United States Office of
Environmental Protection Research and
Agency Development
Municipal Environmental
Research Laboratory
Cincinnati, Ohio 45268
EPA-600/7-77-038
July 1977
PRETREATMENTS AND
SUBSTRATE EVALUATION FOR
THE ENZYMATIC HYDROLYSIS
OF CELLULOSIC WASTES
Interagency
Energy-Environment-
Research and Development
Program Report
-------
RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The nine series are.
1 Environmental Health Effects Research
2 Environmental Protection Technology
3 Ecological Research
4 Environmental Monitoring
5 Socioeconomic Environmental Studies
6 Scientific and Technical Assessment Reports (STAR)
7 Interagency Energy-Environment Research and Development
8. "Special" Reports
9, Miscellaneous Reports
This report has been assigned to the INTERAGENCY ENERGY-ENVIRONMENT
RESEARCH AND DEVELOPMENT series. Reports in this series result from the
effort funded under the 17-agency Federal Energy/Environment Research and
Development Program These studies relate to EPA's mission to protect the public
health and welfare from adverse effects of pollutants associated with energy sys-
tems. The goal of the Program is to assure the rapid development of domestic
energy supplies in an environmentally-compatible manner by providing the nec-
essary environmental data and control technology. Investigations include analy-
ses of the transport of energy-related pollutants and their health and ecological
effects, assessments of. and development of, control technologies for energy
systems, and integrated assessments of a wide range of energy-related environ-
mental issues
This document is available to the public through the National Technical Informa-
tion Service, Springfield. Virginia 22161
-------
EPA-600/7-77-038
July 1977
PRETREATMENTS AND SUBSTRATE EVALUATION FOR THE
ENZYMATIC HYDROLYSIS OF CELLULOSIC WASTES
by
Leo A. Spano
Thomas H. Tassinari
Charles F. Macy
Edward D. Black
Pollution Abatement Division
Food Sciences Laboratory
U.S. Army Natick Research and Development Command
Natick, Massachusetts 01760
Contract No. EPA-IAG-DS-0758
Project Officer
Charles J. Rogers
Solid and Hazardous Waste Research Division
Municipal Environmental Research Laboratory
Cincinnati, Ohio 45268
MUNICIPAL ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
-------
DISCLAIMER
This report has been reviewed by the Municipal Environmental Research
Laboratory, U.S. Environmental Protection Agency, and approved for publica-
tion. Approval does not signify that the contents necessarily reflect the
views and policies of the U.S. Environmental Protection Agency, nor does
mention of trade names or commercial products constitute endorsement or
recommendation for use.
11
-------
FOREWORD
The Environmental Protection Agency was created because of increasing
public and government concern about the dangers of pollution to the health
and welfare of the American people. Noxious air, foul water, and spoiled
land are tragic testimony to the deterioration of our natural environment.
The complexity of that environment and the interplay between its components
require a concentrated and integrated attack on the problem.
Research and development is that necessary first step in problem solu-
tion and it involves defining the problem, measuring its impact, and
searching for solutions. The Municipal Environmental Research Laboratory
develops new and improved technology and systems for the prevention, treat-
ment, and management of wastewater and solid and hazardous waste pollutant
discharges from municipal and community sources, for the preservation and
treatment of public drinking water supplies, and to minimize the adverse
economic, social, health, and aesthetic effects of pollution. This publica-
tion is one of the products of that research; a most vital communications
link between the researcher and the user community.
The objective of this study was to determine the applicability of
various pretreatment methods for conversion of cellulose to glucose and
other sugars. A combination of pretreatments, two-roll milling followed by
liquid ammonia, affected significant increases in the enzymatic hydrolysis
yield of glucose from wood shavings and hammer milled newspaper.
Francis T. Mayo, Director
Municipal Environmental Research
Laboratory
-------
ABSTRACT
Presented are initial studies aimed at determining the applicability of
various pretreatment methods and existing cellulosic wastes to a process for
the conversion of cellulose to glucose and other reducing sugars. In this
process, a cellulase from a mutant of Trichoderma yiride is used.
An extensive literature survey of cellulose pretreatment methods was
conducted followed by experimental work consisting of an evaluation of pulp
and paper mill primary sludge waste and investigation of four pretreatments,
viz., modified hydropulping, two-roll milling, attritor milling and anhydrous
liquid ammonia. Reference point for the sugar yield obtained for a
particular substrate is taken as the value for ball milled newspaper (BMNP)
hydrolyzed at the same time. Some primary sludge discharges from pulp and
paper mills, without further pretreatment, exhibit hydrolysis yields higher
than BMNP. Hydrolysis of hammer milled newspaper that had been treated in
a type of hydropulping mill resulted in saccharification yields that were
approximately 80% of those of BMNP. Preliminary results with an attritor
mill pretreatment of newspaper are sufficiently promising to warrant a more
extended investigation.
Pretreatment using differential speed two-roll mills has significantly
enhanced the susceptibility of diverse cellulosic substrates such as news-
paper, cotton, pine and maple. With processing times of less than ten
minutes on a six-inch (roll diameter) mill, hydrolysis yield improvements
were two and twelve-fold for newspaper and cotton respectively. Power
requirements for processing waste newspaper on a three-inch laboratory mill
are estimated to be 20% less than for a commercial ball mill. Anhydrous
liquid ammonia treatments of hardwood birch and maple sawdusts rendered them
more susceptible to enzymatic hydrolysis than substrates from a softwood
source; e.g., white pine and newspaper. A combination of pretreatments,
two-roll milling followed by liquid ammonia, affected significant increases
in hydrolysis yield for maplewood shavings and hammer milled newspaper.
This report is submitted in fulfillment of contract No. EPA-IAG-DS-0758
by U.S. Army Natick Research and Development Command under the sponsorship
of the U.S. Environmental Protection Agency. This report covers the period
16 July 1975 to 16 July 1976 and work was completed as of 4 August 1976.
IV
-------
CONTENTS
Foreword iii
Abstract iv
Figures vi
Tables vii
Acknowledgments viii
1. Introduction 1
2. Conclusions 2
3. Recommendations 4
4. Background Review 5
Properties of cellulose affected by pretreatment ... 5
Chemical pretreatments 10
Mechanical pretreatments 12
Irradiation 14
5. Experimental Results and Discussion 15
Evaluation of primary sludges 15
Modified hydropulping 18
Attritor milling 18
Two-roll milling 19
Anhydrous liquid ammonia 27
References 33
Appendices
A. Pulp and paper industry delignification processes .... 38
B. Pretreatment evaluation procedures 41
-------
FIGURES
Number Page
1 A Typical Differential Speed Two-Roll Mill .......... 21
2 Effect of Roll Clearance on Mean Relative Yield of Two-
Roll Milled Newspaper ......... 24
3 Effect of Processing Time on Mean Relative Yield of Two-
Roll Milled Newspaper .............. 24
4 Effect of Two-Roll Milling on Enzymatic Hydrolysis of
White Pine and Maplewood Sawdusts 25
5 Effect of Two-Roll Milling on Enzymatic Hydrolysis of
Cotton . 25
6 Effect of Liquid Ammonia Pretreatment on the Hydrolysis
of Avicel . . 29
7 Effect of Liquid Ammonia Pretreatment, Under Varying
Conditions of Temperature, Pressure and Time, on the
Hydrolysis of Yellow Birch Sawdust, 35 mesh ......... 29
8 Effect of Combined Two-Roll Milling and Liquid Ammonia
Pretreatments on the Hydrolysis of Maplewood ........ 30
9 Effect of Combined Two-Roll Milling and Liquid Ammonia
Pretreatments on the Hydrolysis of White Pine ........ 30
10 Effect of Liquid Ammonia Pretreatment on the Hydrolysis
of Hammer Milled Newspaper 32
11 Effect of Combined Two-Roll Milling and Liquid Ammonia
Pretreatments on the Hydrolysis of Hammer Milled
Newspaper 32
-------
TABLES
Number
1 Pulp and Paper Mill Effluent Suspended Solids, Ash Content
and Total Suspended Solids Reduction Effected by
Clarifier 16
2 Estimated Wood Pulp Production in the Major Pulp and Paper
Manufacturing Areas of the U.S. (1975) 16
3 Reducing Sugar Yields of Various Pulp and Paper Mill
Sludges Relative to Ball Milled Newspaper 17
4 Twenty-four Hour Relative Yields for Reiling Machine
Treated Newspaper 19
5 Power Consumption for Laboratory Two-Roll Milling of
Newspaper 26
6 Sedimentation Volume of Two-Roll Milled and Ball Milled
Newspaper 26
-------
ACKNOWLEDGMENTS
The authors wish to express their appreciation to Mr. Charles Rogers of
EPA for his support during this program.
Also, Mr. Angus Wilson and Mr. Patrick Mahoney are recognized for
providing invaluable assistance and the equipment used for the two-roll
milling portion of this study.
SP5 Pamidimakkala Vijayakumar and SP4 Joy Loehr, contributed substan-
tially by performing DNS reducing sugar analyses. In addition, the authors
are indebted to Mr. Philip Hall for assisting in a variety of technical and
administrative functions.
VI 11
-------
SECTION 1
INTRODUCTION
Cellulose, the world's most abundant organic compound, is a replenish-
able resource generated at an estimated annual rate of 100 billion tons.
This is equivalent to 150 Ibs/day for every man, woman, and child in the
World.
Ultimately, most of the cellulose utilized by man becomes part of the
solid waste stream. In the United States, significant amounts of cellulo-
sic materials are found in solid waste derived from industrial, agricul-
tural and municipal sources. Disposal of these cellulosic wastes in an
environmentally acceptable manner is a problem which must be dealt with.
Landfilling will only be practical in the short term as land for this
purpose becomes scarce. An inexpensive, non-polluting incinerator
technology has yet to be developed. The Natick Research and Development
Command (NARADCOM) Process for the Conversion of Cellulose to Glucose
offers a potential solution to this waste disposal problem.
In the process, a mutant of Trichoderma viride is grown on a cellulose
substrate, and the extracellular enzyme complex produced is separated from
the mycelia. Waste cellulose is then hydrolyzed at 50°C, pH 4.8 in the
presence of the fungus derived cellulase yielding a syrup consisting
primarily of glucose with lesser amounts of cellobiose and xylose.
To minimize hydrolysis time on the way toward making process economics
acceptable for commercial application, it is necessary for most cellulosic
wastes to be pretreated. In the past, mechanical and chemical pretreatments
have been tried with improvements noted. However, none have been econom-
ically successful.
NARADCOM, supported by the Environmental Protection Agency Agreement
No. EPA-IAG-DS-0758, has continued pretreatment studies initiated at Natick.
The results contained herein are in fulfillment of this Agreement and stem
from the following objectives: (a) to search for pretreatment methods
which will enhance susceptibility of cellulosic wastes to enzymatic
hydrolysis by J_. viride cellulase while minimizing costs, and (b) evaluate
cellulosic wastes with respect to their potential as acceptable substrates
for the Cellulose Conversion Process.
This report consists of a review of pertinent background information
from the literature and the results and discussion of experiments to date.
-------
SECTION 2
CONCLUSIONS
The results of an extensive literature survey indicate that important
factors in increasing susceptibility of cellulosics to enzymatic hydrolysis
are the extent and type of crystal!inity and lignin content of the substrate
Most pulp and paper mill primary sludges from chemical pulping
operations could be used with no pretreatment to enhance susceptibility
to enzymatic hydrolysis by T_. viride cellulase.
The Reiling Machine, a modified hydropulper, provided only modest
improvements in susceptibility of newspaper to enzymatic hydrolysis with
low power demands and under a variety of operating conditions. Hence,
further experimental studies of this machine were considered not desirable.
Preliminary results indicate that attritor milling significantly
increases susceptibility of newspaper to enzymatic hydrolysis by J_. viride
cellulase.
Two-roll milling of newspaper, cotton, and wood chips has been shown
effective in enhancing susceptibility to enzymatic hydrolysis. This was
accomplished using 3, 6, and 10-inch diameter mills.
Power requirements for the three-inch two-roll laboratory mill are at
least 20% less than for a commercially ball milled sample and are expected
to decrease as scale-up to larger mills proceeds.
The two most important factors discovered to date in determining the
effectiveness of two-roll mill processing are roll clearance and processing
time. Data collected thus far indicate that large-scale mills using short
processing times, and high loadings may be possible.
Laboratory two-roll milled newspaper has a low sedimentation volume
(lower than commercially ball milled newspaper) thus making it conceivable
to slurry at high concentrations in the hydrolysis vessel of a production
scale process.
Studies of anhydrous liquid ammonia treatment of cellulosic materials
provide the potential of a chemical process in which the pretreating agent
can be recycled.
Materials originating from hardwood sources appear more amenable to
ammonia treatment than those from softwood sources.
-------
The possibility of a combined mechanical-chemical pretreatment is
indicated by experiments on two-roll milling followed by treatment with
anhydrous liquid ammonia.
While laboratory data indicate significant advances in the pretreatment
of waste cellulosic materials, considerable further effort is necessary to
develop commercially practical methods which will contribute substantially
to the economic viability of the enzymatic conversion process. Optimum
equipment design, energy requirements, processing techniques and other
system variables will require extensive investigation.
-------
SECTION 3
RECOMMENDATIONS
Continue scale-up of two-roll milling, paying particular attention to
power requirements and capacities as well as effects of moisture, temper-
ature, roll speed ratio, and roll surface.
Continue further liquid ammonia pretreatment studies concentrating on
effects due to substrate composition and optimizing operating conditions.
Investigate other gas phase treatments such as S0£ which might be more
effective in treating unresponsive (to liquid ammonia) substrates such as
softwood.
Expand studies of combination chemical/two-roll milling treatments.
Initiate studies of the effectiveness and economics of attritor millir
Study the nature of chemical and/or physical changes occurring in
cellulosic materials as a result of pretreatment. Important factors to b'
considered include changes in crystallinity, degree of polymerization and
the lignin-carbohydrate complex.
-------
SECTION 4
BACKGROUND REVIEW
PROPERTIES OF CELLULOSE AFFECTED BY PRETREATMENT
The action of the cellulase enzyme complex (C], Cx) on cellulose can be
approximated by the following reaction scheme (1).
Native
Cellulose
Hydrated
Polyanhydroglucose
Chains
V
Cy'S
Cellobiose
3-Glucosidase
V
Glucose
In actuality, the reactions are quite complex with multi-component
enzymes and many reaction pathways to be considered (2, 3).
Susceptibility of cellulose and cellulosic wastes to enzymatic hydrol-
ysis is greatly affected by a number of features of the cellulose-enzyme
system. These have been discussed by Cowling and Brown (4) and are summa-
rized as follows:
1. moisture content
2. size and diffusibility of the enzyme molecules in relation to the
capillary structure of cellulose
-------
3. degree of crystal!inity of the cellulose
4. unit cell dimensions of the crystallites present
5. conformation and steric rigidity of the anhydroglucose units
6. degree of polymerization of the cellulose
7- nature of the substances with which the cellulase is associated
8. nature and concentration and distribution of substituent groups
To a greater or lesser degree it is possible to alter these factors
by physical and/or chemical means prior to hydrolysis in order to facilitate
the action of the enzyme. It is evident that such pretreatment can have
an important bearing on the overall costs of an effective utilization of
waste cellulose through an enzymatic hydrolysis process.
Three aspects of cellulosic materials are considered to be particularly
significant with respect to susceptibility to enzymatic hydrolysis and as
potential bases of effective pretreatment. Highly crystalline; i.e.,
ordered, regions of a substrate are more resistant than the less ordered
amorphous regions. Secondly, the presence of lignin and its manner of
association with the cellulose can interfere with the hydrolysis. Reduction
in the degree of polymerization of cellulose leads to an enhancement of the
enzymatic hydrolysis. The following summary indicates previous work that
has been directed to these subjects.
Crystallinity
By x-ray diffraction analysis, cellulose has four known stable crystal
lattices (5). Cellulose I, the native form, is found in cotton, ramie,
wood, jute, etc., while cellulose II is formed by treating cellulose I with
alkali solutions and is found in viscose process products (i.e., rayon,
cellophane).
Cellulose III is formed by treating cellulose I or II with liquid
ammonia (6) or by decomposition of the ethylamine-cellulose complex (7).
Cellulose IV is prepared by heating cellulose III in glycerin at 250°C (8).
Ball milling has been demonstrated to induce crystalline modifications
in a-cellulose pulps (9, 10). Dry ball milling results in complete destruc-
tion of the crystalline lattice as determined by x-ray diffraction. However,
upon wetting and redrying, reversion to the native cellulose I form occurs in
cases where the ball milling time was insufficient. In the Caulfield and
Steffes report (11) ball milling times of six hours gave a product whose
crystalline structure was entirely cellulose II after being soaked in water
at 40°C and dried. The extent of crystallinity as measured by the crystal-
line index was roughly one-half of the original non-milled cellulose pulp.
-------
After three hours of ball milling, the wetted and dried product's crystalline
phase was a mixture of cellulose I and cellulose II.
It has been proposed that the mercerization process causes a sequence
of metastable crystalline structural changes leading to cellulose II (12).
The native cellulose I form of ramie fibers when treated with caustic soda
at room temperature is transformed into soda cellulose. Upon alkali removal
by washing with water at room temperature, the soda cellulose decomposes to
give water cellulose. As the fibers are dried, the water cellulose changes
to cellulose II.
Clark and Terford (13) have determined by x-ray diffraction the % crys-
tal linity of 23 commercial paper pulps. The groundwood pulps had higher
amorphous contents (about 44%) than the chemical pulps (42-39% amorphous).
This is attributed to the use of mechanical equipment (stone grinders,
refiners) in groundwood production and the resulting breakdown of crystalline
regions during fiber separation. Bleaching was demonstrated to increase
slightly the percent crystallinity of both chemical and groundwood pulps.
Measuring x-ray diffraction peak widths, Parks (14) estimated the size
of ordered regions of a variety of sulfite and kraft pulps. The sulfites
contained small ordered regions which are uniform in perfection of order,
whereas the kraft pulps have intermediate size ordered regions with varying
degrees of perfection.
Anhydrous liquid ammonia affects cellulose and cellulosic materials in
a number of ways.
Of particular interest are the swelling and transitions in crystalline
forms, as determined by x-ray diffraction measurements, that can be brought
about through treatment with anhydrous ammonia (6, 15, 16). Schuerch (17)
summarizes the crystal lattice changes schematically as follows:
liq. NH3
liq- NH3 < -3QQC ammonia
cellulose I > ammonia cellulose I <- > cellulose II
-30°C
boiling water
or dilute acid
> -30°C
-NH3
gas
cellulose III < - amorphous cellulose
high
temperature
cellulose IV
-------
Lewin and Roldan (18) present evidence for a disordered cellulose and an
interpretation that the amorphous cellulose of Schuerch (17) consists of a
mixture of less ordered cellulose I and cellulose III. The ammonia-cellulose
system is sensitive to temperature and pressure and to the manner in which
ammonia is removed. In general, evaporation of the ammonia leads to
retention of the less ordered structure and the presence of water or dilute
acid in the removal process tends towards reconversion to cellulose I.
The occurrence of small amounts of ammonium salts and amides in the
residues of woods treated with liquid ammonia indicate reaction with uronic
acids and uronic acid ester links present in the original wood (19).
An increase of accessibility of different cellulose structures pre-
treated with liquid ammonia was observed by Schleicher e_t al_. (20). Con-
siderable increases in the reaction rate and degree of conversion were
found for the reactions xanthogenation, carboxymethylation, and cyanoethyl-
ation.
The phenomenon of the plasticization of wood, similar to plasticizing
with steam, has been described by Schuerch (17, 21). In many instances,
the shaping of wood may be carried out quite easily and in shorter times
with liquid ammonia than with steam. This is attributable to the ability
of the ammonia not only to solvate the amorphous regions of the cellulose
but also to enlarge and relax the crystal lattice.
Millett et_ aj_., (33) review the use of ammonia, aqueous, gaseous or
liquid in attempts to upgrade the feeding value of lignocellulosic materials.
In general, strong interactions of an acid-base nature between ammonia
and the hydroxyl groups and hydrogen bonds of cellulose are to be expected.
Disruption of the Lignin Carbohydrate Complex
The nature of the association between lignin and carbohydrate in wood
has been debated for many years. Three main theories are prevalent: (a)
hydrogen bonding between constituents, (b) covalent chemical bonds, and (c)
incrustation where the three dimensional lignin network encases the cellu-
lose, thereby preventing easy access of enzyme molecules.
Browne!! and West (22) have presented some evidence for the presence of
a lignin-carbohydrate chemical bond in wood. Through fractionation of ball
milled wood which has been solubilized with ethylene oxide, they were able
to obtain one-half of the carbohydrate essentially lignin-free while three-
quarters of the initial lignin present was found in fractions containing
carbohydrate. They discount the possibility of incrustation since the wood
was entirely solubilized.
On the other hand, Pew and Weyna (23) have been advocates of the incrus-
tation theory. They contend that evidence presented for the existence of
covalent bonds is incomplete and that chemical bonds alone could not explain
the total resistance of wood to cellulolytic enzymes. It is their conten-
tion that even ball milled wood particles as small as several microns con-
8
-------
stitute a "cage" of lignin in which carbohydrate molecules are enmeshed.
The exposed carbohydrate would allow ready dispersal in cellulose solvents
and in lignin dissolving solvents which have the additional tendency to
dissolve amorphous cellulose. A model experiment in which holocellulose
was impregnated and cured with phenolic resin tended to support their view.
With respect to hydrogen bonding, it was concluded based on their
work and that of Lindberg (24) that the strength of hydrogen bonding of
lignin to carbohydrate would be weak due to the highly amorphous character
of lignin.
Consider now some of the more important treatments believed to disrupt
the ligno-carbohydrate complex, however it may be consituted.
Gralen e_t a]_. , (25) have shown that 295 kHz ultrasonic vibrations break
the cellulose-lignin bond of wood (1% aqueous slurry) so that cellulose is
soluble in cuprammonium solvent.
Millett and associates (33) have been able to enhance significantly the
enzymatic saccharifi cation of hardwoods and softwoods using a moist S02
treatment. Although no lignin was removed during the processing, Klason
values for the hardwoods were less than 9% compared to the initial values
of greater than 20%. Similar less dramatic results were seen for the soft-
woods. Apparently some depolymerization of the lignin occurred disrupting
the lignin-carbohydrate complex.
Van and Purves (26, 27) examined delignification of a number of sub-
strates via liquid ammonia. Employing temperatures and pressures up to
10QOC and 900 p.s.i., only small amounts of material were extracted from
the softwood spruce but 25 to 30% of the lignin was removed from the hard-
wood's beech, birch, and maple, and 52% from rye straw.
In the Fe++ + HpO? pretreatment of sweetgum wood, Koenigs (28) has,
without lignin removal, determined that Klason lignin of the untreated wood
was 18.1% as opposed to 8.4% for the treated sweetgum thus indicating an
alteration of the lignin network.
For a brief review of significant commercial and experimental lignin
removal methods, see Appendix A.
Reduction of the Degree of Polymerization
It is well documented in the literature (10, 11, 29) that ball milling
pure cellulose results in substantial decreases in degree of polymerization
(DP). The fine structure of the cellulose to be milled apparently determines
in large measure the extent of degradation to be expected for a given length
of time in the mill. For example, Leopold and Moulik (30) using a vibratory
ball mill, found that kraft pulp is depolymerized more than twice as rapidly
as sulfite pulps. They attributed this behavior to the presence of smaller
and more numerous crystallites in the kraft pulp thus making it less able to
-------
delocalize mechanical energy resulting in destruction of ordered regions as
well as cleavage of covalent bonds.
Leopold and Moulik also observed that 10 hours of dry milling is roughly
equivalent to 50 hours of milling in water (liquid to solid ratio - 200/1)
for lignin containing kraft and sulfite pulps. Kraft pulp degraded almost
four times as fast as sulfite pulp on wet milling which they contend is due
to the greater ability of kraft pulp to be plasticized by water.
Howsmon and Marchessault (10) noted that rotary ball milling acetate-
grade wood pulp in dry air resulted in greater reduction in degree of poly-
merization than a carbon dioxide atmosphere. They theorize that the presence
of oxygen increases the extent of chain cleavage probably by acting as a
radical acceptor of the free radical chains produced by mechanical action.
The peroxy radical probably then initiates further chain scission.
Morehead (31) showed that ultrasonic vibration reduces the DP values
of cellulose but not below the limiting values obtained by acid hydrolysis.
Cotton 1 inters and alpha sulfite pulp were depolymerized extensively
by high voltage cathode rays at dosage levels above 5 x 106 equivalent
roentgens (32). They depolymerized at practically the same rate resulting
in products with nearly average chain length. At a dose of 5 x 108
equivalent roentgens, the cotton linters were converted to water soluble
materials.
CHEMICAL PRETREATMENTS
Chemical pretreatments of cellulosic materials are of potential benefit
in the preparation of substrates for enzymatic hydrolysis. Factors that may
be modified in order to improve susceptibility to hydrolysis include
swelling, degree of polymerization, hydrogen bonding., relative extent of
crystalline and amorphous regions, amount of lignin present and the nature
of its association with cellulose. Disadvantages of chemical pretreatment
are the possible occurrence of undesirable side reactions, as for example,
derivative formation and oxidation. Also, the chemical media themselves may
present problems in their disposal without contributing further to the
contamination of the environment.
Millett et_ jfL , (33) reported that mercerization of cotton and ramie
yielded 40-50% increases in acid hydrolysis rates using constant boiling
hydrochloric acid. However, mercerized hemlock pulps showed no improve-
ment. They also investigated dilute alkali pretreatment (5-10% NaOH) of
a variety of wood species and measured in vitro digestibility by deter-
mining substrate weight loss following incubation with rumen fluid under
prescribed conditions (34). Softwoods (26-35% lignin), in general, were
more resistant to alkali treatment than hardwoods (17-28% lignin), indic-
ative of the effect of lignin content.
10
-------
Baker (35) has produced a series of hard and softwood kraft pulps of
varying residual lignin contents and measured in vitro digestibility. The
extent of delignification required to achieve 60% digestibility (equivalent
to a good quality hay), for Douglas Fir softwood was 73% while only 25% for
white birch hardwood. Once again the relationship of lignin content to
digestibility was demonstrated.
Mandels, Hontz, and Nystrom (36) treated hammer milled newspaper with
2% NaOH at 70°C for 90 minutes and noted moderate improvement with respect
to susceptibility to enzymatic hydrolysis by T_. viride cellulase. If the
treated sample was dried, susceptibility was markedly reduced and in fact,
less than the untreated sample, perhaps indicative of crystalline changes
within the cellulose microstructure.
More impressive results were noted for viscose and cuprammonium regen-
erated newspapers after a 48-hour saccharification. The cuprammonium treat-
ment provided a greater than three-fold improvement in susceptibility while
the viscose newspaper was 2.5 times better than untreated newspaper. Dry-
ing these samples and retesting for susceptibility, an initial lag was
observed but disappeared by 48 hours as the undried and dried samples were
equal.
Andren et al_., (37) tested commercial sulfite and kraft process pulps
for susceptibility to T_. viride cellulase hydrolysis. Both were considered
excellent substrates with the sulfite completely saccharified at 48 hours.
Beremberg rayon (wet) was also evaluated and found to be excellent with a
72% saccharification at 48 hours.
Using a T_. viride derived cellulase, Toyama and Ogawa (38) examined the
effects on decomposition (weight loss) of the alkaline and peracetic acid
pretreatment of rice straw, bagasse and two wood species. Partial deligni-
fication of bagasse and rice straw specimens by boiling in 1% NaOH for 3
hours or alternatively 1 hour at 120QC, showed 55% and 48% weight losses
at 48 hours respectively as a result of enzymatic hydrolysis. Boiling in
20% peracetic acid for 1 hour followed by autoclaving at 120°C in 1% NaOH
resulted in decompositions of 94.1% for the hardwood (Machilus thunbergii)
and 77.9% for the softwood (Cryptomeria japonica) at 48 hours. Residual
lignin contents were 18% for the softwood (originally 32%) and 15% for the
hardwood, down from 30%. Applying the NaOH treatment first followed by
the peracetic acid was not effective in enhancing 48-hour decomposition of
the softwood (only 22% decomposition), even though there was essentially
no difference for the hardwood decomposition.
Koenigs (39) using sweetgum woodmeal slurried with 0.44 mM Fe++ and 1%
H202 controlled at room temperature and pH 4.2 for three days, showed a 50-
55% weight loss after 64-hour treatment with Onazuka cellulase derived
from T. viride. Significant reduction in degree of polymerization and
enhancement of alkali solubility were noted for the treated material. In
addition, hemicellulose loss was high and apparently lignin structure was
altered. It was proposed that this Fe++ catalyzed system may be acting
similar to brown rot fungi, producers of extracellular H202-
11
-------
The action of the Fe++ + H202 system on cattle feed lot waste and its
effect on hydrolysis by X- viride cellulase were examined by Elmund, Grant
and Morrison (40). Feed lot waste slurried at 5% was reacted with 150 mM
H202 and 10 mM ferrous sulfate at 22°C for 6-8 hours. Results from a 24-
hour hydrolysis showed 46% of the cellulose in feed lot waste was converted
to reducing sugar.
Bellamy (41) investigated the 48-hour growth at 55°C of Thermoactinomyces
on pretreated feed lot waste fiber. Mith no pretreatment, only 15-20% of the
cellulose was utilized. Sodium hydroxide treatment (0.05 M, 23°C, 4 hours),
resulted in 74-81% cellulose utilization while treating with anhydrous liquid
ammonia for 10 hours at 23°C and 10 atmospheres netted a 72-80% cellulose
utilization.
Aqueous ammonia treatment has been demonstrated by Han and Callihan (42)
to increase the rice straw utilization by Cellulomonas bacteria from 29% to
57%.
Liquid or gaseous anhydrous ammonia, another strong swelling agent, has
been used on three wood species and evaluated using a 5-day incubation period
in vitro assay (43). Aspen sawdust treated with liquid ammonia for 1 hour
attained 50% digestibility coefficient whereas Sitka spruce and red oak
digestibilities were only 2% and 10% respectively.
Disrupting the lignin-carbohydrate complex without significant removal
of either component can be accomplished with sulfur dioxide. Operating with
moist sawdust (water/wood = 3/1) and gaseous S02 under pressure for 2-3 hours
at 120°C, Mi 11 ett et^ aj_., (33) have obtained impressive results on both
hardwoods and softwoods with Onazuka cellulase (derived from J_. viride).
Almost quantitative conversions of carbohydrate to sugars were attained
after 48-hour saccharification of the hardwoods. Meanwhile, the two soft-
woods were 70-85% converted.
MECHANICAL PRETREATMENTS
The mechanical pretreatment area has received little attention as is
evident from the relatively few publications found in the literature. Pew
and Weyna (23) measured the effects of vibratory ball milling on the diges-
tion (4-6 days) of spruce and aspen sawdusts using T_. viride cellulase in
5% slurries at pH 4.6 and 40°C. As grinding time increased, solubilization
of carbohydrate by enzyme increased to a 96% level for both woods after 8
hours (spruce) and 5 hours (aspen) of ball milling.
Mandels eit a]_., (36) investigated hammer milling, ball milling, fluid
energy milling and colloid milling, finding ball milling best due to its
capacity to size reduce effectively while providing high bulk density and
maximum susceptibility. Among the waste substrates treated were newspaper,
bagasse, rice hulls, rumen fibers, corrugated fiberboard, and computer cards.
Following are significant results of the Mandels1 study with respect to
12
-------
susceptibility of substrates to enzymatic hydrolysis by J_. viride cellulase:
Hammer Milling
This is an impact mill consisting of a rotor to which a set of hammers
are attached. As the rotor turns rapidly, the hammers impact the material
against a breaker plate exerting compressive, shear, and tensive forces. The
process is repeated until the material is small enough to pass through a
screen at the bottom of the machine.
Results with newspaper showed virtually no improvement in susceptibility
using a variety of screen sizes. In fact, for the finer screen sizes, sus-
ceptibility was less than for the newspaper feed material.
Fluid Energy Milling
Particles to be size reduced are entrained in high velocity gas or
vapor streams and impacted against a target plate or opposing entrained
particles. Product is removed using an internal air classifier.
Feeding between 0.036 kg/hr and 2.8 kg/hr and using both the target
plate/nozzle and double opposed nozzle combinations, only modest suscepti-
bility improvements were noted over the untreated newspaper. For this
reason, plus the high operating costs and low production rates, this type
of milling was considered unsatisfactory.
Colloid Milling
This attriting machine consists of two disks revolving in opposite
directions at peripheral speeds between 10 and 50 m/sec and set close to
each other. Feed in the form of a slurry is passed between the disks
where particles impact each other and in the process are reduced in size
and dispersed.
Operating at gap settings between 0.001 and 0.005 inches and with 2%
water slurries of newspaper, only modest improvements in susceptibilities
were observed. This fact along with high operating costs, led the authors
to conclude that colloid milling would not be a satisfactory method of
substrate preparation.
Ball Milling
The distinctive feature of a ball mill is the use of balls (steel,
ceramic, etc.), filling about one-half the volume of a rotating shell. The
feed material is placed within the shell, impacts the balls as tumbling
occurs and in so doing is size reduced.
Ball milling was the most effective pretreatment found in the Mandels'
study. Newspaper after 1 day of milling in a pot mill was 46.8% saccharified
at 24 hours as compared to the hammer milled newspaper control which was only
23% saccharified. The major drawback to ball milling pretreatment is high
power consumption making operating costs excessive.
13
-------
IRRADIATION
The basic effects of ionizing radiation on cellulose have been reviewed
by Arthur (44). In general, radiation initiates an oxidative degradation of
the cellulose molecule, Dehydrogenation, destruction of anhydroglucose units
to yield carbon monoxide and carbon dioxide and cellulosic chain cleavage to
yield an homologous series of products are observed. Oxidative products
include carboxyl and carbonyl compounds.
High voltage cathode ray electrons were used by Saeman et_ aj_., (45)
to treat alpha cellulose wood pulp and cotton linters. Extent of dilute
acid hydrolysis (0.1 N ^$04 at 180°C) was used to determine treatment
effectiveness. An optimum dosage of 108 equivalent roentgens resulted in
overall sugar yields (carbohydrate decomposition included) of 70.2%, and
62.1% for the wood pulp and cotton linters respectively. Untreated wood
pulp and cotton linters gave respective overall sugar yields of only 33%
and 23%.
A combination thermal and linear accelerator high-energy electron beam
radiation treatment was investigated by Millett and Goedken (32) using
cotton linters and a nitration grade softwood sulfite pulp. Sugar production
via dilute acid hydrolysis (0.1 N ^04 at 180°C) was measured to determine
effectiveness of treatment. A dosage of 106 equivalent roentgens, the level
at which irradiation begins to significantly affect cellulose structure, was
applied since it is also the maximum permissible dosage from an economic
standpoint. Best results were obtained by irradiating first followed by
heat treatment at 200°C for periods up to 16 hours. Maximum sugar yield
increased from 22.5% to 28.5%. Irradiation alone at 106 equivalent roentgens
improved sugar yield from 22.5% to only 24.5%.
Even though the hydrolytic activity of cellulose was enhanced by this
dual pretreatment, the authors concluded that the improvement was not suffi-
cient to be of potential commercial significance.
Kelly (46) studied the effects of gamma radiation on the dilute-acid
hydrolysis of cellulose. He found an enhancement of glucose yields due to
the radiation that was too low to be industrially attractive. Imamura
et_ a]_. , comparing 60Co gamma-ray and electron beam irradiation found little
difference in the extent of depolymerization of cellulose by the two types
of radiation (47).
14
-------
SECTION 5
EXPERIMENTAL RESULTS AND DISCUSSION
EVALUATION OF PRIMARY SLUDGES
The spent process waters discharged from pulp and paper manufacture
contain, among other constituents, suspended solids some of which are
biodegradable. These solids contain, depending upon the source, varying
amounts of non-usable short fibers, lignin, tannins, and various inorganics
such as dirt, pigments, and fillers. Through sedimentation, filtration and/
or flotation, suspended solids are separated from the effluent waste stream
and the resulting residue (primary sludge) dewatered for subsequent landfill
disposal or burning.
In Table 1 data compiled from Gehm et_ aj_., (48) is presented on the
expected levels of suspended solids in effluents from various pulp and paper
mill processes, and their ash contents. Note that in special cases such as
fine paper products, it is possible to find as much as 50% ash. However, in
most processes, ash content is less than 15%. Also presented in this table
are values representing the effectiveness of total suspended solids removal
by clarifiers for selected mill processes. Except for deinking operations,
a significant portion of the total suspended solids (80-90%) is being
effectively removed from waste streams and hence the cellulosic fractions
could be made available for subsequent utilization such as enzymatic con-
version to sugars.
Dr. Edward Soltes (St. Regis Paper Co., West Nyack, NY) has provided
estimates of wood pulp production in the major pulp and paper manufacturing
states of this country (Table 2). Assuming that primary sludges account for
3% of the 81,500 ton/day total production, then an estimated 2445 tons/day
(dry basis) are available. In addition, about 75% are derived from chemical
operations (i.e., kraft and sulfite).
Susceptibility data of nine primary sludges submitted by major Northwest
based paper companies is presented in Table 3. These sludges were evaluated
as received (generally 60 to 80% moisture content) according to the incuba-
tion and DNS reducing sugar determination procedures described in Appendix B.
Relative yield is defined as the ratio of total reducing sugar concen-
tration from the hydrolyzed substrate in question to that of ball milled
newspaper. A value greater than or equal to 1.0 is considered good whereas
between 0.69 and 0.99 fair. Less than 0.69 (hammer milled newspaper) the
15
-------
Table 1. Pulp and Paper Mill Effluent Suspended Solids, Ash Content and
Total Suspended Solids Reduction Effected by Clarifier
Product
Tissues
Fiber and Felts
Deinking
Newsprint
Fine Paper
Bleached Kraft
Semi-chemical Pulp
Sulphite Pulp
Suspended Solids
(Ib/ton)
10-50
60-150
200-500
20-40
75-100
5-10
50-100
35-50
% Ash
5-15
10-25
25-50
less than 10
10-50
less than 10
less than 10
less than 10
% Reduction
in Suspended Solids
91
--
69
92
87
81
--
—
Table 2. Estimated Wood Pulp Production (tons/day) in the Major
Pulp and Paper Manufacturing Areas of the U.S. (1975)
Location
Alabama
Florida
Georgia
Louisiana
Maine
Mississippi
Oregon
Washington
Wisconsin
Totals
Kraft
10,345
9,095
12,470
9,560
2,785
4,615
5,215
5,460
1,284
60,829
Sulfite
--
--
--
--
1,445
--
790
3,992
1,462
7,689
Groundwood
500
--
300
570
3,138
250
1,390
739
900
7,787
Semi -chemical
725
200
640
1 , 1 60
—
--
625
825
1,020
5,195
Total
11,570
9,295
13,410
11,290
7,368
4,865
8,020
11,016
4,666
81,500
16
-------
Table 3. Reducing Sugar Yields of Various Pulp and Paper
Mill Sludges Relative to Ball Milled Newspaper
Primary Sludge
1
2
3
4
5
6
7
8
9
10
Processing Source
100% sulfite pulp
90% kraft
10% sulfite
10% bleached
100% kraft
80% kraft
20% groundwood
100% bleached
100% sulfite
75% bleached
100% kraft
5-10% bleached
40% sulfite
60% refiner groundwood
33% sulfite
67% refiner groundwood
hammer milled newspaper
control
40% groundwood
5% sulfite
45% bark & wood-log debris
Rating
good
good
good
good
good
fair
fair
fair
poor
1 hr
1.08
1.20
1.16
1.06
1.13
0.88
1.00
0.87
0.77
0.66
Relati
5 hr
1.18
1.21
1.08
1.13
1.03
0.83
0.82
0.73
0.73
0.57
ve Yiel
12 hr
1.26
1.23
1.09
1.09
1.05
0.87
0.84
0.71
0.66
0.51
d
24 hr
1.44
1.28
1.24
1.19
1.10
0.96
0.85
0.71
0.62
0.52
substrate is deemed poor. This rating system is based upon economic esti-
mates for enzymatic process viability.
Most chemically derived sludges were good substrates and could be used
without pretreatment to enhance susceptibility to enzymatic hydrolysis. As
the content of groundwood increased in the sludges, the susceptibility
decreased and approached hammer milled newspaper which is mostly groundwood.
Even less susceptible was sludge number 10 containing 45% bark and wood-log
debris.
Based on evidence found in the literature, the results obtained here are
reasonable. Susceptibility improvements for groundwood over native wood
17
-------
would be expected due to decreased crystalline content as a result of mechan-
ical processing. The good results for sulfite and kraft derived sludges
correlate well with data previously reported for sulfite and kraft pulps.
Reactivity enhancements for sludges with high chemical fiber contents over
high groundwood fiber sludges are probably due to differences in lignin con-
tent and the extent and types of molecular order in the cellulosic fraction
of each sludge.
MODIFIED HYDROPULPING
Reiling Industries, Inc. under Contract DAAG-17-76-C-0014 attempted to
produce a better and less expensive method of pretreating cellulosic materi-
als to make them more effective as a substrate in the enzymatic hydrolysis
of cellulose to soluble sugars. This study was confined to waste newspaper.
The Reiling mill is a simplified version of the "Pulping Machine"
described in S. G. Stapley e^ a\_. , expired U.S. patent no. 2,525,772 dated
May 31, 1950. It consists of a horizontal tank with face plates at either
end. Each face plate is marred with a thick rough weld line applied radial-
ly. The plates rotate in opposite directions causing the cellulosic materi-
als, when mixed with water, to form a vortex in the center of the tank. The
motion causes the substrate fibers to separate.
The mill was operated in the following manner. One hundred and ten
pounds of water were added to the tank and the mill was put into operation--
while 4.4 pounds of whole newspaper were added. The temperature rise and
electric power consumption were noted for each run with samples taken at
various times during each run. Various additives such as sulfamic acid,
corn oil, enzyme broth and steel nailpoints (10 mm long by 2.5 mm in diame-
ter) were added at the start of each run in an effort to improve the
efficiency of the mill. Hot water was added in place of the cold tap water
in one run. The mill speed was increased from 647 rpm to 828 rpm in another
run in an effort to increase the mill efficiency. Table 4 summarizes the
data from the various runs.
This study shows the Reiling mill capable of producing a product equal
to approximately 80% of the product produced by ball milled newspaper at an
energy level of approximately 1/2 k.w.h. per pound of dry newspaper. Chemi-
cal additives such as wetting agents, nailpoints, enzyme broth, corn oil,
sulfamic acid or even the absence of printing ink had little effect on the
efficiency of the Reiling mill. Changes in physical conditions such as
temperature of the water and speed of the mill had little effect on the
efficiency of the mill.
ATTRITOR MILLING
Union Process Inc. of Akron, Ohio was contacted to process newspaper,
both wet and dry, through an attritor mill.
-------
Table 4. 24-hr Relative Yields for Reiling Machine Treated Newspaper
24-hr Relative Yield for
Various Processing Times
Operating Conditions
Newspaper with nail points
Newspaper with Tween 80 (20 g)
Unprinted newspaper
Newspaper in hot water (130°F)
Newspaper w/sulfamic acid (1/2 Ib.)
Newspaper (agitation - 828 rpm)
Newspaper (agitation - 647 rpm)
1 hr
0.65
0.85
0.67
0.62
0.61
0.68
0.83
2 hr
0.77
--
0.72
0.70
0.62
0.75
0.83
3 hr
—
0.84
0.66
0.59
0.60
0.91
0.84
4 hr
0.70
0.88
0.66
0.59
0.60
0.73
0.89
An attritor is a vertical cyclinder containing small steel or stone
balls. A steel shaft having steel extensions is rotated in the tank con-
taining the balls. Grinding is obtained by the motion of the balls on the
newspaper added to the mill. A no. 1-S pilot laboratory model attritor was
used. It has a 1 1/2 gallon grinding tank with 50 Ibs. of 3/8" steel balls.
A load of 400 grams of hammer milled newspaper was used for dry grinding
and a 4% concentration of hammer milled newspaper in hot water was used
for wet grinding (140 grams of hammer milled newspaper with 3,360 grams of
hot water was used to make the 4% slurry).
Preliminary data indicates that processing hammer milled newspaper in
an attritor mill might yield aVi excellent product for the enzymatic con-
version of cellulose to glucose. However, this preliminary study lacks
data regarding energy costs, process optimization and reproducibility.
Much additional work using newspaper and other substrates processed in the
attritor is necessary before any valid conclusions can be formulated.
TWO ROLL MILLING
Background
The roller crusher was probably first used, in the form of three rolls,
to crush grain and beans and later to crush sugar cane as early as the 15th
century. Rolls were first made to be used in a horizontal form in the
eighteenth century and were generally driven by water wheels. There are many
types of roll crushers. Their essential features are that they consist of
19
-------
at least one roll rotating on Us principal axis and ripping material parti-
cles between its surface and another surface to compress and break them.
Roll crushers may consist of one, two, three, four or six or more cylinders,
which are usually horizontal and revolve either towards each other or toward
a plate. If they consist of more than one roll, the diameters and speeds of
the rolls may differ. The distance between the rolls is usually adjustable.
The action of the roll crusher is very similar to that of the jaw crusher.
Instead of jaws moving in a horizontal direction relative to each other, the
cylinder surfaces move in combined horizontal and vertical movements so that
the material being crushed is continuously and steadily induced into the
smaller part of the mouth by the circular movement of the cylinders and the
friction between the material and the cylinder surfaces. Advantages of roll
crushers are many.
(1) It will handle wet as well as dry material.
(2) The product can be controlled so that there is a high yield with
a narrow size range.
(3) The power consumption of the two-roll crushers is low compared with
that of many other machines such as jaw and gyratory crushers (49).
(4) Roll surfaces can be heated or cooled by the circulation of fluid
within the interior of the rolls.
One of the basic pieces of equipment used in the rubber and plastics
industries is the two-roll mill which is quite similar in design and con-
struction to the two-roll crusher (50). It was for many years the only
recognized mixing equipment for extremely viscous and elastic materials. A
two-roll mill is used in the rubber industry to perform such operations as
mixing, warm-up, sheeting, strip feeding, washing, refining and cracking.
A two-roll mill is used in the plastics industry primarily to warm-up and
blend granular resin particles and to form sheets for further processing.
A mill consists of two cast-iron tempered surface rolls, sometimes with
other metals for additional surface protection, placed horizontally in
bearings and held in place by mill housings. The rolls are set close togeth-
er and are adjustable by mill screws. The mill rolls are driven by a bull
gear powered through a reduction drive connected to a large motor. The other
end of the rolls are connected by pinion gears. Figure 1 shows a typical
differential speed two-roll mill. The rolls are usually cored, except that
mills used in the plastics industry are frequently bored to provide for more
accurate cooling and heating. The operation of a typical two-roll mill
consists of placing crude rubber or premixed stocks between the rolls and
as it is heated up the stock forms a band around the roll.
There are many types of mills used in the rubber and plastics industries
such as mixing mills, strip mills, warm-up mills, sheeting mills, cracker
mills, washer mills, refiner mills and other special mills each having
specific roll speed ratios and roll surfaces unique to the type of processing
performed on each. Three major factors must always be considered aside from
the matter of established operational procedures. These factors involve the
20
-------
Drive P1i
Connecting Pinion
Connecting
Gear
Guide
Drive Gear
Reduction
Gear Drive
Gear flex
Coupling /
Motor
Figure 1. A Typical Differential Speed Two-Roll Mill
21
-------
determination of the proper batch size and processing time, the need for
consistent reproducibility and compound formulation.
There has been much work and many theories formulated about what happens
to rubber when processed on a two-roll mill. It is generally agreed among
researchers that during the first passage through the rolls, the rigid mass
is stretched and ruptured, resulting in a tearing of the bonds of the
micelles (51). Since rapid stretching is exothermic and rubber is a poor
conductor, the temperature increases rapidly with the time of processing.
Processing on a two-roll mill causes a ten-fold decrease from the initial
molecular weight of natural rubber (52). The rate of degradation is greatest
during the early stages and slows down eventually to almost zero, even
though the shearing forces are still an appreciable fraction of those
initially applied. Rubber molecules therefore suffer scission and breakdown
until they reach a limiting molecular weight under normal masticating con-
ditions, in the range of 70,000 to 100,000 for natural rubbers.
Many plastics, resins and fibers can be transformed into a rubber!ike
state by raising the temperature and adding plasticizers. Degradation under
mechanical treatment has been noted for a great variety of natural and
synthetic polymers.
Two-roll milling of cellulosic materials covered by this report
follows none of the known procedures for the rubber and plastics industries
but is a combination of several known methods. In preliminary work at
Natick, only available laboratory equipment for rubber and plastics
processing was used. Initial trials were made using various test substrates
on a 3" x 8" two-roll mill made by Wm. R. Thropp & Sons Co., and a fixed
roll speed ratio of 1.4 to 1 (23 rpm x 16 1/2 rpm). Next, a Farrell-
Birmingham Co. 6" x 13" two-roll mill, and a fixed roll speed ratio of 1.4
to 1 (32 1/2 rpm x 23.2 rpm) was used. The only other available two-roll
mill was a Farrell-Birmingham Co. 10" x 20" mill, with a variable speed
drive. Each roll is driven by a D.C. motor which obtains its power from an
A.C.-D.C. generator thus allowing wide variability in roll speed ratios.
The mill rolls are flat rather than crowned, with very small roll clearances.
The material is taken off the mill with a scraper after a specified proc-
essing time. The mill rolls can be heated or cooled as desired.
Experimental
Since the discovery midway through this program that two roll milling
enhances cellulose susceptibility to enzymatic hydrolysis, significant
progress has been made in translating favorable pilot scale milling results
to full scale operation although much additional effort is needed. This
preliminary data will be useful in determining future program direction.
Perhaps the most encouraging result so far is that two-roll milling
has been shown to be effective using 3, 6 and 10-inch diameter two-roll
mills on diverse cellulosic substrates such as newspaper, wood and cotton.
This point will be clearly illustrated as the following data is presented
concerning some significant milling parameters.
22
-------
Roll Clearance--
The distance between the rolls of the mill (roll clearance) is very
critical in determining enzymatic hydrolysis susceptibility of the product.
In Figure 2 mean relative yield is plotted versus roll clearance for a
hammer milled newspaper treated on the 10-inch mill for six minutes. Mean
relative yield is the arithmetic mean of relative yields at the 4, 12 and
24 hour points on the hydrolysis curves. It is obvious that as the roll
clearance decreases, susceptibility increases. This relationship would be
expected to hold for other substrates besides newspaper and is probably a
reflection of increasing pressure and tearing action as the rolls are brought
closer together.
Processing Time--
Figure 3 shows the effect of processing time at various roll clearances
on mean relative yield for a hammer milled newspaper on the 10-inch mill.
For roll clearances of less than 0.040 inches, susceptibility increases with
increasing processing time.
This behavior is similar to that noted for ball milling in that sus-
ceptibility improves with increasing processing time. However, whether it
reaches a limiting value similar to ball milling (36) is unknown at this
time. Additionally, the data indicates that processing times of 10 minutes
or less can result in susceptibilities which could only be achieved by hours
of ball milling.
Substrate Composition--
Figure 4 illustrates the substantial susceptibility improvements
attained through two-roll milling (0.010 roll clearance) of maple and white
pine wood chips. Three and twelve-fold improvements were noted for the
white pine and maple respectively.
It should be pointed out that the presence of lignin is apparently
affecting the results since the maplewood was milled for only 1 minute
whereas the white pine had three minutes of milling and yet the maplewood
was equally as reactive. This is not surprising in light of findings by
other investigators using different pretreatments (see background review).
In general, they observed that hardwoods were more readily susceptible to
digestion by rumen fluids (enzymatic system) than softwoods.
Absorbent Cotton processed on the three and six-inch mills has ex-
hibited twelve-fold increases in susceptibility over unmilled cotton.
Figure 5 shows data for the six-inch mill. The authors believe that these
impressive results are indicative of crystallinity changes and molecular
cha.in scission processes occurring as the cotton is milled. Similar changes
may have been observed by other researchers following ball milling of alpha
cellulose pulp (background review). Alteration of lignin structure cer-
tainly cannot be responsible for the increased reactivity since there is no
lignin in cotton.
Power Data--
Electrical power data was collected on the three-inch laboratory mill
using newspaper. A cumulative watt meter was connected between the line
23
-------
1.20
1.7.
PO
O 0.020 inches
A 0.030 inches
0.040 inches
0.60
0.010 0.020 0.030
Roll Clearance (inches)
Figure 2. Effect of Roll Clearance on Mean
Relative Yield of Two Roll Milled Newspaper.
Roll Speeds: 25 fpm and 35 fpm; Rolls chilled;
1.25 Ib. of newspaper; Roll diameter: 10 inches.
4 6
Processing Time (minutes)
Figure 3. Effect of Processing time on Mean
Relative Yield of Two Roll Milled Newspaper.
Roll Speeds: 25 fpm and 35 fpm; Rolls preheated
to 130°F; 1.25 Ib. newspaper; 0.020 inches roll
clearance: Roll diameters = 10 inches.
-------
ro
en
30-
25-
20-
Key:
O treated white pine sawdust
untreated white pine sawdust
treated maplewood
untreated maplewood
BMNP
12 16
Time (hrs)
20
24
Figure 4. Effect of Two Roll Milling on
Enzymatic Hydrolysis of White Pine and Maple-
Wood Sawdusts. Roll diameters: 10 inches;
Roll speeds: 25 and 35 fpm; Sample weight:
1.25 lb.; Processing times: 1 min for
maplewood, 3 min for pinewood; Roll clearance:
0.010 inches.
Key:
treated cotton
untreated cotton
BMNP
HMNP
Time (hrs)
Figure 5. Effect of Two Roll Milling on Enzymatic
Hydrolysis of Cotton. Roll diameters: 6 inches;
Roll speeds: 36 and 51 fpm; Sample weight: 50 g;
Roll clearance: 0.005 inches; Processing time:
2 min.
-------
and the A.C. motor which operates the rolls.
From the very limited power consumption data of Table 5, it appears
that as roll clearance is decreased, power consumption and substrate
reactivity increases. For a product of comparable reactivity, power re-
quired per pound is at least 20% less than that for commercially ball milled
newspaper as estimated by Nystrom (53). In addition, it is expected from
conversations with equipment manufacturers, that this power figure should
decrease as larger production scale two-roll mills are evaluated.
Table 5. Power Consumption for Laboratory Two-Roll Milling of Newspaper
Roll
Clearance
(inches)
Processing
Time
(minutes)
Sample
Weight
(g)
Mean
Relative
Yield
Power
(kwh) (kwh/lb.
0.008 2
0.010 2
30
30
1.05
0.93
0.0576
0.0461
0.872
0.697
Sedimentation Volume--
Five gram samples of commercially ball milled and two-roll milled
newspaper (six-inch mill at 0.010 inch roll clearance) were dispersed
thoroughly in distilled water to give a total volume of 100 ml. The slurry
was then shaken for 10 minutes, total volume measured and water added to
bring it back to 100 ml. It was shaken for another fifteen minutes, volume
was measured in a graduated cylinder and found unchanged. The mixture was
allowed to settle in the cylinder and the volume occupied by the solids
measured at various times. The results are presented in Table 6.
Table 6. Sedimentation Volume of Two-Roll Milled and Ball Milled Newspaper
Substrate
Sedimentation Volume (ml)
1 hr. 17 hrs. 144 hrs.
Ball Milled Newspaper
Two-Roll Milled Newspaper
38.5
31.0
39.5
31.5
39.5
31.5
The data clearly shows that the wet density of two-roll milled news-
paper is greater than ball milled newspaper. This is important to the
cellulose conversion process, since it implies that slurries of greater
concentration will be possible in production scale hydrolysis vessels
26
-------
thereby reducing reactor volume and hence capital costs over substrates
which can only be slurried at lower concentration levels.
ANHYDROUS LIQUID AMMONIA
To study the disruptive effects of liquid ammonia on the lattice
structure and chemical bonding of cellulosic materials, experiments were
begun on a number of substrates. These include softwood and hardwood
sawdusts, newspaper and a lignin-free cellulose, Avicel PH102 (American
Viscose). In some cases ammonia treatment was performed following two-roll
milling of the material.
Liquid ammonia experiments were carried out in two ways: 1) in an open
Dewar flask at atmospheric pressure and a temperature of -55°C, 2) in a Parr
Instrument Co. Pressure Reactor, Model No. 4562 at temperatures and pressures
of 25°C, 10 atmospheres and 60°C, 24 atmospheres.
The anhydrous liquid ammonia used was 99.99% purity (Matheson Gas.
Products Co.). For the open Dewar experiments, ammonia gas was condensed by
passing over a stainless steel coil cooled to -60°C by means of a Model
C-60 Cryocool Unit (Neslab Instruments Inc.). When using the pressure
reactor, the gaseous ammonia was first condensed into a 300 ml flask im-
mersed in a dry ice-alcohol bath at -70°C and then delivered through a tube
into the precooled reactor by applying a pressure of dry nitrogen to the
surface of the liquid ammonia. The reactor was then sealed and allowed to
come to room temperature or heated in a hot water bath. Volume of the
reactor was 450 ml and sufficient liquid ammonia was used (usually 150 ml)
to insure that the liquid phase was still present at the higher temperatures.
In the experiments thus far atmospheric moisture and oxygen have not been
rigorously excluded.
Materials to be treated were usually oven dried overnight at 103°C and
10 gm samples were taken for any particular run. At the conclusion of a
run, the reactor was vented and the remaining ammonia allowed to evaporate
at room temperature overnight or until the odor of the gas was no longer
detectable.
Effect of the ammonia treatment was assessed by enzymatic hydrolysis
employing the cellulolytic enzyme preparation derived from a mutant of the
fungus Trichoderma viride (54). After evaporation of the ammonia, the 10 gm
samples were divided in two and hydrolyzed as 5% slurries by the procedure
given in Appendix B. The concentration of reducing sugars obtained was
determined by the spectrophotometric dinitrosalicylic (DNS) acid method (55)
and in some instances also by liquid chromatography (56). The DNS method
determines the total of reducing sugars while the chromatographic method
determines only the xylose, glucose and cellobiose. -
Avicel, PH102, a commercial microcrystalline cellulose obtained from the
dilute acid hydrolysis of cotton, was taken as representative of a lignin-
free cellulose. It was held in liquid ammonia for 3 hours at 60°C, 24
27
-------
atmospheres pressure. Enzymatic hydrolysis of this material gave the
results presented in Figure 6. Data for ball milled newspaper (BMNP),
hydrolyzed at the same time, are also given for comparison. It is inter-
esting that the initial rate of hydrolysis of the ammonia treated Avicel
is intermediate between the untreated control and the BMNP, which is
generally considered to be more nearly amorphous because of the ball milling.
This could be indicative of a disordering of the highly crystalline Avicel
by the action of the ammonia.
Figures 7, 8 and 9 present hydrolysis data for two hardwood samples,
yellow birch and maple and the softwood white pine.
Figure 7 was obtained with yellow birch sawdust that passed a 35 mesh
sieve. It shows an increasing effect of ammonia pretreatment with increasing
severity of experimental conditions. The points at 26 hours are the sums of
xylose, glucose and cellobiose values determined by liquid chromatography.
The hydrolysis curve for 60°C, 24 atmospheres closely paralleled that of
ball milled newspaper. In an experiment to determine the importance of the
length of time of treatment, 1/2 hour of contact with liquid ammonia gave
approximately the same result after 24 hours of hydrolysis as did 3 hours of
contact at 60°C and 24 atmospheres. The chromatographic data indicated
that at the higher temperature and pressure relatively more glucose was
produced at the expense of xylose and cellobiose than under room temperature
conditions.
In Figure 8 data are presented for a sample of maplewood that had
received only one minute processing time on a 10-inch diameter two-roll
mill. A two-fold increase in production of reducing sugar is shown for
the ammonia pretreated sample. Again it appears that 1/2 hour of treatment
with liquid ammonia is as effective as 3 hours. The 24-hour hydrolysis
value for BMNP was midway between the maplewood values. Satisfactory
agreement between the 24-hour DNS values and liquid chromatography values
was also found in this instance.
Experiments with white pine sawdust, 35 mesh, demonstrate the often
observed variability in the behavior of hardwoods and softwoods towards
particular chemical and/or physical treatments. In this instance, improve-
ment in enzymatic hydrolyzability was approximately three-fold less than in
the case of the hardwood birch sawdust, 35 mesh, Figure 9. Also, treatments
at room temperature, 10 atmospheres and 60°C, 24 atmospheres and for 3 hours
or 24 hours under the room temperature conditions gave little or no differ-
ence in the hydrolysis results. Presumably, an explanation for the differ-
ences in hard and softwood behavior lies in the higher lignin content of the
pinewood as well as differences in wood structures which can limit accessi-
bility.
Included in Figure 9 are data for white pine shavings that had been
ground in the two-roll mill prior to treatment with liquid ammonia at 60°C,
24 atmospheres. The milling alone provided a somewhat higher value than
that from ammonia pretreatment alone. Treating of the milled material with
ammonia increased the reducing sugar yield by approximately 10%.
28
-------
ro
30.
25--
Key:
2BMNP
Avicel
^7 Avicel, NH3£, 60°C, 24 atm, 3 hrs
30-
2S..
20--
en
3
15--
u
3
•O
CD
D;
10-.
10 15
Time (hrs)
20
25
Key:
9
A
A
yellow birch sawdust 35 mesh (YBS)
YBS, -55°C, 1 atm, 3 hrs, NH3£
YBS, 25°C, 10 atm, 3 hrs, NH3£
YBS, 60°C, 24 atm, 3 hrs, NH3£
liquid chromatograph
results
10 15
Time (hrs)
20
25
Figure 6. Effect of Liquid Ammonia Pretreatment
on the Hydrolysis of Avicel.
Figure 7. Effect of Liquid Ammonia Pretreatment,
Under Varying Conditions of Temperature, Pressure
and Time, on the Hydrolysis of Yellow Birch Sawdust,
35 mesh.
-------
CO
o
E
-\
cn
cr.
c
TJ
ID
a:
30-
25
20-
15-
10-
Key:
OTRM Maple
^ TRM Maple, 60°C, 24 atm, % hr,
VTRM Maple, 60°C, 24 atm, 3 hr,
untreated maplewood
NH3£
NH3£
10 15
Time (hrs)
20
25
30'
25--
'=r> or,
E 20-
i-
fO
D.
Z!
oo
O! 15
35 mesh (WPS)
NH3£,
Key:
§ white pine sawdust
WPS, -55°C, 1 atm,
A WPS, 25°C, 10 atm, 3 hrs,
C> WPS, 60°C, 24 atm, 3 hrs,
O WPS, 25°C, 10 atm, 24 hrs, NH^
WTRM - white pine shavings, WP
ATRM - WP, 60°C, 24 atm, 3 hrs, NH.£
NH3«.
NH3£
10 15
Time (hrs)
25
Figure 8. Effect of Combined Two Roll Milling
and Liquid Ammonia Pretreatments on the Hydrolysis
of Maplewood.
Figure 9. Effect of Combined Two Roll Milling
and Liquid Ammonia Pretreatments on the Hydrolysis
of White Pine.
-------
Figures 10 and 11 present data from initial experiments on the pre-
treatment of newspaper samples.
In Figure 10, a 12% increase in hydrolysis yield over the control is
seen for the shredlike hammer milled newspaper after a 1 1/2 hour, 60°C,
24 atmospheres ammonia pretreatment. The results for the non-treated,
very finely powdered ball milled newspaper are shown for comparison.
A combination of two-roll milling and liquid ammonia pretreatment
appears to have a significant effect as seen in Figure 11. Initially,
hammer milled newspaper was ground in the two-roll mill. The additional
ammonia treatment appears to effect a 22% increase in yield of reducing
sugar after a 24-hour hydrolysis. Results for a commercially ball milled
newspaper, no further pretreatment, are shown for comparison.
31
-------
£
£
!^
O->
13
OO
D-)
ID
OJ
Key:
OTRMNP
A BMNP
TR>INP
10 15
Time (hrs)
Figure 10. Effect of Liquid Ammonia Pretreatment
on the Hydrolysis of Hammer Milled Newspaper.
5 10 15
Time (hrs)
Figure 11. Effect of Combined Two Roll Milling
and Liquid Ammonia' Pretreatments on the Hydrolysis
of Hammer Milled Newspaper.
-------
REFERENCES
1. Reese, E. T., R. G. H. Siu5 and H. S. Leyinson. The Biological
Degradation of Soluble Cellulose Derivatives and Its Relationship
to the Mechanism of Cellulose Hydrolysis. J. Bact. 59:485-497.
1950.
2. Hajny, G. J., and E. T. Reese, eds. Cellulases and their Applications.
Advances in Chemistry Series No. 95. Am. Chem. Soc., Washington, D.C.,
1969. 470 pp.
3. Reese, E. T., and M. Mandels. Enzymatic Degradation Ch. XVIII in High
Polymers, Vol. V, 2nd Ed. Cellulose and Cellulose Derivatives, Part V.,
N. M. Bikales and L. Segal, eds. John Wiley and Sons, Inc., New York,
N.Y., 1971. pp. 1079-1094.
4. Cowling, E. B. Physical and Chemical Constraints in the Hydrolysis of
Cellulose and Lignocellulosic Materials. Biotech, and Bioeng. Symp.
No. 5:163-182, 1975.
5. Harper, D. The Application of X-Ray Techniques in the Pulp and Paper
Industry with Particular Reference to Cellulose Structure (A Review).
Pulp and Paper Mag. of Canada, 371-376, Aug., 1963.
6. Clark, G. L., and E. A. Parker. An X-Ray Diffraction Study of the
Action of Liquid Ammonia on Cellulose and Its Derivatives. J. of Phy.
Chem., 41(8):777-786, 1937.
7. Segal, L., L. Loeb, and J. J. Creely. An X-Ray Study of the Decomposi-
tion Product of the Ethyl amine-Cellulose Complex. J. Polymer Sci., 13:
193-206, 1954.
8. Loeb, L., and L. Segal. Preparation of Cotton Cellulose IV from Cotton
Cellulose III. J. Polymer Sci., 14(73):121-123, 1954.
9. Forziati, F. H., W. K. Stone, J. W. Rowen, and W. D. Appel. Cotton
Powder for Infrared Transmission Measurements. J. of Res., Nat. Bur.
of Standards, 45(2):109-113, 1950.
10. Howsmon, J. A., and R. H. Marchessault. The Ball Milling of Cellulose
Fibers and Recrystallization Effects. J. Appl. Polymer Sci., 1(3):
313-322, 1959.
33
-------
11. Caulfield, D. F., and R. A. Steffes. Water-Induced Recrystallization
of Cellulose. TAPPI, 52(7):1361-1366, 1969.
12. Mukherjee, R. R., and H. J. Woods. An X-Ray Study of the Transition
from Water-Cellulose to Cellulose II in Ramie, Jute and Sisal. Textile
Inst. Journal, 42:T309-T313, July/Aug., 1951.
13. Clark, G. L., and H. C. Terford. Quantitative X-Ray Determination of
Amorphous Phase in Wood Pulps as Related to Physical and Chemical
Properties. Anal. Chem., 27(6):888-895, 1955.
14. Parks, L. R. Classification of Pulps According to Supermolecular
Structure of Cellulose. TAPPI, 42(4):317-319, 1959.
15. Barry, A. J., F. C. Peterson, and A. J. King. X-Ray Studies of
Reactions of Cellulose in Non-Aqueous Systems, I. Interaction of
Cellulose and Liquid Ammonia. J. Am. Chem. Soc., 58(2):333-337, 1936.
16. Hess, K., and J. Gundermann. The Influence of Liquid Ammonia on
Cellulose Fibers (Formation of Ammonia-Cellulose I, Ammonia-Cellulose
II and Cellulose III) Ber., 706:1788-1799, 1937.
17. Schuerch, C. Wood Plasticization. Forest Products J., 14(9):377-381,
1964.
18. Lewin, M., and L. G. Roldan. The Effect of Liquid Anhydrous Ammonia
on the Structure and Morphology of Cotton Cellulose. J. Polymer Sci.,
Part C, No. 36:213-229, 1971.
19. Wang, P. W., H. I. Bolker, and C. B. Purves. Ammonolysis of Uronic
Ester Groups in Birch Xylan. Can. J. Chem., 42:2434-2439, 1964.
20. Schleicher, H., C. Daniels, and B. Philipp. Changes of Cellulose
Accessibility to Reactions in Alkaline Medium by Activation with
Ammonia. J. Polymer Sci., Symp. No. 47:251-260, 1974.
21. Schuerch, C. Wood Forming and Shaping. U.S. Patent 3,282,313. 4 pp.
22. Browne!!, H. H., and K. L. West. The Nature of the Lignin-Carbohydrate
Linkage in Wood. Pulp and Paper Mag. Canada, T374-T383, Aug., 1961.
23. Pew, J. C., and P. Weyna. Fine Grinding, Enzyme Digestion and the
Lignin-Cellulose Bond in Wood. TAPPI, 45(3):247-256, 1962.
24. Lindberg, J. J. Solubility and Hydrogen-Bond Formation of Lignins.
Papieri ja Puu (Finland, in English), 42:193-196, 1960.
25. Gralen, N., and S. Berg. Treatment of Wood with Ultrasonic Waves.
J. Polymer Sci., 6(4):503-507, 1951.
34
-------
26. Van, M. M., and C. B. Purves. Attempted Delignifications with Sodium
Bicarbonate-Carbon Dioxide, and with Anhydrous Liquid Ammonia under
Pressure. Can. J. Chem., 34:1582-1590, 1956.
27. Van, M. M., and C. B. Purves. Extraction of a Lignin Fraction from
Maplewood by Liquid Ammonia. Can. J. Chem., 34:1747-1755, 1956.
28. Koenigs, J. W. Hydrogen Peroxide and Iron: A Proposed System for
Decomposition of Wood by Brown-Rot Basidiomycetes. Wood Fiber, 6(1):
66-80, 1975.
29. Hess, K., H. Kiessig, and J. Gundermann. X-Ray and Electron-Microscopic
Investigations of the Process of Grinding of Cellulose. Z. Physik
Chem., B49:64-82, 1941.
30. Leopold, B., and S. K. Roy Moulik. Mechanical Degradation of Chemical
Wood Pulps. TAPPI, 51(8):334-339, 1968.
31. Morehead, F. F. Ultrasonic Disintegration of Cellulose Fibers Before
and After Acid Hydrolysis. Textile Research J., 20:549-553, 1950.
32. Millett, M. A., and V. L. Goedken. Modification of Cellulose Fine
Structure-Effect of Thermal and Electron Irradiation Pretreatments.
TAPPI, 48(6):366-371, 1965.
33. Millett, M. A., A. J. Baker, and L. D. Satter. Physical and Chemical
Pretreatments for Enhancing Cellulose Saccharification. Biotech, and
Bioeng. Symp. No. 6:125-154, 1976.
34. Mellenberger, R. W., L. D. Satter, M. A. Millett, and A. J. Baker. An
In Vitro Technique for Estimating Digestibility of Treated and Untreated
Wood. J. Animal Science, (30):1005-1011, 1970.
35. Baker, A. J. Effect of Lignin on the In Vitro Digestibility of Wood
Pulp. J. Animal Science, 36(4):768-771, 1973.
36. Mandels, M., L. Hontz, and J. Nystrom. Enzymatic Hydrolysis of Waste
Cellulose. Biotech, and Bioeng., 16:1471-1493, 1974.
37. Andren, R. K., M. H. Mandels, and J. E. Medeiros. Production of Sugars
from Waste Cellulose by Enzymatic Hydrolysis: Primary Evaluation of
Substrates. 19-23 May 1975. Applied Polymer Symposia, 28:205-220, 1975.
38. Toyama, N., and K. Ogawa. Sugar Production from Agricultural Woody
Wastes by Saccharification with Trichoderma viride cellulase. Biotech.
and Bioeng. Symp. No. 5:225-244, 1975.
39. Koenigs, J. W. Hydrogen Peroxide and Iron: A Microbial Cellulolytic
System. Biotech, and Bioeng. Symp. No. 5:151-159, 1975.
35
-------
40. Elmund, G. K., D. W. Grant, and S. M. Morrison. Cellulase Production
in Dual Culture Fermentation of Feed Lot Waste Paper presented at
76th Annual Meeting of the American Society for Microbiology, Atlantic
City, N.J., 2-7 May, 1976.
41. Bellamy, W. D. Single Cell Proteins from Cellulosic Wastes. Biotech.
and Bioeng., 16:869-880, 1974.
42. Han, Y. W., and C. D. Callihan. Cellulose Fermentation: Effect of
Substrate Pretreatment on Microbial Growth. Applied Microbiology,
27(1):159-165, 1974.
43. Millett, M. A., A. J. Baker, W. C. Feist, R. W. Mellenberger, and L. D.
Satter. Modifying Wood to Increase Its In Vitro Digestibility. J.
Animal Science, 31(4):781-788, 1970.
44. Arthur, J. C., Jr. Radiation Effects on Cellulose. In: Energetics and
Mechanisms in Radiation Biology, G. 0. Phillips, ed. Academic Press,
New York, N.Y., 1968. pp. 153-182.
45. Saeman, J. F., M. A. Millett, and E. J. Lawton. Effect of High-Energy
Cathode Rays on Cellulose. Ind. and Eng. Chem., 44(1):2848-2852, 1952.
46. Kelly, J. A. Radiolytic Hydrolysis of Cellulose EPA - 670/2-73-030,
U.S. Environmental Protection Agency, Cincinnati, OH, 1973. 20 pp.
47. Imamura, R., T. Veno, and K. Murakami. Depolymerization of Cellulose
by Electron Beam Irradiation. Bull. Inst. Chem. Res., Kyoto Univ.,
50(l):51-63, 1972.
48. Gehm, H. W., F. E. Lamarche, D. G. Kitson, J. L. Bryant, J. A. Brown,
and J. N. Franklin. Services. In: Pulp and Paper Manufacturer-Volume
II, R. G. MacDonald and J. F. Franklin, eds. McGraw Hill Book Co.,
New York, N.Y., 1969. pp. 324-330.
49. Lowrison, G. C. Crushing and Grinding. CRC Press, Inc., Cleveland, OH,
1974. 286 pp.
50. Taylor, P. C. Mills and Milling. In: Machinery and Equipment for
Rubber and Plastics, Vol. 1., R. G. Seaman and A. M. Merrill, eds.
Bill Brothers Publishing Corp. for India Rubber World, New York, N.Y.,
1952. pp. 13-52.
51. Golloy, W. Mastication and Plasticity. In: Chemistry and Technology
of Rubber, C. C. Davis and J. T. Blake, eds. Reinhold Publishing Co.,
New York, N.Y., 1937. pp. 150-180.
52. Bristow, G. M., and W. F. Watson. Mastication and Mechanochemical
Reactions of Polymers. In: The Chemistry and Physics of Rubberlike
Substances, L. Bateman, ed. John Wiley and Sons, New York, N.Y.,
1963. pp. 417-447.
36
-------
53. Nystrom, J. Discussion of Pretreatments to Enhance Enzymatic and
Microbiological Attack of Cellulosic Materials. Biotech, and Bioeng.
Symp. No. 5:221-224, 1975.
54. Mandels, M., J. Weber, and R. Parizek. Enhanced Cellulase Production
by a Mutant of Trichoderma viride. Applied Microbiology, 21(1):152-154,
1971.
55. Miller, G. L. Use of Dinitrosalicylic Acid Reagent for Determination
of Reducing Sugar. Anal. Chem., 31(3):426-428, 1959.
56. Palmer, J. K. Liquid Chromatography for Monitoring the Conversion of
Cellulosic Wastes to Sugars. Applied Polymer Symposia, 28:237-246,
1975.
57. Jahn, E. C. The Lesser Delignification Processes. In: Wood Chemistry,
Vol. II, L. E. Wise and E. C. Jahn, eds. Reinhold Publishing Corp.,
New York, N.Y., 1952. pp. 1021-1030.
58. McGregor, G. H. Manufacture of Sulfite Pulp. In: Pulp and Paper
Manufacture, Vol. I - Preparation and Treatment of Wood Pulp, J. N.
Stephenson, ed. McGraw Hill Book Co., New York, N.Y., 1950. pp. 303-
313.
59. Rydholm, S. A. Pulping Processes. Interscience Publishers, New York,
N.Y., 1965. pp. 401-991.
60. Tomlinson, G. H. Manufacture of Alkaline Process Pulps-Part I. In:
Pulp and Paper Manufacture, Volume I - Preparation and Treatment of
Wood Pulp, J. N. Stephenson, ed. McGraw Hill Book Co., New York, N.Y.,
1950. pp. 364-388.
61. Aronovsky, S. I., and D. F. J. Lynch. Pulping Bagasse with Alcoholic
Nitric Acid. Ind. and Eng. Chem., 30:790-795, 1939.
62. Aronovsky, S. L, and R. A. Gortner. The Cooking Process-IX Pulping
Wood with Alcohols and Other Organic Reagents. Ind. and Eng. Chem.,
28(9):1270-1275, 1936.
63. Schuerch, C. Solubility Effects in Lignin, Alcoholysis Reactions.
J. Am. Chem. Soc., 73:2385-2386, 1951.
64. Clermont, L. P. Delignification of Aspen Wood with Aqueous Sulfolane
Solutions. TAPPI, 53(12):2243-2245, 1970.
37
-------
APPENDIX A
DELIGNIFICATION PROCESSES
PULP AND PAPER INDUSTRY
In the pulp and paper industry, the major objective in chemically
treating wood is to produce a long fibered paper product of high strength
and varying degrees of whiteness depending upon end use. To accomplish
these aims, chemical processes such as bleaching and delignification are
used. Excellent reviews of the numerous bleaching and delignification
processes are provided by Jahn (57), McGregor (58), Rydholm (59) and
Tomlinson (60). A brief summary follows:
Sulfite
In this delignification process, wood chips are cooked at 130-150°C in
pressurized vessels containing water, sulfur dioxide, and bisulfites of
various bases. Lignin combines with the sulfur dioxide and/or bisulfite and
in so doing is solubilized. Some hemicelluloses are hydrolyzed and a portion
of the wood cellulose degraded. Typical pulp yields are 40-50% of the
weight of the wood charged to the digester. Numerous by-products are formed
during the sulfite cooking process including fermentable sugars, carbon
dioxide, acetic and formic acids, methanol, and cymene.
Soda
Wood chips are cooked in
solutions of 17 to 25 percent
for periods ranging from 2 to
Typical yields are from 43 to
portion of the hemicelluloses
waste liquor contains cooking
fragments but is missing the
in the sulfite process.
Kraft
pressurized vessels containing aqueous
sodium hydroxide on a dry wood weight basis
6 hours and at temperatures of 165 to 175°C.
48 percent of original weight. A significant
are solubilized and lignin is removed. The
chemicals., fermentable sugars and lignin
troublesome secondary reaction by-products found
In the kraft or sulfate process, the same operating conditions as the
soda process are utilized. The only difference is that a mixture of sodium
hydroxide and sodium sulfide is charged to the reaction vessel. As a result,
higher strength pulp in greater yield with the same degree of delignification
is obtained. This process and the sulfite process are the two most widely
used commercial chemical pulping processes.
38
-------
Semi-chemical
This involves a two-stage production process. The first is a mild
chemical action which loosens and partly removes lignin; the second step
completes the separation of fibers by'mechanical means. The first stage
quite commonly involves the use of the neutral sulfite process - sodium
sulfite buffered with sodium carbonate or sodium bicarbonate to maintain
neutral pH. Modified kraft or sulfite processes are also used. The second
stage involves the use of mechanical equipment such as refiners or rod
mi 11 s.
With all semi-chemical processes, high yields (60-70%) can be expected
as less hemicellulose and cellulose is attacked than in chemical pulping
processes. On the other hand, less lignin is removed.
Bleaching
The importance of bleaching in pulp manufacture is to impart whiteness
or brightness to the final product. This can be done in two ways - lignin
bleaching or lignin removal. In lignin bleaching, chromophores of lignin and
other impurities are destroyed without removal of substance. If greater color
stability and higher brightness is desired, multistage processing including
an alkali extraction stage is used to oxidize and remove the lignin with
minor cellulose loss. Hence this type of bleaching represents a continuation
of the cooking processes using much more selective and at the same time
expensive chemicals.
Among the most commonly used bleaching agents are elemental chlorine,
chlorine dioxide, hypochlorites, and chlorites. Others include permanga-
nates, and peroxides with reducing agents such as zinc hydrosulphite used
extensively for the reduction of color in groundwood pulps.
Other
Aqueous solutions of nitric acid (2-8%) have been found effective in
delignifying agricultural residues (rice straw, bagasse, etc.), but have
produced low yields (37-44%) due to partial removal of cellulose and
hemicellulose resulting from hydrolysis. The process involved oxidation
and nitration of the lignin into fragments which are removed by an alkali
extraction step.
Through the use of ethyl alcohol in various proportions with aqueous
nitric acid, it is possible to increase yield to 40-50% due mainly to the
inhibitory action of the alcohol on carbohydrate hydrolysis by nitric acid
(61).
Favorable aspects of alcoholic nitric acid pulping of bagasse include
(a) low pulping temperatures (80°C), (b) atmospheric pressure, (c) short
pulping time (2-4 hours), (d) low acid concentration, and relatively good
yields.
39
-------
Aronovsky and Gortner (62) looked at aqueous solutions of aliphatic
monohydroxy and polyhydroxy alcohols and found that n-butyl alcohol, n-amyl
alcohol, isoamyl alcohol and ethylene glycol with an acid or aluminum
chloride catalyst will delignify wood chips. Using aspen, a cooking time of
1.5 to 2.0 hours at 188°C, and a 1:1 solution of butyl alcohol in water,
yields of 48-55% were obtained with a lignin content to 6-10%.
The steps involved in delignification using alcohols are believed to be
alcoholysis causing partial depolymerization followed by dissolution. If the
solvent power is poor, only the lower molecular weight lignin fragments will
dissolve; the remainder to undergo condensation reactions and repolymeriza-
tion. Schuerch (63) has shown that the ability of solvents to dissolve or
swell isolated lignins increases as the hydrogen bonding capacities of the
solvents increase and as their solubility parameters approach a value of
eleven.
Sulfolane (tetramethylene sulfone), a highly polar solvent with sub-
stantial chemical and thermal stability, has been used in aqueous solutions
to delignify aspen wood chips. Using a 30 vol. % solution of sulfolane in
water, Clermont (64) was able to attain rapid delignification. He obtained
a 67% yield with a 6.4% residual lignin content after cooking for 1 hour at
175QC under pressure.
Yields were dependent on the amount of water in the cooking liquor and
the initial pH of same. With increasing amounts of water, more lignin and
also more carbohydrate was removed.
40
-------
APPENDIX B
PRETREATMENT EVALUATION PROCEDURES
The evaluation of any particular pretreatment is carried out in two
phases, 1) the enzymatic hydrolysis of the substrate under investigation and
2) the analysis of the hydrolysate for reducing sugars by the dinitro-
salicylic (DNS) acid method (55).
The enzymatic hydrolysis is carried out on 5% slurries (dry weight
basis) of substrates in the presence of enzyme preparations of J_. viride,
adjusted to constant enzyme activity, usually 1.0 filter paper units/ml.
Sodium citrate buffer (0.05 m) is used to control pH to approximately 4.8
and small amounts of merthiolate (.01%) are added as preservative.
Hydrolyses are carried out in a vibrating incubator at 50QC with small
aliquots removed for sugar analysis at 1, 4, 12 and 24 hours.
The DNS analyses for reducing s.ugar are carried out spectrophoto-
metrically at 550 nanometers. Measured values of transmittance are con-
verted to concentration values by reference to a standard curve obtained
from solutions of known glucose content.
41
-------
TECHNICAL REPORT DATA .
(Please read Instructions on the reverse before completing)
REPORT NO
EPA-600/7-77-038
FLE AND SUBTITLE
Pretreatment And Substrate Evaluation For The Enzymatic
Hydrolysis Of Cellulosic Wastes
6. PERFORMING ORGANIZATION CODE
3. RECIPIENT'S ACCI
REPORT DATE
July 1977 (Issuing Date)
Leo A. Spano, I homas
F. Macy, and Edward D. Black
TT. Tassinari, Charles"
I. PERFOF
) PERFORMING ORGANIZATION NAME AND ADDRESS
Pollution Abatement Division
Food Sciences Laboratory, U.S. Army
Natick Research & Development Command
Natick, Massachusetts 01760
10. PROGRAM ELEMEI*
EHE624; A.P.C.6240, Task 9.2
11. CONTRACT/GRANT NO.
EPA-IAG-DS-0758
12. SPONSORING AGENCY NAME AND ADDRESS
13. TYPE OF REPORT AND PERIOD COVERED
Municipal Environmental Research Laboratory—Gin., OH
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
14. SPONSORING AGENCY CODE
EPA/600/14
15. SUPPLEMENTARY NOTES
Project Officer: Charles J. Rogers
is. ABSTRACT Presented are initial studies aimed at determining the applicability of
various pretreatment methods and existing cellulosic wastes to a process for the
conversion of cellulose to glucose and other reducing sugars. In this process, a
cellulase from a mutant of Trichoderma viride is used.
Pretreatment using differential speed two-roll mills has significantly enhanced the
susceptibility of diverse cellulosic substrates such as newspaper, cotton, pine and
maple. With processing times of less than ten minutes on a six-inch (roll diameter)
mill, hydrolysis yield improvements were two and twelve-fold for newspaper and cotton
respectively. Power requirements for processing waste newspaper on a three-inch
laboratory mill are estimated to be 20% less than for a commercial ball mill. Anhy-
drous liquid ammonia treatments of hardwood birch and maple sawdusts rendered them
more susceptible to enzymatic hydrolysis than substrates from a softwood source, e.g..
white pine and newspaper. A combination of pretreatments, two-roll milling followed
by liquid ammonia, affected significant increases in hydrolysis yield for maplewood
shavings and hammer milled newspaper.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.IDENTIFIERS/OPEN ENDEDTERMS
c. COSATI Field/Group
Cellulose
Enzymes
Hydrolysis
Biomass
Enzyme Hydrolysis
Municipal Sol id Waste
Glucose Production
Pretreatment
6A
13. DISTRIBUTION STATEMENT
Release to Public
19. SECURITY CLASS (This Report)'
Unclassified
21. NO. OF PAGES
50
20. SECURITY CLASS (This page)
Unclassified
22. PRICE
EPA Form 2220-1 (9-73)
42
i-V.S GOVERNMENT PRINTING OFFICE: 1977— 757-056/6478
------- |