.

13.

14.

Improvement

25 wt % substrate loading

24 hours hydrolysis residence time

85% cellulose conversion

90% cellulose conversion

75% of base-case steam explosion utilities

Xylose utilization

Enzyme load reduction to 10 FPU/g

Figure 6-11. Ethanol Selling Price versus Process Improvement Implementation

45
S=~I !;-II-----------------~~~ TR-2093

- .

xylose fermentation, enzyme reuse,

and increased conversion. The other

cost components have a less

significant but decreasing trend as

each improvement is implemented.

Utilities increase as conversion is

increased due to a decrease in

available residual biomass for

onsite steam production.

6.2.4 Selling Price versus

Substrate Cost

Key

___Raw material costs

_ .. _ .._ Depreciation cost

__ - - -_ Utilities costs

_ . . _ Operating costs

___ Overhead costs

--,

-I

I

';-1

50 ----I

70,....-----------------, 0'

60

5

Run number

Figure 6-12. Cost Components for

Cases of Curve 1,

Figure 6-10.

The base-case process assumes a

feedstock of chipped aspen ~ood at a

cost of 7si/wet lb (50% moisture) or

$30/dry ton. Changes in collection

radius or changes in the physical

size reduction requirements for the

process pretreatment step may alter

this price. Research may be able to

provide a less costly feedstock, for

example, through reduction in growth

time.

10

<Il 20 C/)

To study the effect of feedstock cost on the selling price of ethanol, simulations were performed in which the feedstock cost was varied from 01:. to

1.s~/ wet lb for four process designs. These designs are:

1. Base-case design

2. 60% conversion plus increase in cellulose conversion to 90% plus

decrease in enzyme requirement to 15 FPU/g of cellulose

3. Case 2 plus xylose fermentation

4. Case 3 plus 50% reduction in steam explosion costs and enzyme production

section capital plus increased hydrolysis solids loading to 25 wt %

Figure 6-13 shows the selling price versus the substrate cost for these four

designs. At zero feedstock cost, the base-case selling price is $1.52/gal.

From these simulations, the required feedstock cost to produce a desired selling price can be determined for each of the designs. For example, at a target

price of $1.0/gal, the feedstock cost would need to be 0.099, 0.318, and

o. 768~/ wet lb ($4.0, $12.7, and $30.7/dry ton) for designs 2, 3, and 4,

respectively.

46
_'IIIIIt I•.

.~Ii---- TR-2093

300 .----------------------,

0.5 1.0 1.5

Aspen wood cost (¢/lb)

50 ~..L....__..L-_...L_ ..o.___L._ ...I

o

z» 200 CIl

OJ

Q)

o

.~

OJ

c::

100

Key

Base case

Alternate case 1

Alternate case 2

Alternate case 3

Figure 6-13. Ethanol Selling Price

versus Aspen Wood

Cost (wet basis).

See text for explanation of alternative

cases.

47
S=~II.I --------------------------.==.:..-:=..:~ TR-2093

SECTION 7.0

DISCUSSION

After careful appraisal of all the assumptions that went into the development

of the base-case flowsheet, the current research results that determined these

assumptions, and the results of the parametric analysis, it is apparent that

future research should follow two major pathways. First, verification on a

larger scale of the base-case process verificat ion is needed both to demonstrate that the unit operations of enzyme hydrolysis, enzyme production, and

enzyme recycle will perform as anticipated on a larger-than-Iaboratory scale

as well as determine what problems may occur during the operation of the integrated process. Second, basic laboratory research is needed to improve the

base-case unit operations in order to improve process economics as demonstrated by the parametric analysis.

7.1 BASE--eASE PROCESS VERIFICATION

The following issues are among those that need to be addressed to verify the

base-case economics.

Agitator power requirements: The power requirements for agitation of the cellulosic slurries have been estimated according to procedures by Chemineer

Inc. These requirements should be verified for the highly viscous slurries to

be encountered in the enzyme hydrolysis, enzyme production, and enzyme recycle

sections. In the enzyme production section, due to the metabolic requirements

of the fungi, oxygen transfer limitations from the gas to liquid phases may

dictate that the power requirements be greater by as much as an order of magnitude (Maiorella 1983).

Materials handling: Rotary wash filters are included in the flowsheet for

cellulosic separation in the enzyme production, enzyme hydrolysis, and enzyme

recycle sections. It is assumed that 70% moisture could be attained in the

filter cake. The filtration properties of cellulose and cellulose plus

mycelia slurries may alter the handling requirements and should be evaluated

in terms of equipment and performance on a larger scale.

Enzyme product ion: The ability to produce an enzyme titre of 30 FPU/mL in

13.5 days batch cycle time needs to be verified with the use of the process

cellulosic feedstock as carbon source. This performance has been obtained in

the laboratory with a fed-batch system using the pure cellulosic carbon

source, Solka Floc. Preliminary investigations indicate similar results may

be obtained with water-washed steam-exploded substrates (Sciamanna 1983).

Fed-batch enzyme production also needs to be evaluated on a larger scale. In

particular, due to the high capital cost of equipment in the base-case enzyme

product ion sect ion, studies in fed-bat ch enzyme product ion need to be performed using less expensive equipment, i.e., carbon steel, atmospheric vessels

with external heat exchangers. If atmospheric equipment is to be used, the

adequacy of chemical sterilization will need to be determined.

48
S=~II.' ----------------------------~---~~~~ TR-2093

Steam explosion: Due to proprietary information, the exact steam requirements

for steam explosion are not readily apparent. Process development studies

will determine the minimum requirements with inclusion of the maximum feasible

heat reuse. A recent study reports inhibition of the cellulase enzymes by a

toxin present in steam-exploded biomass (Sinitsyn 1982). Water washing of the

substrate after steam explosion removed the inhibition. The effects of steam

explosion on the hydrolysis of biomass and the subsequent fermentation of soluble sugars should be assessed because it may be necessary to water wash the

steam-exploded feedstock prior to hydrolysis as well as enzyme production, and

to acclimate the yeast to whatever toxins are present.

Enzyme hydrolysis: The base-case process assumes that 80% conversion of the

cellulose can be achieved in 48 hours with an enzyme load of 25 FPU/g of cellulose, at a substrate loading of 20 wt % (with staged-cascaded feed). This

needs to be verified on a larger scale due to potential rheological problems

encountered with viscous cellulosic slurries and due to potential problems

with enzyme inactivation due to product inhibition. Researchers at the University of California at Berkeley laboratories have indicated that cellulase

prepared from the RUT-C30 organism contains high levels of the ~-glucosidase

component (Tangnu 1981). Recent conversations with some Berkeley laboratory

staff members have indicated that this ~-glucosidase component may have a very

short half-life (Sciamanna 1983). In an industrial-scale operation,

~-glucosidase enzyme may need to be supplemented from another source. This

may be particularly the case when enzyme recycle via adsorption is incorporated, as studies have shown little adsorption of the ~-glucosidase component

on cellulosics (Orinchowskyj 1982).

Enzyme recovery: Operating conditions for enzyme hydrolysis may vary

depending on the level of incorporation of enzyme recycle via adsorption.

This is because the various cellulase components adsorb to different extents

on cellulose. Fresh enzyme broth would thus have a different component composition than the equivalent broth produced from, for example, 40% fresh

makeup enzyme and 60% recycled enzyme via adsorption (based upon filter paper

activity). This may cause a significant variation in hydrolysis results.

Larger scale simulations of the integrated process, with and without enzyme

recycle, should be performed to determine any changes in operational

performance.

7.2 BASIe LABORATORY RESEARCH

The following are recommendat ions for further research to improve the basecase process economics, based on conclusions drawn from the parametric analysis.

Of primary importance is the need for a correlation of cellulose conversion

versus pretreatment, feedstock, and hydrolysis operating conditions. Many

attempts to model or correlate the enzymatic hydrolysis reaction can be found

in the literature, but to date none of the models considered all the major

parameters or they have been derived from experiments performed at operating

conditions too far removed from that required for an economical process. A

simple correlation would facilitate more useful process modeling by enabling a

prediction of conversion efficiencies, and thus process economics, for

49
S=~II.I --------------------------------~:......=:~:...::!...- TR-2093

variations in the base-case process. A more detailed mechanistic model that

would describe the microscopic events of enzyme hydrolysis could, in addition,

define research pathways toward the goal of process improvement.

Research directed toward reduction in enzyme production costs would be of

major benefit to process economics. Fundamental research on strain improvement of Trichoderma reesei should continue to allow production of cellulase in

a less costly manner. Alternate fungal and bacterial strains should also be

considered, because T. reesei may not be the optimal cellulase source.

Process research and engineering of the enzyme production section may lead to

a decrease in the high capital and operating costs.

In addition to further process studies on the recycle of cellulase via adsorption onto incoming cellulosics, other methods of enzyme recycle should be

researched that may allow a greater percentage recovery and an equal recovery

for each of the cellulase components. Ultrafiltration equipment is continually decreasing in price and could provide an alternate means of cellulase

recycle. One study indicates that enzyme adsorbed onto residual solids may be

recovered with washing techniques (Sinitsyn 1983).

A major improvement would be the development of organisms that have the

ability to ferment five-carbon as well as six-carbon sugars to ethanol. In

this manner, substrate utilization would be increased and the plant capacity

of the front end (steam explosion through hydrolysis) would be decreased due

to the production of additional ethanol via fermentation of the xylose.

Further fundamental and process research on pretreatment is required. Pretreatments that allow high conversion efficiencies in enzymatic hydrolysis

have been identified, but processing costs are. high. Research should be

directed toward the development of new, cost-effective pretreatments as well

as toward cost reduction of current methods of pretreatment.

Lignin has the potential to be a valuable by-product. Research is required to

identify methods of lignin recovery and better define its uses. Removal of

lignins prior to hydrolysis may have the following advantages: (a) separation

of the potentially valuable lignin by-product from cellulose; (b) removal of

the inert lignin from the hydrolysis slurry, which at high substrate loadings

would improve hydrolysis agitation; and (c) removal of lignins as a potential

site for cellulase adsorption.

Removal of lignins prior to hydrolysis may have another benefit. An effective

pretreatment such as steam explosion may render the cellulose totally accessible to enzymatic hydrolysis within a reasonable reaction time. Therefore,

limits on conversion percentage may be due to product inhibition rather than

slow reaction rates caused by inaccessible regions of cellulose. If this were

the case and the lignins were removed, cellulose conversion may be enhanced

with some form of cellulose recycle. Cellulose recycle may also aid in enzyme

recycle by allowing the reuse of the enzyme adsorbed to the recycled

cellulose.

One study indicated that higher levels of effective cellulase activity were

obtained when whole culture broth from enzyme production was used rather than

culture filtrate alone (University of Arkansas 1982). Most studies that form

50
S=~II.i -...::..::...:e..-....:.....;..~ TR-2093

the basis for the assumptions of the base-case process presented here were

performed with culture filtrate. Use of whole culture broth should be studied

because it may enhance the levels of cellulase activity obtained per batch as

well as simplify the enzyme production section by removal of the filtrationwash step.

Finally, research should continue in alternative enzyme hydrolysis processes. Due to the combining of processing steps, the simultaneous saccharification and fermentation (SSF) process and the direct conversion process have

great potential toward reduction of costs and process complexity. Continuous

fermentation of sugars to ethanol as they are formed also would relieve inhibition of cellulase by glucose.

51
S=~II.I --------------------------=.:::........=.~ TR-2093

SECTION 8.0

CONCLUSIONS

A base-case selling price of etganol of $2.13/gal has been demonstrated by the

simulation model for a 50 x 10 gal/yr plant. This price is not competitive

with the current market price for ethanol production from ethylene or starch

and sugar crops, or via a proposed acid hydrolysis process (Wright 1982).

However, the parametric analysis of this study indicates that significant

reductions to this price can be attained through process research and

engineering.

Due to the degree of uncertainty of the process performance for the proposed

operating conditions at an industrial scale (as described in Section 7.0),

uncertainty in the feedstock costs, and lack of detail in the simulation model

for the back end process (of the fermentation, purification, and waste treatment sections), the actual costs per gallon of ethanol as determineq by this

simulation model should be considered only as approximations and as a method

for identifying research areas with significant impact on costs. However, the

results may be comparable with those of the acid hydrolysis process proposed

by Wright (1982). because the simulation models were similar with respect to

the back end of the plant and economic assumptions. In the referenced report,

the base-case cost for the high-temperature acid hydrolysis process of

$2.15/gal was reduced to $1.45/gal or as low as $1.10/gal when xylose fermentation was included. In both cases, . aspen wood cost $20/wet ton, and furfural was not recovered for by-product credit. The parametric analysis of the

current study of an enzyme hydrolysis process showed a potential decrease in

ethanol cost to as low as 99i/gal with a combination of process improvements

(Figure 6-10) at a feedstock cost of $15/wet ton. For comparison for aspen

wood priced at $20/wet. ton (Ii/wet lb) these improvements produce a decrease

in price to as low as $1.12/gal (see Figure 6-13).

The most significant cost reductions can be achieved through incorporation of

the enzyme recycle section for 60% enzyme recovery and fermentation of

xylose. Combination of these two improvements alone reduces the base-case

price to $1.40/gal (curve 1 of Figure 6-10). Reduction in enzyme loading per

gram of cellulose will also cause a significant price reduction if the cellulose conversion is unaffected and the required hydrolysis residence time is

not greatly increased. To a lesser but still significant degree, price reduction can be achieved with an increase in hydrolysis solids loading, an

increase in cellulose conversion, and a decrease in enzyme production and pretreatment capital and operating cost.

Ethanol price has the potential for further reduction by improvements not

included in the derivation of Figure 6-10. These include additional byproduct credits (lign1ns and furfurals formed during steam explosion), and

improvements in cellulase-producing organisms that may provide higher enzyme

titres, shorten batch production times, or remove the requirements for a cellulose carbon source.

In conclusion, the parametric analysis of this study points out key areas of

research that will significant ly improve the base-case process economics.

52
TR-2093 S=~II.*i1 -~ . -------------------------------~---­

Careful scrutiny of the simulation model and the process flowsheet also point

out key areas of research still to be performed that would verify the basecase process performance on an industrial scale. Most important is the demonstration of this analysis that this particular enzymatic hydrolysis process

may allow ethanol production at a price competitive with current ethanol

production technologies and with the acid hydrolysis cellulose-to-ethanol

process.

53
S=~II.I ----------------------~~~ TR-2093

SECTION 9.0

REFERENCES

Wenzl, H. F. J., 1970, The Chemical Technology of Wood, New York: Academic

Press.

Burr, H. K., and A. J. Stamm, 1947, "Diffusion in Wood," J. Phys. and Colloid

Chern., Vol. 51, p. 240.

Mandels, M., and D. D. Y. Ryu, 1980, Enzyme Microb. Technol., Vol. 2, p. 91.

Reese, E. T., R. G. H. Siu, and H. S. J. Levinson, 1950, J. Bacterial.,

Vol. 59, p, 485 •

Montencourt, B. S., and D. E. Eveleigh, 1977, Appl. Environ. Microbiol.,

Vol. 34, p, 777.

Emert, G. H., and R. Katzen, 1979, "Chemicals from Biomass by Improved Enzyme

Technology," presented at the ACS/CSJ Joint Chemical Congress, April 1-6,

Honolulu, HI.

University of Arkansas, 1982, Develop and Demonstrate the Cellulose Process,

Subcontract No. XR-O-9248-1, Fayetteville, AR: University of Arkansas.

Jenkins, D. M., and T. S. Reddy, June 1979, Economic Evaluat ion of the MIT

Process for Manufacture of Ethanol, Columbus, OH: Battelle Columbus

Laboratories.

Brooks, R., T. Su , M. Brennan, and J. Frick, 1979, "Bioconversion of Plant

Biomass to Ethanol," Third Annual Biomass Energy Systems Conference Proceedings, Golden, co: Solar Energy Research Institute. Conference held on

June 5-7, 1979.

Fan, L. T., Y. H. Lee,

Engineering, Vol. 14,

p, 101.

and D. H. Beardmore, 1980a, Advances in Biochemical

edited by A. Fiechter, Berlin: Springer-Verlag,

Fan, L. T., Y. H. Lee,

Engineering, Vol. 14,

p, Ill.

and D. H. Beardmore, 1980b, Advances in Biochemical

edited by A. Fiechter, Berlin: Springer-Verlag,

Orichowskyj, Stephen T., Charles R. Wilke, and Harvey W. Blanch, 1982, Recovery of Cellulase Enzymes by Countercurrent Adsorption, M.S. Thesis,

Berkeley, CA: University of California at· Berkeley. Also published as

Lawrence Berkeley Laboratory Report No. 15153.

Wright, J. D., 1982,

Fuel Production,

Institute.

of Cellulose for Alcohol

Solar Energy Research

54
TR-2093

Wald, S. A., H. W. Blanch, and C. R. Wilke, 1981, Enzymatic Hydrolysis of Rice

Straw for Ethanol Production, M.S. Thesis, Berkeley, CA: University of

California at Berkeley.

Maiorella, B. L., 1983, oral communicat ions, Department of Chemical Engineering' University of California at Berkeley, Berkeley, CA.

Sciamanna, A., 1983, personal communication.

Sinitsyn, A. P., L • S. Clesceri, and H. R. Bungay , 1982, "LnhLb i t Lon of Cellulases by Impurities in Steam-Exploded Wood, It Appl. Biochem. and Bf.o t ech; ,

Vol. 7, pp. 455-458.

Tangnu, S. K., H. W. Blanch, and C. R. Wilke, 1981, "Enhanced Production of

Cellulase, Hemicellulase, and ~-glucosidase by Trichoderma reesei

(RUT-C30)," Biotech. Bioeng., Vol. 23, No.8, p. 1837.

Kelsey, R. G., and R. Schafizadeh, 1980, Biotech. Bioeng., Vol. 23, p. 1025.

Chum, H. L., L. J. Douglas, D. A. Feinberg, H. A. Schroeder, forthcoming,

Evaluation of Pretreatments of Biomass for Enzymatic Hydrolysis of

Cellulose, SERI/TR-231-2183, Golden, co: Solar Energy Research Institute.

Chern Systems Tnc , , 1982a, Process Simulation of Ethanol from Cellulose via

Enzyme Hydrolysis, SERI Subcontract No .. XK-1-1034-1, Tarrytown, NY: Chern

Systems Inc.

Chern Systems Inc., 1982b, Process Design and Economics for Ethanol from Corn

Stover via Dilute Acid Hydrolysis, SERI/STR-231-17 45, Golden, co: Solar

Energy Research Institute.

Iotech Corporation, Apr. 1980, 0 timization of Steam Ex losion Pretreatment,

U.S. Department of Energy, DOE ET 23050-1.

Perez, Javier, C. R. Wilke, and H. W. Blanch, 1981, Enzymatic Hydrolysis of

Corn Stover Process Development and Evaluation, M.S. Thesis, Berkeley, CA:

University of California at Berkeley. Also published as Lawrence Berkeley

Laboratory Report No. 14223.

Blanch, H. W., 1983, personal communications.

Hendy, N., C. R. Wilke, and H. W. Blanche, 1981, "Enhanced Cellulase Production Using Solka Floc in a Fed-Batch Fermentation, II Biotechnology Letters,

Vol. 4, No. 12, pp , 785-788.

Wilke, C. R., B. Maiorella, A. Sciamanna, K. Tangnu, D. Wiley, and H. Wong,

June 1980, "Enzymatic Hydrolysis of Cellulose, Theory and Applications,"

submitted to the Workshop on the Cooperative Research Project of Food

Production, Preservation, and Conversion of Lignocellulosic Waste Products,

Paris, France. Also published as Lawrence Berkeley Report No. 13669.

SRI International, October 1984, itA Biomass Opportunities Program," Project

No. 1134, Final Report.

55
S=~II.I---------------- ----b~~ TR-2093

APPENDIX A

ALTERNATIVE ENZYMATIC HYDROLYSIS PROCESSES

It is beyond the scope of this report to include a parametric analysis and

evaluation of alternative enzymatic cellulose-to-ethanol processes. Many of

these alternatives are just in the early stages of laboratory research. However, they have the potential to provide significant cost reduct ion, so a

brief discussion of the flowsheets is provided below.

Enzymatic hydrolysis processes can be grouped into three classes. The first

class, which was evaluated in this report, is based on separate hydrolysis and

fermentation stages. The next class has often been referred to as simultaneous saccharification and fermentation (SSF) and combines hydrolysis and

fermentation in one vessel. This is accomplished by introduction of a glucose-fermenting organism into the hydrolysis vessel along with the biomass

feed and cellulase. The final class, referred to as direct conversion processes, combines all three process steps of enzyme production, hydrolysis,

and fermentation in one vessel.

SSF processes have the advantage of reducing equipment costs due to the combination of two processing steps. The required degree of asepsis in the

hydrolysis reaction is reduced due to the presence of ethanol and anaerobic

conditions of yeast fermentation. Hydrolysis rates, sugar yields, and ethanol

yields are potentially higher due to continuous removal of the glucose and

cellobiose inhibition of the cellulase enzymes. A major disadvantage of the

SSF concept is that the operating conditions must be a compromise between

those .for cellulase enzymes and those for the fermenting organisms. For

example, fungal cellulases have a temperature optimum between 45 0 and 50 0C,

but Saccharomyces cerevisiae optimally operates at 30 0C.

An SSF process was developed by Gulf Oil and Chemical Company and later transferred to the University of Arkansas (Emert 1980). The basic steps of this

process are shown in Figure A-I. A mutant strain of Trichoderma reesei is

used as the cellulase source. The enzyme is added to the hydrolysis vessels

as whole culture broth, with no concentration or filtration, along with the

pretreated biomass. Yeast (Saccharomyces cerevisiae and Candida brassicae are

the preferred yeasts) is added either as a cake or recycled as a cream.

Direct conversion processes further simplify process design by combining the

enzyme production step with hydrolysis and fermentation. This is achieved

with use of an organism or compatible mixture of organisms that will grow on

cellulose and convert it to sugars and ethanol. Current problems associated

with direct conversion are that growth and reaction rates are relatively low,

and significant amounts of by-products may be produced at the expense of

ethanol.

Figure A-2 shows the Battelle design of the MIT direct conversion process. A

mixed culture of Clostridium thermocellum and Clostridium thermosaccharolyticum is used. ~. thermocellum cellulase hydrolyzes both cellulose to glucose and hemicellulose to xylose. This organism also ferments glucose to

ethanol but cannot utilize xylose. C. thermosaccharolyticum is added to

56
S=~II.: ------.-------------------------=.~~~ TR-2093

ferment xylose and is environmentally and biologically compatible with

c. thermocellum.

More detailed descriptions can be found in the literature on direct conversion

processes developed by MIT (Jenkins 1979) and GE/CRD (Brooks 1979).

57

TR-2093

APPENDIX B

ENZYMATIC HYDROLYSIS EXAMPLES

To provide a background for the discussions and assumptions of this study, a

few examples of enzymatic hydrolysis are presented here. Many mechanistic

models of the action of cellulases on cellulose have been proposed, and an

excellent review is provided by Fan (1980a, 1980b). However, none of these

models encompass all the factors that may affect conversion. Some of the more

comprehensive models may not be applicable to the conditions of the base-case

process of this study. As an example, Chem Systems developed a complex empirical formula to describe the percentage of conversion for varying operating

conditions based on laboratory data using Dartmouth acid-pretreated biomass

and hydrolysis solids loadings under 10 wt %. This formula may not apply to

the current base-case process that employs steam explosion as pretreatment and

hydrolysis loading of 20 wt %.

At high specific enzyme activity

(63 FPU/g biomass or approximately

150 FPU/ g cellulose in Figure B-1),

enough ~-glucosidase is present in

the cellulase system of..!.. reesei to

convert the cellobiose to glucose at

long reaction times. Cellobiose

accumulates initially during the

period of high cellulose hydrolysis

rate. Figure B-2 shows an example

of the hydrolysis of steam-exploded

corn stover with low enzyme load

(10 FPU/g biomass, or approximately

25 FPU/g cellulose). Although cellobiose is consumed continuously

after the initial few hours, a significant amount remains after 48

hours. Glucose production proceeds

more slowly and does not appear to

have reached the maximum after 48

hours..

24 48

Time (h)

Example of Enzyme

Hydrolysis at High

Enzyme Loading (63

FPU/g solids). Substrate is steamexploded corn stover.

Source: Perez 1981

Xylose

OI------I--....:::::r=--u~-......-----_t..Jo_.J

o 8

Figure B-1.

40

Figure B-1 is an example of a typical hydrolysis of steam-exploded corn stover

using T. reesei cellulase. Glucose formation is extremely rapid in the initial stages of reaction due to the ease of hydrolysis of the amorphous cellulose. Glucose production steadily decreases as the remaining cellulose

becomes less accessible (more crystalline or shielded by lignin). At high

reaction times, glucose formation

may reach a maximum at less than

100% conversion due to deactivation

of the enzymes (thermal instability

or product inhibition) or due to inaccessibility of the remaining

cellulose..

:J'

§

c:

o

20

C

Q)

o

c:

o

o

59
S=~II.J ----------------------~~~ TR-2093

The interactions between enzyme

load, substrate load, and 48-hour

conversion are illustrated for acidtreated corn stover in Figure B-4.

As substrate load is increased, the

48-hour conversion decreases, possibly as a result of product

inhibit ion at the higher substrate

loadings. As the enzyme load is

decreased, this trend becomes more

significant. This can be explained

by product inhibition due to cellobiose accumulation. At lower

Figure B-3 shows the relationship

between the percentage of cellulose

conversion at 48 hours and the

enzyme load using steam-exploded

corn stover. Maximum conversion of

82% occurs at approximately

30-40 FPU/g. Additional enzyme will

not increase the 48-hour conversion

but would perhaps allow a reduction

in residence time. Increased

residence time may shift the maximum

to lower enzyme loads. .

Cellobiose

Xylose

00 24

Time (h)

Figure B-2. Hydrolysis of SteamExploded Corn Stover

at Low Specific Cellulase Activity

Source: Perez 1981

c:

o

~40 c:

Q)

(J

c:

o

o

60

80

60.--------------·---------.1"'---------~".--___.

c:

o

'§

Q)

>

c:

o

o

Solids (FPU/g)

Figure B-3. Glucose Conversion versus Specific Activity. Acid-treated corn

stover (Calif.), cellulase from RUT C30 (11/80)

Source: Perez 1981

60
S=~I TR-2093 !!li ~-----------------------------.,.------

enzyme loadings, more cellobiose will accumulate due to insufficient

~-glucosidase activity.

Figure B-4 shows the relationship between hydrolysis residence time, substrate

load, and enzyme load. At high enzyme load (circles), maximum conversions may

be reached at shorter residence times as substrate load is decreased. This is

illustrated by the fact that at 5 wt % substrate and 7 FPU/g enzyme, there is

no conversion increase between 24 and 48-hour residence time. As substrate

load increases, more time is required to reach the maximum conversion. As the

enzyme load is decreased, this trend becomes more significant.

An example of the effect of pretreatment can be seen in Figure B-5. The

source of this figure did not indicate the percentage of conversion at

24 hours, and the exact cellulose content of the steam-exploded and acidtreated corn stover was unknown. However, at 24 hours the steam-exploded corn

stover produced 1.8 times the glucose of the acid-pretreated corn ~tover.

The parametric analysis in Section 6.0 of this study indicated a significant

reduction in cost of ethanol production when 60% of the enzyme is recycled via

countercurrent adsorption. The hypothesis that at least 60% of the filter

paper activity can be recycled in this fashion is based on laboratory studies

carried out at the University of California at Berkeley (Orinchowskyj 1982).

16 20 24

DNS

Steam-exploded corn stover

8

5

Substrate (wt %)

60 .~

-

35

30

40

c

0

-(I}

Q) ::J' > 20 c

0

0 C/I

Cl>

C/I OJ 0

0 20 Hours :::J 15

24 48 FPU/mL C/)

CD

• 0 7

£ 6. 1

Figure B-4. Example of Interaction

of Substrate Loading

(FPU/mL), Reaction

Time, and Enzyme Loading on Hydrolysis of

Acid-Treated Corn

Stover

Source: Perez 1981

Figure B-5. Effect of Pretreatment

on Hydrolysis of Corn

Stover. Data for

acid-treated corn

stover from LBL report

LBL-14223.

Source: Orinchowskyj

1982

61
TR-2093 S=~II.I -----------------------------

Figure B-6 shows an example of the disappearance of cellulase activity from

solution when contacted with a 5 wt % solution of steam-exploded corn

stover. The majority of adsorption occurs within the first two minutes of

contact. The relative amounts of adsorption of the various cellulase

components are consistent with their affinity for the substrate;

~-glucosidase, which acts on the soluble cellobiose, adsorbs the least whereas

the C1 activity, which attacks the crystalline cellulose, adsorbs the most.

Filter paper activity (FPA) is an estimation of the combined cellulase

activities, and the amount of adsorpt ion is between that of the Cx and C1

activities.

At low cellulose conversions, the majority of this activity remains adsorbed

onto the solids and enzyme recycle first requires desorption. Washing with

wat er was shown to desorb the Cx act i vity but not C1 (Orinchowsky j 1982).

Desorption can be achieved with addition of urea, but an analysis of such a

process shows unfavorable economics due to the cost of urea (Wilke 1982).

At high cellulose conversions, however, such as those achieved with steamexploded biomass, a major portion of the enzyme activities is released. Figure B-7 indicates that after 24 hours approximately 80% of the original FPA is

present in solution. Theoretically, this activity can be readsorbed onto

fresh hydrolysis biomass feed and, in this manner, recycled back to the

hydrolysis vessel.

Langmuir isotherm constants can be determined from adsorption data such as

those shown in Figure B-6. These constants are specific for the particular

enzyme preparation, substrate, and pretreatment, and they describe the

adsorption of the enzyme according to the equation:

Eads

-Emax

KE

(1 + K) ,

where

maximum level of enzyme adsorption (units/g)

= enzyme adsorbed onto solids at equilibrium (units/g)

enzyme activity in solutuion (units/mL)

constant (mL/units).

Using the langmuir isotherm constants for steam-exploded corn stover of

K = 0.329 mL/FPU and Emax = 133 FPU/g, and assuming a conservative estimate

that 65% of the FPA available is in solution after hydrolysis, simulations of

a two-stage countercurrent adsorption reactor conducted at SERI indicates that

as much as 97% of this soluble FPA may be reads orbed onto the fresh hydrolysis

feed. Thus, in excess of 60% of the original FPA can be recycled and reused.

At large percentages of enzyme recycle via adsorption, the steady-state cellulase composition will differ from that of native cellulase due to the difference in affinity of the various components for the solid substrate, as shown

62
TR-2093 =~ i'*l:---------------,----- -~

100

in Figure B-6. For example, a mixture of 60% recycled enzyme and 40%

make-up enzyme (based on FPA) may

contain as little as 40% of the

original ~-glucosidase component.

This may have a pronounced effect on

the hydrolysis kinet ics due to the

inhibition caused by the accumulation of cellobiose. Figure B-3

shows the accumulation of cellobiose

at 48-hour hydrolysis time versus

the ~-glucosidase loading in international units (IU) per gram. Figure B-9 shows how the cellulose conversion at 24 hours is decreased as

the ratio of ~-glucosidase to cellulase is decreased for a range of

enzyme-to-substrate loadings. In

order to maintain a sufficient level

of ~-glucosidase in the hydrolysis

reactors, either a certain minimal

amount of fresh cellulase with sufficient ~-gluosidase activity must

be supplied, or ~-glucosidase must

be supplemented from another

source. In the study at the

FPA

20 30 50

Time (min)

Adsorption of Cellulase

Enzyme Activities onto

5 wt % Steam-Exploded

Corn Stover at 150 C and

Enzyme Load of 25 FPU/g

Solids

10

.------------------~ oc

:'.

-r

Figure B-6.

100

c 80 .Q

"0

C/)

c

2=' 60

'5

t5

l'll

""iii

c

'6:1 40 s

a

0

20

C,

FPA

Cellobiase

Enzymes during Hydrolysis

40 48

Ol------L-----.L----~ ..L__ L.___ ___l

o 8 16 24 32

Time (h)

Adsorption and Desorption of Cellulase

of Steam-Exploded Corn Stover

Source: Orinchowskyj 1982.

Figure B-7.

63
S=~II.I-__------------------- TR-2093

10 0

::::J 8 E!

c:

0

.~

6 C

Q)

0

c:

0

0

(I)

en

0

:0

.9

(jj

o 2

60 r-----------------__.

e...

c: 40 0

·00

Q) Ratio of cellulase to cellobiase >

c:

0 A 1:1.1 0

Q)

en 0 1:1.9 0 20 0

:3 Reaction time: 24 h

a

0

0 20 40 60 85

Specific activity (IU/g)

=

Cellobiase (IU/g)

Figure B-8. Cellobiose Concentration Observed

after 48 hours of

Hydrolysis versus

Specific Cellobiase

(P-glucosidase)

Activity

Source: Perez 1981

Figure B-9. Glucose Production

versus Specific Cellulase Activity for Different Ratios of

Cellobiase

(p-Glucosidase) to

Cellulase

Source: Perez 1981

University of California at Berkeley, bench-scale simulation of a two-stage

countercurrent adsorption reactor and hydrolysis vessel indicated that a

steady-state level of ~-glucosidase could be obtained if make-up enzyme from

RUT C30 was at least 35%, based on FPA.

64
Document Control I' SERI Report No. [ 2. NTIS Accession No.

Page SERI/TR-231-2093 4 Title and Subtitle

Ethanol Production by Enzymatic Hydrolysis:

Parametric Analysis of a Base-Case Process

7. Authcrts)

Steven H. Isaacs

9. Performing Organization Name and Address

Solar Energy Research Institute

1617 Cole Boulevard

Golden, Colorado 80401

12. Sponsoring Organization Name and Address

15. Supplementary Notes

3. Recipient's Accession No.

5. Publicatton Date

May 1984

6.

8. Perlorming Organization Rept. No.

10. Project/Task/Work Unit No.

1372.35

11 Contract (C) or Grant (G) No.

(el

(G)

'3. Type 01 Report & Period Covered

Technical Report

14.

16. Abstract (Limit: 200 words)

A process design is presented for the production of 50 million gallons per year

of fuel ethanol from cellulose through enzymatic hydrolysis. The base-case

selling price for ethanol of $2.13/gal is estimated for this process utilizing

steam explosion pretreatment of aspen wood feedstock, fed-batch enzyme production

with Trichoderma reesei, separate hydrolysis and fermentation, and vacuum distillation. A parametric sensitivity analysis is performed with a computer simulation model to study the influence of selling price on key process parameters,

which include enzyme recycle, enzyme loading, residence time, cellulose conversion, xylose utilization, and feedstock use.

17. Document Analysis

a. Descriptors Cellulose; Economics ; Enzymatic Hydrolysis ; Enzymes ; Ethanol ;

Fermentation ; Flowsheets ; Glucose ; Parametric Analysis

b. identifiers/Open-Ended Terms

c. UC Categories

61a

'8. Availability Statement

National Technical Information Service

U.S. Department of Commerce

5285 Port Royal Road

Springfield, Virginia 22161

Form No. 0069 {3-25-82l

19. No. of Pages

76

20. Price

A05