Acid and enzymatic hydrolysis of autohydrolyzed lignocellulosic substrates
by David Allen Lamar
A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in
Chemical Engineering
Montana State University
© Copyright by David Allen Lamar (1987)
Abstract:
Four biomass residues (barley straw, wheat straw, lodge pole pine, and Douglas fir) were analyzed for
effects of an autohydrolysis pretreatment on lignin extractibility and cellulose hydrolysis, both acid and
enzymatic catalyzed. A pure cellulose substrate, Chromedia, was also used for hydrolysis tests.
The conditions used for the autohydrolysis were 205 °C and 10 minutes. These conditions were those
found to be optimal for lignin extractibility from wheat straw during previous work at this laboratory.
The extractibility of lignin (by an ethanol-water solvent) following pretreatment was very similar for
barley straw and wheat straw. A total of about 75% of the lignin was removed during the
autohydrolysis and subsequent solvent extraction. The amounts of lignin removed from the two woods
were also very similar with about 30% of the lignin being removed.
Experiments were performed to determine the effects of lignin content and substrate morphology on
acid hydrolysis of lignocelluloses. These experiments involved hydrolyses on lignin-free substrates and
substrates containing lignin. The delignification procedure resulted in a substrate that was no more
hydrolyzable than a non-pretreated substrate with the lignin intact. Acid hydrolyses on ball-milled
Chromedia revealed that as the amorphous content of the substrate increases the rate of hydrolysis also
increases.
Pretreated and non-pretreated substrates were hydrolyzed using both sulfuric and hydrochloric acids
and enzymes to determine the pretreatment effect on degree of hydrolysis. Pretreatment of wood
substrates resulted in only slightly increased carbohydrate conversion via acid hydrolysis over that
observed for non-pretreated woods. No increase in hydrolysis rate was observed for pretreated straw
substrates when acids were used as the catalytic agents. When mixed enzymes were substituted for
acids in wheat straw hydrolysis, the cellulose conversions increased dramatically for pretreated
substrates, with values in excess of 90% observed.
A theory based upon the solubility of reaction products is presented to explain higher cellulose
conversions with mixed enzymes versus acid hydrolysis results. This theory leaves open the possibility
that autohydrolysis pretreatment renders all substrates investigated more subject to hydrolysis. A C ID AND ENZYMATIC HYDROLYSIS OF AUTOHYDROLYZED
L I GNOC ELLULOSI C SUBSTRATES
by
D a v id A l l e n
Lam ar
A t h e s i s s u b m itt e d i n p a r t i a l f u l f i l l m e n t
o f t h e r e q u ir e m e n t s f o r t h e d e g r e e
of
M a s t e r o f S c ie n c e
in
C h e m ic a l
E n g in e e r in g
MONTANA STATE U N IV E R S IT Y
B ozem an, M o n ta n a
A ugust 1987
P N
main
Ufa.
JlfiIS
APPROVAL
o f a th e s is submitted by
David A lle n Lamar
This th e s is has been read by each member o f the th e s is committee
and has been found to be s a tis fa c to ry regarding content, English usage,
form at, c ita tio n , b ib lio g ra p h ic s ty le , and consistency, and is ready
fo r submission to the College o f Graduate S tudies.
/ 7
D a te T j
M
S ?
Chairperson, Graduate C d p itte e
Approved fo r the Major Department
Date;
o
Jinn
Heajd, Major Department
Approved fo r the College o f Graduate Studies
Date
Graduate Dean
STATEMENT OF PERMISSION TO USE
In
presenting
requirements
fo r
th is
th e s is
in
p a r tia l
f u lf illm e n t , o f
a m a sters's degree a t Montana S tate
U n iv e rs ity ,
the
I
agree th a t the L ib ra ry s h a ll make i t a v a ila b le to borrowers under ru le s
of
the
L ib ra ry .
B rie f
quotations
from
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are
allow able
w ith o u t special perm ission, provided accurate acknowledgement o f source
is made.
Permission f o r extensive quotation from o r reproduction o f th is
th e s is may be granted by my major p ro fe sso r, o r in h is absence, by the
Dean o f L ib ra rie s when, in the opinion o f e ith e r, the proposed use o f
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f o r s c h o la rly purposes.
Any copying o r use o f the
m a teria l in th is th e s is fo r fin a n c ia l gain sh a ll not be allowed w ith o u t
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Signature
iv
ACKNOWLEDGEMENTS
The fa c u lty and s t a f f o f the Engineering Department deserve and
have my g ra titu d e f o r the help and support th a t they gave to me.
I
would e s p e c ia lly lik e to thank Dr. Daniel S h a ffer, my frie n d and advisor,
f o r the advice and guidance th a t he gave me w hile I was a t Montana State
U n iv e rs ity .
My good frie n d s Steve J e tte and Ron Nakaoka were a great
help in the completion o f th is e f f o r t .
itu d e and love to my fa m ily ,
s a c rific e s
Karen,
Most o f a ll I owe eternal g ra t­
Frank and Nathan, w ith o u t whose
and encouragement the completion o f th is degree would not
have been p o s s ib le .
Last, I would lik e to thank the Novo Corporation
fo r g iv in g me the enzymes used during th is study.
V
TABLE OF CONTENTS
APPROVAL
.......................................
ii
STATEMENT OF PERMISSION TO U S E ............................................................ i i i
ACKNOWLEDGEMENTS...................................................................................
IV
TABLE OF CONTENTS...................................................................................
v
LIST OF T A B LE S ............................................................................................v i i
LIST OF FIGURES..........................................................................................v i i i
ABSTRACT...................................................................................................
INTRODUCTION
...........................................
Research O bjectives
ix
I
...................................................................
2
STRUCTURE OF LIGNOCELLULOSE...............................................................
.3
C e l l u l o s e ......................................................................
4
Hemi c e ll u l o s e ...............................................................................
5
Lig n in ...............................................................................................
6
S tru ctu re as I t Relates to H ydrolysis
..................................
6
PRETREATMENTS TO ENHANCE HYDROLYSIS ................................................
11
EXPERIMENTAL
...........................................................................................
14
S u b s tra te s .......................................................................................
14
C h a ra cte riza tio n o f Substrates ................................................
15
A s h ...........................................................................................
15
M o is t u r e ...............................................................................
15
vi
TABLE OF CONTENTS—Continued
I
------------------------
E x t r a c t i b l e s ........................................................... ...
16
L i g n i n ...................................................................
17
C ellulo se and Hemic e llu lo s e ............................................
19
A lpha-C ellulose A nalysis ........................................
20
Beta- and Gamma-Cellulose A n a ly s is ...................
21
A utohydrolysis and Lignin E x tra c tio n ....................................
22
A u t o h y d r o ly s is .......................
22
D e lig n if ic a t io n ...............................................
26
B all M illin g . .......................................................................
26
Dry B all M i l l i n g ................................................................
27
Wet B all M i l l i n g ...............................................................
27
Acid and Enzymatic H y d r o l y s i s ................................................
27
Acid H y d r o ly s is ...................................................................
28
Enzymatic H ydrolysis
........................................................
29
.......................................................................
31
A utohydrolysis o f Substrates ....................................................
31
RESULTS AND DISCUSSION
Barley Straw
........................................................................
32
Douglas F ir and Lodge Pole P in e ....................................
34
Acid H ydrolysis E x p e r im e n ts ..............................
37
Barley S t r a w ...............................
Douglas F ir and Lodge Pole P in e ............................ ...
37
.
37
Wheat S tr a w ...........................................................................
43
C hrom edia...............................................................................
46
Enzymatic H ydrolysis Experiments ............................................
49
CONCLUSIONS.......................................
60
SUGGESTIONS FOR FUTURE RESEARCH ........................................................
61
REFERENCES CITED
62
APPENDIX
...................................................................................
...................................
65
vi i
LIST OF TABLES
1.
Major Component Composition o f L ig noce liu lose ....................
2.
Weight Percent Composition o f Substrates
............................
31
3.
Weight Percent Ash o f Ethanol-Benzene Extracted Substrates
32
4.
Comparison o f A utohydrolysis Experiments f o r Barley Straw
and Wheat S tr a w ...............................................................................
5.
.
38
. ................................................
39
Carbohydrate Conversion Results o f H2SO4 H ydrolysis o f
Wood S u b s tra te s .................... ...
8.
Comparison of Douglas F ir Lignin E x tr a c tib ility by Two
S o lv e n t s ...........................................................................................
9.
36
Carbohydrate Conversion Results o f Acid H ydrolysis on
Straw S u b s t r a t e s ................................................................... ...
7.
35
Summary o f A utohydrolysis Experiments on Wood
S u b s t r a t e s .......................................................................................
6.
3
40
Carbohydrate Conversion Results o f Autohydrolyzed
Dioxane-Water Extracted Douglas F ir ........................ . . . .
10. C ellulo se A nalysis A fte r Acid H ydrolysis
41
............................
42
11. Carbohydrate Conversions o f Douglas F ir Substrates by
HCl
...............................................
44
12. Carbohydrate Conversions o f Wheat Straw Substrates by
HCl
. . ...........................................................................................
45
13. Comparison o f Amorphous C ellulo se Content versus Amount
o f C ellulose Converted
...........................................
47
14. C ellulo se Analyses on M ille d and Non-Milled Chromedia . .
48
15. Results o f Acid H ydrolysis on Chromedia ................................
48
16. Results o f Enzymatic Hydrolyses on Wheat Straw
57
................
17. Results o f the Acid H ydrolysis Development Experiments
.
65
v iii
LIST OF FIGURES
1.
Basic S tru ctu re o f a Plant C ell . . . .
................................
4
2.
Chemical S tru ctu re o f C ellulose . , ........................................
5
3.
S tru c tu re o f a P ortion o f L i g n i n ............................................
7
4.
An Acid Catalyzed H ydrolysis Reaction ....................................
8
5.
Funnel Setup used fo r Lig nin Determination
. . ..................
18
6.
Sample Basket Apparatus ...............................................................
23
7.
Setup used fo r A utohydrolysis and E xtra ctio n Experiments
24
8.
E ffe c ts o f A utohydrolysis and Dioxane E x tra c tio n on Aspen
9.
Woodmeal Lig nin ...............................................................................
33
A Proposed Mechanism fo r an Enzymatic H ydrolysis
51
. . . .
10. E ffe c ts
o f C ellulase A c t iv it y fo r a 6 hour H ydrolysis .
.
52
11. E ffe c ts
o f Time on a C ellulase H ydrolysis ................. .
.
53
12. E ffe c ts
o f Cellobiase A c t iv it y on a 6 hour H ydrolysis .
.
55
13. E ffe c ts
o f Time on a C e llo b ia se -C e llu la se H ydrolysis
.
56
.
.
ix
ABSTRACT
Four biomass residues (b a rle y straw , wheat straw , lodge pole pine,
and Douglas f i r ) were analyzed fo r e ffe c ts o f an a u to h yd ro lysis p re tre a t­
ment on lig n in e x t r a c t i b i l i t y and c e llu lo s e h y d ro ly s is , both acid and
enzymatic cata lyzed.
A pure c e llu lo s e su b stra te , Chromedia, was also
used f o r h y d ro ly s is te s ts .
The co n d itio n s used fo r the a u toh ydro lysis were 205 °C and 10 min­
utes. These co n d itio n s were those found to be optimal f o r lig n in e x tra c t­
i b i l i t y from wheat straw during previous work a t th is la b o ra to ry .
The e x t r a c t i b i l i t y o f lig n in (by an ethanol-w ater so lve n t) fo llo w in g
pretreatm ent was very s im ila r fo r ba rle y straw and wheat straw . A to ta l
o f about 75% o f the lig n in was removed during the a u toh ydro lysis and
subsequent solve nt e x tra c tio n . The amounts o f lig n in removed from the
two woods were also very s im ila r w ith about 30% o f the lig n in being
removed.
Experiments were performed to determine the e ffe c ts o f lig n in content
and sub strate morphology on acid h y d ro ly s is o f lig n o c e lIu lo s e s . These
experiments involved hydrolyses on lig n in - fr e e substrates and substrates
co n ta in in g lig n in . The d e li g n i f i ca tio n procedure re s u lte d in a substrate
th a t was no more hydrolyzable than a non-pretreated sub strate w ith the
lig n in in ta c t. Acid hydrolyses on b a ll- m ille d Chromedia revealed th a t
as the amorphous content o f the substrate increases the ra te o f hyd ro lysis
also increases.
P retreated and non-pretreated substrates were hydrolyzed using
both s u lfu r ic and h yd ro ch lo ric acids and enzymes to determine the pre­
treatm ent e ffe c t on degree o f h y d ro ly s is . Pretreatment o f wood substrates
re su lte d in only s lig h t ly increased carbohydrate conversion v ia acid
h y d ro ly s is over th a t observed fo r non-pretreated woods. No increase in
h y d ro ly s is ra te was observed fo r pre tre a te d straw substrates when acids
were used as the c a ta ly tic agents. When mixed enzymes were s u b s titu te d
f o r acids in wheat straw h y d ro ly s is , the c e llu lo s e conversions increased
d ra m a tic a lly f o r p retre ate d su b strates, w ith values in excess o f 90%
observed.
A theory based upon the s o lu b ilit y o f re a ctio n products is presented
to exp lain higher c e llu lo s e conversions w ith mixed enzymes versus acid
h y d ro ly s is re s u lts . This theory leaves open the p o s s ib ilit y th a t autoh y d ro ly s is pretreatm ent renders a ll substrates in v e s tig a te d more subject
to h y d ro ly s is .
I
INTRODUCTION
The re a liz a tio n th a t petroleum supplies are not lim itle s s has awak­
ened the U.S. to the need o f an a lte rn a tiv e hydrocarbon source fo r fu e l
and chemical fee d-stocks.
A search has been focused on determ ining a
hydrocarbon source th a t does not compete w ith food supplies o r oth er
valuable raw m a te ria ls .
An ideal source o f hydrocarbon might be waste
o r by-product biomass from a g ric u ltu ra l operations o r wood product in ­
d u s trie s .
The u t iliz a t io n o f th is biomass would b e n e fit the above in ­
d u s trie s by converting low value m a te ria ls in to more valuable commodities.
The energy requirement o f the U.S. is roughly 76 q u a d r illio n BTU's
(Quads) per ye a r.
The generation o f th is energy would re q u ire 40 m illio n
b a rre ls o f o i l per day.
A major goal is to replace expensive petroleum
used in energy production w ith low cost biomass [ I ] .
P rese ntly, two to three Quads o f the U.S. energy requirements are
supplied by biomass u t iliz a t io n .
Combustion o f fo re s t products is the
prim ary source fo r th is energy.
A conservative estim ate o f the energy
th a t w i l l be supplied by u t iliz a t io n o f biomass by the end o f th is century
is 15 Quads [ I ] .
Biomass fo r energy could be supplied from d ir e c t and in d ir e c t
sources.
D ire c t sources might include farms developed to grow pla nts
s o le ly fo r energy uses, w hile in d ir e c t sources would includ e a g ric u l­
tu ra l
and fo re s t waste o r by-products mentioned above.
The in d ir e c t
sources are o f prim ary in te re s t in th is study.
I t is estim ated th a t 278 m illio n dry tons o f a g ric u ltu ra l by-products
and 108 m illio n dry tons o f unused m ill and logging residues are produced
annually [ I ] .
T h e o re tic a lly , i f these m a te ria ls were converted to glucose
and the glucose fermented to a lc o h o l, approxim ately 30 b i l l i o n gallons
o f ethanol
could be produced per ye a r.
This ethanol would meet the
e n tire c u rre n t in d u s tria l demand and provide ethanol fo r gasoline blending
2
as w e ll.
Most o f the present in d u s tria l grade ethanol is now produced
from petroleum-based feedstocks.
This use o f biomass would th e re fo re
reduce the demand fo r petroleum [ I ] .
The above estim ate o f ethanol from IignocelluT ose requires a 90
percent conversion o f the c e llu lo s e to glucose.
B a rrie rs e x is t in I i g -
n o cellulose s th a t prevent such high conversion o f c e llu lo s e to glucose.
E x is tin g technologies on ly provide about a 50 percent c e llu lo s e conver­
sio n . A hig her conversion is d e sira b le to econom ically produce c e llu lo s e derived products.
Research O bjectives
The f i r s t o b je c tiv e o f th is in v e s tig a tio n is to te s t three lig n o c e llu lo s ic substrates fo r degree o f lig n in removal and enhanced hyd ro lysis
o f t h e ir c e llu lo s e to glucose a fte r a novel pretreatm ent.
The substrates
to be in v e s tig a te d are ba rle y straw , lodge pole pine, and Douglas f i r .
These m a te ria ls w i l l be pre tre a te d by au toh ydro lysis a t c o n d itio n s found
to be optimum fo r lig n in removal from wheat straw during previous work
a t th is la b o ra to ry .
The second o b je c tiv e is to in v e s tig a te reasons th a t might explain
the low acid h y d ro ly s is y ie ld s observed w ith p re tre a te d wheat straw .
Meeting th is o b je c tiv e e n ta ils applying new h y d ro ly tic c a ta ly s ts and/or
co n d itio n s to the hydrolyses in an attem pt to increase y ie ld s .
3
STRUCTURE OF LIGNOCELLULOSE
Forest and a g ric u ltu ra l residues c o n s is t o f several prim ary compo­
nents.
These components include c e llu lo s e , hemic e llu lo s e , lig n in , pro­
te in , and miscellaneous e x tr a c tib le s .
Table I summarizes the percent
composition o f in d iv id u a l components by w eight.
Table I .
Major Component Composition o f L ig n o ce llu lo se [2 ].
Component
C ellulo se
Hem icellulose
Lignin
E x tra c tib le
and P rotein
%
Composition
45-50
20-25
20-30
0- 1:0
Together these components make the basic s tru c tu ra l u n it o f b io ­
mass, the p la n t c e ll.
A c e ll, in simple terms, is composed o f two basic
p a rts , the lumen and the c e ll w a ll.
o f the c e ll.
The lumen contains the liv in g m atter
Once dead the c e l l 's lumen is e ith e r void space o r f i l l e d
w ith e x tra c tib le s
[2 ] .
The c e ll w all serves as a mechanical d iv id e r
between in d iv id u a l c e lls .
R ig id ity o f a p la n t s ta lk is the d ir e c t re s u lt
o f i t s c e ll w a lls .
The c e ll w all also consists o f two p a rts , the prim ary w all and the
secondary w a ll.
The prim ary w all is very th in in comparison w ith the
secondary w a ll.
The secondary w all con sists o f three d is t in c t layers
termed the o u te r (S1) , middle (S2) and in n e r (S3) laye rs [3 ] .
Surrounding
the prim ary w all and separating adjacent c e lls is the middle lam e lla.
Figure I [1] is a re pre senta tion o f the basic s tru c tu re o f a p la n t
c e ll.
C ellulo se is m ainly in the m ic r o f ib r ils , shown as lin e s in the
diagram.
O rie n ta tio n o f the f i b r i l s
is
d iffe r e n t in the respective
p o rtio n s o f the c e ll w a ll.
The amount o f c e llu lo s e in the p la n t is highest in the secondary
w all and decreases toward the middle la m e lla .
Hemic e llu lo s e has i t s
4
• Secondary wall
Primary wall
Middle lamella
Figure I .
Basic S tru ctu re o f a Plant Cell [ I ] .
highest
percentage
lumen.
H em icellulose
c e llu lo s e
spaces
in
f ib r ils .
between
the
middle
and lig n in
Lignin
the
lam ella
and decreases
form a m a trix
and hemic e llu lo s e
c r y s ta llin e
regions
of
th a t
toward
the
surrounds
the
found in
the
are also
the
m ic r o f ib r ils ,
the
amorphous (n o n -c ry s ta llin e ) regions [2 ].
C ellulose
C e llu lo se
is
a
lin e a r
serving as the monomer.
bonds.
Figure 2 is
polymer
of
D-anhydroglucose
molecules
These monomers are lin ke d by /? -l-4 -g lu c o s id ic
a schematic o f a c e llu lo s e m olecule.
C ellulose
degree o f p o lym eriza tion ranges from 3,500 to 14,000 glucose u n its when
in a n a tiv e form. The average length o f c e llu lo s e molecules range from
2,500 nm to 5,000 nm [2 ,5 ].
The
lin e a r
molecules
lay
one
on
another
forming
bundles
of
molecules, f i b r i l s , th a t are held tog eth er by la te ra l hydrogen bonding.
The la rg e number o f hydrogen bonds re s u lt in c r y s ta llin e
regions o f
about 60 nm in length th a t comprise 67 to 90% o f the c e ll w a ll.
Since
the c e llu lo s e molecule is longer than 60 nm, the molecules pass through
several c r y s ta llin e and amorphous regions [2 ].
F ib r ils are surrounded
by a sheath o f hem icellulose and lig n in [5 ].
The apparent morphology o f c e llu lo s e
depends on the methods o f
a n a lysis and also the source o f the c e llu lo s e .
At present c e llu lo s e is
5
Figure 2.
Chemical S tru ctu re o f C e llu lo s e .
categorized in to
fo u r d is t in c t types,
c e llu lo s e
I,
II,
m ,
and IV.
Each group is based on the aggregation o f molecules in the c r y s ta llin e
s o lid .
Degree o f c r y s t a l li n i t y decreases w ith c e llu lo s e type ( i . e . ,
C ellulo se I is more c r y s ta llin e than C ellulo se I I ,
class
and so f o r t h ) .
The
to which a p a r tic u la r c e llu lo s e belongs depends on the method
used to produce the pure c e llu lo s e . The class is id e n tifie d by the xray d if f r a c t io n p a tte rn o f the sample [4 ].
C ellulo se in i t s n a tive form is classed C ellulo se I .
is
a c e llu lo s e
th a t
has been regenerated
C ellulose I I
from s o lu tio n
at
ambient
temperatures o r one th a t has been mercerized w ith c a u s tic s o lu tio n in
excess o f 15% sodium hydroxide.
morphologies.
several
C ellulo se I and I I are the most common
Treatment o f c e llu lo s e w ith anhydrous ammonia o r one o f
d iffe r e n t amines produces c e llu lo s e
III.
Heat treatm ent o f
c e llu lo s e I I
o r regeneration o f c e llu lo s e from s o lu tio n s at elevated
temperatures re s u lts in c e llu lo s e IV [4 ] .
Hemicel Iulo se
Hem icellulose is a polymer o f simple sugar molecules lik e c e llu ­
lose, though i t con sists o f more than one type o f sugar.
The backbone
o f the polymer is a lin e a r chain co n tainin g D-xylose sugar u n its lin ke d
to g e th e r by
/? - l- 4 - g lucosidic bonds.
is not a lin e a r homopolymer.
U nlike c e llu lo s e ,
hem icelIulose
I t contains side chains branching from
6
the main chain o f D-xylose sugars.
the
xylose
contain
molecules
v ia
1-3
The branches are u s u a lly bonded to
g ly c o s id ic
1-4 and 1-6 g ly c o s id ic bonds.
glucose,
glucose
xylose,
galactose,
and galactose.
mannose,
lin k s ,
they
can also
The side chains can contain
arabinose,
Composition
but
of
and uronic acids o f
a p a r tic u la r
hemic e llu lo s e
va rie s from source to source, not only w ith p la n t species, but also
w ith clim a te and lo c a tio n o f the p a r tic u la r p la n t.
za tio n o f hemic e llu lo s e
exceeds 200 [ 1, 2] .
Degree o f polym eri­
ranges from 100 to 200 molecules but ra re ly
Hem icellulose does not form c ry s ta ls lik e c e llu lo s e and is found
on ly in an amorphous s ta te .
It
is
u s u a lly in in tim a te contact w ith
p la n t lig n in s .
I t is thought th a t the hemic e llu lo s e and lig n in
chem ically bonded to g e th e r.
are
Lignin
Lig n in is a h ig h ly complex, three-dim ensional polymer o f various
phenolic acids connected by ether lin ka g e s.
U nlike the o th e r compo­
nents discussed so fa r , lig n in has no set p a tte rn o f s tru c tu re .
Figure
3 [1] presents a possible s tru c tu re o f lig n in .
Lig n in
to g e th e r.
acts
as
the
cement
th a t
holds
the
c e llu lo s e
fib r ils
I t is an in te g ra l p a rt o f a system th a t gives p la n ts t h e ir
stre ngth and r i g i d i t y .
This polymer not only acts as a cement but also
as a p ro te c tiv e s h ie ld against elements th a t would otherw ise destroy
the p la n t by a tta c k in g the c e llu lo s e .
lig n in
[6].
can l i m i t
the m icro bial
In v e s tig a tio n s have shown th a t
degradation o f p la n t polysaccharides
S tru ctu re as I t Relates to H ydrolysis
A l o t o f work has been done to determine how the chemical
physical
s tru c tu re
of
carbohydrate components.
IignoceT lulose
in h ib its
h y d ro ly s is
of
and
the
The fo llo w in g is an overview o f t h is work and
some o f the conclusions th a t have been drawn.
Many d iffe r e n t aspects o f p la n t s tru c tu re and chemical make-up
7
Figure 3.
S tru ctu re o f a P ortion o f Lig nin [ I ] .
have been id e n tifie d as detrim ental to the h y d ro ly s is o f biomass.
are the hemic e llu lo s e - lig n in b a r r ie r ,
c r y s t a l li n i t y
These
o f the c e llu lo s e ,
surface area, degree o f polym erization o f c e llu lo s e , and pore size d is ­
t r ib u t io n .
Some o f these p ro p e rtie s
act s y n e r g is tic a lly
to prevent
h y d ro ly s is .
here.
The ra m ific a tio n s o f in d iv id u a l aspects w ill be discussed
H ydro lysis o f c e llu lo s e can be catalyzed by e ith e r acids o r enzymes.
Although chem ically the re s u lts are the same, the mechanisms are q u ite
d if f e r e n t .
A successful acid catalyzed h y d ro ly s is (see Figure 4) takes
place when the oxygen atom o f the /M ,4 - g ly c o s id ic bond is attacked by
a hydrogen io n .
This a tta ck re s u lts in a p o s itiv e charge on the oxygen
which then p u lls e le ctron s from the oxygen-carbon bond re s u ltin g in a
p a r tia l p o s itiv e charge on the carbon atom.
Non-bonding ele ctron s o f a
water oxygen atom are a ttra c te d to th is p a r tia l p o s itiv e charge.
Elec­
tro n s from the o rig in a l carbon-oxygen bond form a bond w ith the a tta ckin g
hydrogen io n , and hydrogen-oxygen bonding e le ctron s from the water mole­
cule form a bond between the carbon atom and the a tta c k in g oxygen atom
re le a sin g a hydrogen atom.
This series o f events re s u lts in the breaking
o f the /? -l,4 -g ly c o s id ic bond by the a d d itio n o f a water molecule in
between two glucose monomers o f the c e llu lo s e molecule.
A proposed
mechanism fo r an enzyme catalyzed h y d ro ly s is is presented la te r in th is
work.
CH2OH
Figure 4.
CH 2OH
An Acid Catalyzed H ydrolysis Reaction.
Hemic e llu lo s e and lig n in are believed to be chem ically bonded in
biomass.
These
two
components
p ro te c ts the c e llu lo s e [3 ].
form
a sheath
th a t
surrounds
and
The I ig n in -h e m ic e llu lo s e b a rrie r prevents
con tact o f h y d ro ly tic agents (such as enzymes and acid) and c e llu lo s e
molecules, thus preventing h y d ro ly s is .
Removing e ith e r hemicellu lo s e ,
lig n in ,
the
or
both,
improves
access
to
c e llu lo s e
increasing
the
h y d ro !iz a b iI i t y o f the c e llu lo s e [7 ,8 ,9 ,1 0 ,1 1 ].
It
has been proposed th a t c e ll u l y t i c
themselves to lig n in molecules.
enzymes ir r e v e r s ib ly attach
This adsorption removes the a ffe c te d
enzymes from the re a ctio n m ixture [1 2 ].
Increasing enzyme charge to
the h y d ro ly s is m ixture only increases h yd ro lysis up to a p o in t.
This
im p lie s th a t enzyme adsorption is not the only in h ib ito r y mechanism o f
Iig ni n .
Several
chem ically
researchers have shown th a t some aromatic compounds can
in h ib it
enzymatic
h y d ro ly s is
Aromatic compounds having an in h ib ito r y
from
the
breakdown
lig n o c e llu lo s e .
of
lig n in
molecules
As an example,
of
hem icellulose
a ffe c t are products derived
or
hot
water
e x tra c ts
of
wheat e x tra c t norm ally contains
p-
coumaric a cid , f e r u lic acid , and v a n illic acid [1 4 ].
have been shown to be detrim ental
[1 3 ].
to d ig e s tio n ,
These compounds
and hence enzymatic
h y d ro ly s is , o f c e llu lo s e in rumenic animals [6 ].
The h e m ice llu lo se -1 ig n in b a rrie r is
not the on ly c h a ra c te ris tic
preventing conversion o f c e llu lo s e to glucose.
Evidence o f th is can be
9
ascertained from the h yd ro lysis o f c o tto n .
no lig n in
Cotton contains v ir t u a lly
and only small amounts o f hem icellulose
[3 ].
The lack o f
lig n in and low amounts o f hem icellulose should render cotton c e llu lo s e
h ig h ly
Several
susce p tib le to
h y d ro ly tic
a tta c k by e ith e r enzymes o r acids.
in v e s tig a to rs in I t a ly have shown th a t th is
is not the case.
A fte r a 24-hour enzymatic h yd ro ly s is they found on ly a 22% conversion
o f c e llu lo s e to sugar [1 5 ].
This in d ic a te s th a t the hem icellulose-
lig n in b a r r ie r is not always the major in h ib it o r o f h y d ro ly s is .
Saddler e t a l . [ 8] and Fan e t a l . [16] have studied the e ffe c ts o f
c e llu lo s e
c r y s t a l li n i t y
on degree
of
h y d ro ly s is .
They separately
reported th a t c r y s t a l li n i t y may represent a major block to c e llu lo s e
conversion.
They
found
th a t
h ig h ly
c r y s ta llin e
c e llu lo s e
re s is ts
h y d ro ly s is , whereas amorphous c e llu lo s e is re a d ily hydrolyzed.
experiments
sub strate
show
th a t
as
c r y s t a l li n i t y
a
h y d ro ly s is
increases.
proceeds,
This
the
suggests
T h eir
,unhydrolyzed
th a t
amorphous
regions o f c e llu lo s e are p r e fe r e n tia lly hydrolyzed lea vin g c r y s ta llin e
sub strate in ta c t.
As the c r y s t a l li n i t y increases, the ra te o f conver­
sion slows [1 0 ,1 6 ].
Although these researchers have placed c r y s t a llin ­
i t y high on the l i s t o f b a rrie rs to h y d ro ly s is , they agree th a t i t is
not the g re a te st block to h yd ro ly s is [8 ,1 0 ].
T h e ir data c o rre la tio n s
seem to in d ic a te th a t surface area has the la rg e s t a ffe c t on hyd roly­
s is .
G re th le in [17] has suggested th a t the enzyme-catalyzed h yd rolysis
o f c e llu lo s e is not dependent on the c r y s t a llin it y
in c e llu lo s e .
He
belie ves surface area is the most im portant c h a ra c te ris tic c o n trib u tin g
to
the
ra te
of
surface
area
is
h y d ro ly s is .
d ir e c t ly
The enzymatic
h y d ro ly s is
dependent on the pore
size
fo r
a given
d is tr ib u tio n .
There may be a larg e surface area, but pore size may l i m i t the amount
o f surface accessible to enzymatic a tta c k .
G re th le in discusses a pore
size o f 51 X based on the size o f h y d ro ly tic enzymes.
Acid molecules are g e n e ra lly very small in comparison w ith enzyme
molecules.
an e ffe c t
Therefore, pore size lim ita tio n s should not have as great
on the
ra te
o f acid
h y d ro ly s is .
Several
researchers at
Dartmouth College have found the ra te constants fo r acid h y d ro ly s is o f
10
c e llu lo s e to glucose fo r a wide v a rie ty o f su b strates.
These constants
are a ll w ith in the same order o f magnitude at a given temperature and
acid co n ce n tra tio n .
The substrates varie d in c r y s t a l li n i t y ,
surface
area, and pore size d is tr ib u tio n [1 8 ].
Degree o f po lym eriza tion
o f c e llu lo s e
some b e lie ve has an e ffe c t on h y d ro ly s is .
chain
molecules
are
less
s h o rte r chain molecules.
o f c e llu lo s e
re s u lts
accessible
to
is
a c h a ra c te ris tic
th a t
T heir b e lie f is th a t long
h y d ro ly tic
agents
than
are
This lowered access to the g lu c o s id ic bonds
in slowed h y d ro ly s is rates and less conversion.
These researchers have shown th a t as chain length increases, hyd ro lysis
ra te decreases [1 9 ].
There is a general consensus th a t the hemic e llu lo s e - lig n in b a rrie r
slows the h y d ro ly s is o f c e llu lo s e , e s p e c ia lly when enzymes are used as
a c a ta ly s t.
A disagreement
c r y s t a l li n i t y
and
purpose o f th is
surface
research
e x is ts
area
p e rta in in g
e ffe c ts
study was to
to
c e llu lo s e
in v e s tig a te
the
degree th a t
h y d ro ly s is .
The
the e ffe c t o f a
pretreatm ent on degree and ease o f conversion o f c e llu lo s e to glucose.
This researcher was aware o f c r y s t a l li n i t y and surface area as in h ib i­
to ry fa c to rs , but the degree o f in d iv id u a l importance was not in v e s ti­
gated.
11
PRETREATMENTS TO ENHANCE HYDROLYSIS
D iffe re n t treatm ents p r io r to a h y d ro ly s is have been studied to
e ffe c t an increase in the s u s c e p tib ility o f lig n o c e llu lo s e to h yd ro ly­
t i c agents.
Physical
These pretreatm ents can be chemical o r physical in nature.
pretreatm ents
are p rim a rily
th a t reduce p a r tic le s iz e .
m illin g
o r g rin d in g
operations
Chemical pretreatm ents in vo lve su b jecting
the lig n o c e llu lo s e to a chemical agent.
Some pretreatm ents invo lve a
combination o f physical and chemical elements.
A novel method o f using physical treatm ents sim ultaneously w ith
h y d ro ly s is has been studied a t the U n iv e rs ity o f Montana.
T h e ir process
involved a simultaneous wet b a ll m illin g and enzymatic h y d ro ly s is o f
various c e llu lo s e sub stra te s.
This process continuously produces a c tiv e
s ite s f o r h y d ro ly tic agents to a tta ck [7 ].
Although physical p re tre a t­
ments may enhance the h yd ro lysis o f lig n o c e lIu lo s e s , they are very energy
in te n s iv e o p era tions.
b ility
o f th is
Therefore,
High energy consumption makes the economic fe a s i­
type o f pretreatm ent doubtful
f o r com m ercialization.
the prim ary work o f developing a pretreatm ent process has
been focused on chemical pretreatm ents.
combinations
of
chemical
and physical
Some studies are looking at
d is ru p tio n
of
lig n o c e llu lo s ic
s tru c tu re .
As stated above, chemical pretreatm ents in vo lve s u b je c tin g lig n o ­
c e llu lo s e to a chemical agent.
Some o f the more common agents are s o l­
u tio n s o f acids o r a lk a li, organic so lve n ts, and hot w ater, both liq u id
and vapor.
A prim ary advantage o f chemical pretreatm ents, over physical
pretreatm ents, is the a b i li t y o f some chemicals to remove lig n in from
the s u b s tra te .
Chemical treatm ents can also swell the carbohydrate-
lig n in m a trix , thus decreasing the mass tra n s fe r lim ita tio n s in h y d ro l­
y s is .
A lk a lin e s o lu tio n s (o f which NaOH is the most common) are thought
to increase h yd ro lysis by sw e llin g the s u b stra te .
Although water can
swell lig n o c e llu lo s e , i t does so p r im a rily by e n terin g regions between
c r y s ta llin e
u n its .
Sodium Hydroxide expands the c e llu lo s e m a trix by
12
breaking bonds in the c r y s ta llin e u n its o f the c e llu lo s e re s u ltin g in
an increase in the amount o f amorphous c e llu lo s e
[2 0 ].
Gharpuray et
a I • have shown th a t ca u stic removes both lig n in and hem icellulose in
a d d itio n to i t s
s w e llin g a ctio n
[1 0 ].
This component removal allows
g re a te r access to the c e llu lo s e molecules by in cre asing surface area
and pore s iz e .
One problem w ith c a u s tic pretreatm ents is the consump­
tio n o f a lk a li during the pretreatm ent.
This loss o f reagent de tra cts
from the economics o f a process using c a u s tic pretreatm ents [ 21] .
D ilu te acid pretreatm ents a t elevated temperatures have been shown
to increase the h yd ro lysis ra te o f the pretreatm ent re sid ue .
This type
o f pretreatm ent could be c a lle d a p re h yd ro lysis since i t hydrolyzes the
hem icellulose fr a c tio n ,
hem icellulose
e x is ts .
is
thus removing i t
removed,
from the m a trix .
I ig n in -h e m ic e llu lo s e
bonding
Since the
no
longer
This c o n d itio n allow s the lig n in to be removed by s o lv a tio n i f
so d e sire d .
G re th le in e t a l . noted however th a t d e li g n i f i ca tio n was
unnecessary [1 8 ].
The d is ru p tio n o f the carbohydrate-1ignin m a trix by
d ilu t e acid was s u ff ic ie n t to render the residue h ig h ly susce ptible to
an enzymatic h y d ro ly s is . Acid pretreatm ents increase the pore volume
a llo w in g
g re a te r
p re tre a te d
pe n e tra tio n
su b stra te s.
of
c e llu la s e
enzyme
molecules
in to
The increased access o f enzymes re s u lts
in
higher ra tes o f c e llu lo s e to glucose conversion.
Engineers a t the U n iv e rs ity o f Arkansas have studied the use o f
moderate tem perature, d ilu t e acid p re h yd ro lysis in an acid hyd ro lysis
process.
This
s u b s ta n tia l
product degradation.
concentrated
acid
pretreatm ent
h yd ro lysis
hydrolyzed hem icellulose
degradation product,
used.
hydrolyzes
o f the
if
c e llu lo s e
products
o f glucose,
re sid ue .
would be la rg e ly
w ith o u t
follow ed by a
The e a s ily
converted to
its
on ly a concentrated acid step was
F u rfu ra l and 5-hydroxymethyl fu r fu r a l
degradation
hem icellulose
The p re h yd ro lysis is
fra c tio n
f u r f u r a l,
the
(HMF), the acid catalyzed
are poisonous to yeast c e lls
th a t
would ferment sugars to ethanol o r o th e r products [ 22] .
A j o i n t p ro je c t between the U n iv e rs ity o f Pennsylvania and General
E le c tric Corporation has developed a solvent d e lig n ific a tio n p re tre a t­
ment.
This pretreatm ent involves a hot aqueous ethanol treatm ent o f a
13
s u b s tra te .
This re s u lts in a s o lid c o n s is tin g o f c e llu lo s e , p a r t ia lly
degraded hemic e llu lo s e , and lig n in
t ic a lly
hydrolyzed and re s u lts
[2 3 ].
This residue is then enzyma­
in c e llu lo s e conversions o f 80 to 90%
[I].
The pretreatm ent schemes th a t seem most prom ising are those th a t
in vo lve a steam o r high temperature water treatm ent.
are known as autohydrolyses.
These processes
The term autohydrosis comes from the fa c t
th a t in the presence o f water a t an elevated temperature acetyl groups
in
hemi c e llu lo s e
(are thought to )
break down and form a c e tic a cid .
Therefore, an automatic acid catalyzed h y d ro ly s is takes place in the
pretreatm ent
m ixtu re .
During
so lva ted.
A b e n e fit o f autoh ydro lysis is th a t considerable amounts o f
be
removed
hydrolyzed [2 4 ].
rendered
w h ile
the
in to
a form th a t
hemic e llu lo s e
is
is
are
and the
can
is
hemic e llu lo s e s
hydrolyzed
lig n in
lig n in
an autoh ydro lysis
e a s ily
sim ultaneously
Rupture o f the p ro te c tiv e lig n in - c e llu lo s e linkage is
s u ff ic ie n t to a llo w a r e la tiv e ly easy h y d ro ly s is o f the c e llu lo s e .
re sid ua l
c e llu lo s e
from
autoh ydro lysis
can
then
be
The
enzym atically
converted to glucose w ith y ie ld s o f 90 to 95% [2 5 ].
A pretreatm ent re la te d to a u toh ydro lysis is the steam explosion o f
lig n o c e llu lo s e .
In
th is
process
a substrate
is
subjected
to
high
pressure steam («500 p s ig ), cooked a t th is temperature fo r a sho rt time
(«5 seconds), and then the m ixture is flashed to atmospheric pressure.
This explosive fla s h in g g re a tly d is ru p ts the s tru c tu re o f the c e llu lo s e
in cre asing
the
pore
volume and surface
area o f
the
su b stra te .
process th a t uses th is pretreatm ent is the Iotech process.
A
In th is
procedure, the steam explosion is follow ed by an enzymatic h yd rolysis
w ith c e llu lo s e conversions o f about 90% and hemic e llu lo s e conversions
of. approxim ately
80%.
The lig n in
need not be e xtra cte d
from the
sub strate but can be l e f t to be removed a t the end o f the h y d ro ly s is .
Of a ll the pretreatm ents and processes discussed here, the use o f
a d ilu t e
acid
pretreatm ent,
a u to h y d ro ly s is ,
o r the
steam explosion
technique o f the Iotech process seem the most com mercially prom ising.
14
EXPERIMENTAL
This sectio n describes the substrates th a t were used and the ex­
perim ental procedures th a t were follow ed during th is study.
Substrates
Substrates used fo r th is
in v e s tig a tio n were wheat straw , ba rley
straw , lodge pole pine chips, Douglas f i r chips, and Chromedia.
Wheat
straw was a spring wheat o f the Pondera v a rie ty and the ba rle y straw
was C la rk 's b a rle y .
Two bales o f each o f these substrates were obtained
from Larry Van Dyke o f Manhattan, Montana.
in November o f 1982.
The straws were harvested
Lodge pole chips were supplied by the Brand-S
Lumber Company o f L ivin g sto n , Montana.
W illow Creek Lumber Company,
also o f L iv in g s to n , provided the Douglas f i r chips.
v a r ie tie s o f woods were sawmill
tim b er in December o f 1983.
The chips o f both
residues from the harvesting o f liv e
I t was approxim ately fo u r weeks between
the tim b er harvest and the a c q u is itio n o f the chip s.
A ll fo u r o f the
above substrates were stored in sealed p la s tic bags u n til used.
s u b stra te , Whatman Chromedia, was used in several experiments.
A fifth
This
m a teria l was e s s e n tia lly a pure c e llu lo s e in powdered form designed fo r
use as a column packing in chromatography. The Chromedia was manufactured
by W & R B a!ston, LTD o f England.
A ll o f the su b stra te s, except the Chromedia, were prepared fo r use
as fo llo w s .
hours.
F ir s t, the m a te ria ls were a i r d rie d fo r approxim ately 48
Next, the substrates were m ille d in a W iley hammer m ill equipped
w ith a I -mm discharge screen.
The m ill was. stopped p e rio d ic a lly and
allowed to cool to prevent overheating o f m aterial being ground so th a t
sub strate would re ta in i t s natural in t e g r it y .
The m ille d m a terial was
then screened to is o la te the 35 to 60 mesh fr a c tio n .
AU ASTM and TAPPI
standard methods required th is size fra c tio n fo r a n a ly s is .
was used in a ll
This fra c tio n
subsequent c h a ra c te riz a tio n s and experiments.
A fte r
g rin d in g and screening, the substrates were stored in containers open
to the atmosphere.
15
C ha ra cte riza tio n o f Substrates
Substrates
used
fo r
th is
research
pretreatm ent experiments were performed.
ized
by
r e la tiv e
amounts
of
ash
were
characterized
before
The m a te ria ls were cha racte r­
content,
m oisture,
e x tr a c tiv e s ,
lig n in , c e llu lo s e and hemic e llu lo s e .
Ash
The ash content in the substrates was determined using a s lig h t
v a ria tio n
[2 6 ].
in the ASTM D 1102-56 Standard Test Method f o r Ash in Wood
This procedure involved weighing about 2 grams o f the substrate
in a ta re d , p re v io u s ly ig n ite d
(a t 600°C) p o rce la in c ru c ib le and l i d .
The m a teria l was weighed to the nearest 0.1 m illig ra m .
and detachable
I id
were placed
temperature below IOO0C.
in
The c ru c ib le
an ashing oven w ith
The oven was slow ly heated to
a s ta rtin g
the 600°C
ashing temperature and allowed to remain there fo r 30 minutes.
At the
end o f th is period the l i d was placed on the c ru c ib le and the c ru c ib le
was then
tra n s fe rre d
to
a dryin g
oven to
co o l.
(The dryin g oven
temperature was maintained between IOS0C and IlO 0C.)
were cooled fu r th e r in
m illig ra m .
a d e sicca to r and weighed to
The c ru c ib le s
the nearest 0.1
The percent ash was corrected fo r m oisture in the i n i t i a l
sample and reported to the nearest 0. 01%.
M oisture
The m oisture a n alysis used ASTM D 1102-56 Standard Test Method fo r
Ash in Wood [2 6 ].
it
This te s t s p e c ific a lly analyzes f o r ash content but
also describes a procedure fo r determ ining m oisture content.
The
standard method e n ta ile d weighing a sample o f substrate to the nearest
0.1 m illig ra m using a tared glass weighing v ia l.
The v ia l containing
the sample was placed in an oven c o n tro lle d between IOS0C to IlO 0C.
Two d iffe r e n t procedures were used from th is p o in t.
The f i r s t
m a teria l
procedure follow ed the standard method by drying the
fo r 2 hours and then tra n s fe rrin g the v ia l
d e s ic c a to r.
unstoppered to a
The v ia l was allowed to cool and was then stoppered and
reweighed to the nearest 0.1 m illig ra m .
The v ia l was unstoppered and
16
returned to the oven f o r one hour, then cooled in the d e s ic c a to r and
reweighed.
obtained.
It
This procedure was repeated u n til
was found th a t
a ft e r
about
fo u r
samples had reached a constant w e ig h t.
hours
of
oven drying
the
Therefore, f o r convenience a
second method o f m oisture content was used.
was kept in the oven ove rnig ht
a constant weight was
For th is method, the v ia l
(e ig h t to twelve hours),
fe rre d from the oven to a d e sicca to r f o r c o o lin g .
then tra n s ­
When the v ia l was
co o l, i t was stoppered and weighed to the nearest 0.1 m illig ra m .
With
e ith e r
c a lc u la te d
samples.
as
method
the
the
Weight
amount o f
loss
m oisture
between
the
in
d rie d
the
and
sample was
the
undried
The m oisture is reported to the nearest 0.1%.
E x tra c tib le s
The e x tr a c t!bles were defined as the components in the lig n o c e llu loses so lu b le in benzene-ethanol, e th a n o l, and w ater.
These components
are gums, re s in s , waxes, and in some substrates, ca te ch o l.
fo r
e x tr a c tiv e s
used
TAPPI
T 12 os-75,
Preparation
The te s t
of
Wood fo r
Chemical A nalysis (In c lu d in g Procedures o f Removal o f E x tra c tiv e s and
D eterm ination o f M oisture Content) [2 7 ].
Approxim ately 80 grams of, the sub strate was weighed to the nearest
0.01
gram.
The substrate
was then
placed
in to
a 250 ml
e x tra c tio n thim ble w ith a coarse f r i t t e d glass d is c .
placed
in
a Soxhlet e x tra c tio n
apparatus
and the
Soxhlet
This thim ble was
solve nt re s e rv o ir
charged w ith 350 ml o f benzene and 175 ml o f 95% e th an ol.
v o ir was placed in a V a ria c -c o n tro lle d heating mantle.
The re se r­
The Variac was
adjusted so the b o il-u p ra te o f the e x tra c tio n solve nt re s u lte d in 6 to
7 re flu x e s per hour.
The e x tra c tio n was continued fo r 6 hours.
At the
end o f th is p e rio d , the substrate was suctioned to remove as much o f
the so lve n t as p o s s ib le .
The substrate was then washed w ith fresh
ethanol and suctioned d ry.
A second e x tra c tio n was performed by charging the re s e rv o ir w ith
95% ethanol w h ile m aintaining 6 to 7 re flu x e s per hour as before .
This
17
e x tra c tio n on ly proceeded f o r 4 hours.
The sub strate was then suc­
tion ed dry and washed w ith deionized w ater.
was s p l i t in to two s im ila r p o rtio n s .
At th is time the sample
The two p o rtio n s were placed in
500 ml
Erlenmeyer fla s k s
added.
They were kept a t SO0C fo r one hour w ith fre que nt s t ir r in g v ia
a glass rod.
and 500 ml o f b o ilin g deionized water was
The substrate was f ilt e r e d
allowed to a i r d ry .
from the w ater e x tra c t and
A m oisture a n alysis was performed on the a ir d rie d
e xtracte d sub strate (e x tra c t-fre e s u b s tra te ).
bles
was determined
m a teria l
by
and e xtracte d
a simple weight
m a te ria l.
The amount o f e x tra c ti -
loss
between
E x tra c tib le s
were
the
s ta r tin g
reported
on a
m o is tu re -fre e basis to the nearest 0. 1%.
Lignin
The next c o n s titu e n t analyzed f o r was lig n in .
The method used was
a v a ria tio n o f ASTM D 1104-56 Standard Test Method f o r H oloce llulose in
Wood as described by Browning [2 8 ].
The method involved weighing about
2.5 grams o f the e x tra c t-fre e substrate to the nearest 0.1 m illig ra m .
The sample was placed in a 30 ml coarse p o ro s ity f r i t t e d glass f i l t e r ­
ing
fu n n e l.
c h lo rin e
The funnel
gas was slow ly
was placed
in
passed through
a bath o f
ic e
the
(see
sample
water w hile
Figure
5 ).
A fte r 3 minutes, the c h lo rin e gas flo w was in te rru p te d so the sample
could be s tir r e d w ith a glass rod.
continued fo r 2 more minutes.
funnel to cover the sample.
dioxane suctioned o f f .
The c h lo rin e gas flo w was then
Enough 1,4-dioxane was added to the
The ic e water was then drained and the
Next the sample was washed tw ice w ith a SO0C
s o lu tio n o f 5% monoethanol amine in 1,4-dioxane.
Each wash was allowed
to remain f o r 2 minutes before being suctioned o f f .
The sample was
then washed tw ice w ith room temperature 1, 4-dioxane.
The
sample was tra n s fe rre d
to
a 60 ml
fr itte d
glass
Buchner
f i l t e r i n g funnel o f coarse p o ro s ity and washed tw ice w ith room tempera­
tu re deionized w ater.
o r ig in a l
clo g g in g .
fu n n e l.
This
The sample was then tra n s fe rre d back to the
procedure
prevented
the
fr itt e d
discs
from
The funnel was reassembled in to the c o n fig u ra tio n necessary
fo r use o f the c h lo rin e .
The gas was passed through the sample fo r 3
18
6 m m GLASS TUBE
rPM RUBBER STOPPER
52 mm (ID ) GLASS TUBE
IO cm
FRITTED GLASS CRUCIBLE
■26 mm
^ ll
RUBBER STOPPER
RUBBER
RFS RUBBER
CEMENT
STOPPER
6 mm GLASS
TU B E
12 mm GLASS TUBE
S U C TIO N
Figure 5.
FLA S K
Funnel Setup used fo r Lig nin Determination.
19
minutes and the washing, procedure repeated.
This sequence o f c h lo rin a tin g and washing was continued u n til the
sub strate
no longer
underwent a c o lo r change upon monoethanol amine
s o lu tio n a d d itio n o r u n til the substrate was w h ite .
is
th a t
associated w ith
the 5% ethanol amine wash.
The c o lo r change
The sample was
washed w ith dioxane u n til neutra l («7 pH) and then washed tw ice w ith 30
ml a liq u o ts o f d ie th y l
e th e r.
A fte r the eth er wash, the sample was
washed w ith deionized water and tra n s fe rre d to a tared P e tri dish fo r
a i r d ry in g .
The lig n in
weight loss
sample.
between the
content was determined as the m o istu re -fre e
e x tra c t-fre e
substrate
and the
d e lig n ifle d
C ellulo se and Hem icellulose
The percentage o f to ta l c e llu lo s e was determined by assuming th a t
the mass not
c e llu lo s e .
accounted
fo r
by the
lig n in ,
m oisture,
and ash was
The procedure used to analyze fo r the r e la tiv e amounts o f c e llu ­
lose and hem icellulose was TAPPI standard method T 203 os-74, Alpha-,
Beta-,
and Gamma-Cellulose in
Pulp
[2 9 ].
It
was assumed th a t the
a lp h a -c e llu lo s e fra c tio n was c r y s ta llin e c e llu lo s e , the b e ta -c e llu lo s e
was amorphous c e llu lo s e , and the gamma-cellulose was hem icellulose.
This method suggests use o f 1.5 gm o f d e lig n ifle d pu lp, weighed to
the nearest 0.1 m illig ra m .
However, i t was not p o ssible to obtain 1.5
gm o f pulp during an actual experimental run due to the small amounts
o f sample th a t could be generated.
sm a lle r amount o f sample used.
The reagents were adjusted fo r the
The fo llo w in g procedure was based on
1.5 grams o f sample.
The sample was placed in a 125 o r 250 ml Erlenmeyer fla s k w ith 100
ml o f 5.21 ± 0.005 N carbonate-free sodium hydroxide (NaOH).
was
placed
on
a magnetic
s tir r e r
suspension o f the pulp in the NaOH.
insure th a t no a ir was drawn in to
and
s tir r e d
to
insure
The fla s k
complete
S tir r in g was done in a manner to
the m ixtu re.
The fla s k was then
placed in a water bath w ith the temperature maintained a t 25 ± 0.2°C.
20
When 30 minutes had passed, 100 ml o f deionized water was added to the
pulp-NaOH m ixtu re .
The d ilu te d m ixture remained in the water bath fo r
30 m inutes.
The m ixtu re, w hile in the bath, was s tir r e d o cca sio n a lly
w ith a glass s t ir r in g rod.
A fte r the 60 minute p e riod ,
the mixture, was f ilt e r e d
coarse p o ro s ity Buchner f i l t e r i n g fu n n e l.
through a
The f i r s t 10 to 20 ml o f the
f i l t r a t e was discarded and the re s t o f the f i l t r a t e was c o lle c te d fo r
la t e r a n a ly s is .
A lpha-C ellulose A nalysis
ten m i l l i l i t e r s
Ten m illit e r s o f the c le a r f i l t r a t e
and
o f 0.5 N potassium d ichromate (K2Cr2O7) s o lu tio n were
placed in a 250 ml Erlenmeyer fla s k .
T h irty m illit e r s o f concentrated
s u lfu r ic acid (H2SO^) were slow ly added to the m ixture w h ile the fla s k
was s w irle d .
The re s u ltin g
F if t y m ill i t e r s
s o lu tio n
was kept hot f o r
15 minutes.
o f deionized water were then added and the fla s k was
cooled in a room temperature water bath.
A blank was made using 12.5 ml o f deionized water and 12.5 ml o f
the 5.21 N (17.5% by weight) NaOH.
F if t y m illit e r s
o f concentrated
s u lfu r ic acid were slow ly added to the blank.
The pulp
filtr a te
ammonium s u lfa te
and blank were t it r a t e d
C F e rrio n ')
p o te n tio m e tric
end
by
N ferrou s
A phenanthroline (C12H8N2) -fe rro u s s u lfa te
in d ic a to r s o lu tio n was also used to
confirm the
p o in t.
s o lu tio n
Ferrous
ammonium
s u lfa te
unstable and was standardized before each use.
accomplished
0.1
(Fe(NH4) 2 (SO4) 2) s o lu tio n w ith the end p o in t d e te r­
mined p o te n tio m e trical I y .
(FeSO4)
w ith
a
p o te n tio m e tric
titr a tio n
S tandardization was
w ith
0.1
N potassium
d ichromate s o lu tio n .
The a lp h a -c e llu lo s e percentage was ca lcu la te d as fo llo w s :
A lp h a -c e llu lo s e % = 100 -
where
is
6.85 (V2__=_Vi) x N x 20
V1 = T itr a tio n o f pulp f i l t r a t e , m i l l i l i t e r s
V2 = Blank t i t r a t i o n , m i l l i l i t e r s
N = Exact n o rm a lity o f the fe rro u s ammonium s u lfa te s o lu tio n
21
A = T itr a tio n o f the pulp f i l t r a t e used in th e o x id a tio n ,
m illilit e r s
W = Oven-dry weight o f the pulp specimen, grams
Beta- and Gamma-Cellulose A nalysis
F if t y m ill i t e r s
o f the pulp
f i l t r a t e were p ip e tte d in to a 100 ml graduated c y lin d e r equipped w ith a
ground glass stopper.
F if t y m illit e r s o f 3 N s u lfu r ic acid were added
to the c y lin d e r and w ith the stopper in place the c y lin d e r was in ve rte d
several times to insure complete m ixing.
The c y lin d e r was placed in an
BO0C water bath fo r several minutes to aid the coagulation o f betac e llu lo s e ,
which p re c ip ita te s
e ith e r allowed to
p r e c ip ita te .
upon a c id if ic a tio n .
The c y lin d e r was
stand ove rnig ht o r was c e n trifu g e d
to
s e ttle
the
F if t y m ill i t e r s o f the c le a r supernatant liq u id was p ip itte d o f f ,
being c a re fu l th a t none o f the p re c ip ita te was removed.
placed in a 250 ml Erlenmeyer fla s k .
d ichromate
s u lfu r ic
were
added to
the
acid were c a r e fu lly
fla s k .
The liq u id was
Ten m illit e r s o f 0.1 N potassium
Then 90 ml
poured in to
the
fla s k
of
concentrated
w h ile
s w irlin g .
This s o lu tio n was kept hot f o r 15 minutes and then cooled in a room
temperature w ater bath.
A blank was prepared in the same manner except
a s o lu tio n o f 12.5 ml o f deionized w ater, 12.5 ml o f 17.5% NaOH, and 25
ml o f 3 N s u lfu r ic
liq u id .
acid was s u b s titu te d
fo r the c le a r supernatant
These s o lu tio n s were t it r a t e d by the same method used du ring the
a lp h a -c e llu lo s e
d e term inatio n.
The r e la tiv e
fra c tio n s
o f beta- and
gamma-cellulose were ca lcu la te d as fo llo w s :
Gamma-cellulose % = 100 - — ^
^ x 20
Zo
where
X
W
V3 = T itr a tio n o f the s o lu tio n a ft e r p r e c ip ita tio n o f
b e ta -c e llu lo s e , m i l l i l i t e r s
V4 = Blank t i t r a t i o n , m i l l i l i t e r s
B e ta -c e llu lo s e
%
= 100 - ( a - c e llu lo s e % + 7-c e llu lo s e %).
22
A uto hydrolysis and Lig nin E xtra ctio n
The
c a rrie d
a u to h yd ro lysis
out in
ca p a c ity .
an Autoclave
lig n in
e x tra c tio n
pretreatm ents
Engineers ZipperClave w ith
were
a one l i t e r
The autoclave has two 500-watt heating bands and was rated
to 2000 psi a t 450°F.
pressure vessel
Temperature
Model
and
to
in s id e
8530 d ig it a l
An a ir driven a g ita to r s h a ft extended in to the
s tir
the
the
re a ctio n
m ixture during
autoclave was monitored
an experiment.
using a Cole Palmer
thermometer and a Type K (Cromel-Alumel)
thermo­
couple in s ta lle d in a s ta in le s s stee l therm owell.
Substrate was held in e ig h t small packets th a t were made o f 200
mesh s ta in le s s stee l w ire c lo th fastened w ith f l a t pieces o f aluminum
(Figure 6) .
These packets were held in a ra d ia l
metal d is c s .
The aluminum closures were f i t t e d
the d is c s .
arrangement by two
in to holes d r ille d in
The two discs were separated by a 1/2 ID s ta in le s s steel
pipe (Figure 6) .
This pip e, w ith the e ig h t packets o f su b stra te , was
mounted on the a g ita to r sh a ft o f the autoclave.
The packets could then
be ro ta te d during the run to minimize mass tra n s fe r lim ita tio n s th a t
might a ffe c t e ith e r the autoh ydro lysis o r the lig n in e x tra c tio n .
A 500 ml Parr bomb was plumbed to the autoclave and served as a
high pressure steam generator.
Plumbing from the bomb to the autoclave
was low pressure s ta in le s s steel tub ing w ith Swagelock f i t t i n g s .
This
lin e had an Autoclave Engineers low pressure valve w ith high tempera­
tu re packing (model number 6V81U4TG) to c o n tro l the in je c tio n o f steam.
The steam lin e
was heated w ith heat tape to prevent co o lin g o f the
steam during steam in je c tio n s .
Cooling o f the autoclave was a tta in e d
by venting to a cold tra p submerged in an ice bath.
Venting was con­
tr o lle d by a valve o f the same type used in the steam lin e .
Figure 7
is a diagram o f th is experimental set up.
Autohydroly sIs
The tared w ire mesh packets were f i l l e d
s tra te and weighed to 0.1 m illig ra m .
w ith e x tra c t-fre e
sub­
Each packet held about one gram;
th e re fo re , approxim ately e ig h t grams o f substrate were pre tre a te d per
23
Agitator
shaft
L
" I
Wire cloth
"envelopes"
\
Figure 6.
/
. ■- ,
I
\
■ . ........J
Sample Basket Apparatus.
Air driven
agitator
Valve
Pressure
gauge
Valve
Parr
bomb
Thermocouple
Sparge
tube
Sample
basket
Dewar's
Ilask
Figure 7.
Bomb
heater
I liter
autoclave
Setup used fo r Autohydrolysis and E xtraction Experiments
25
run.
The packets were assembled and mounted on the a g ita to r s h a ft.
E ith e r 300 o r 350 ml o f deionized water were charged to the Parr bomb
and heated to a pressure o f 1200 psig (1500 psig f o r some ru n s).
autoclave was charged w ith
ISO0C.
600 ml o f deionized w ater and heated to
This volume was required to
thermowell were submerged.
The
insure th a t the sample and the
A temperature o f ISO0C was chosen because
s im ila r work by Waymen and Lora in d ica te d th a t a t and below 150°C the
su b strate does not undergo any s ig n ific a n t change [3 0 ].
Once the bomb was a t the desired pressure and the autoclave at
150°C, the a g ita to r was s ta rte d and adjusted to 300 rpm.
then released in to the autoclave.
The in je c tio n o f high pressure steam
allowed fo r very ra p id heat-up o f the s u b stra te .
was desired
so
a sce rta in e d .)
th a t
accurate
Steam was
(This ra pid heating
tim e-at-tem perature
e ffe c ts
could
be
When the autoclave reached operating temperature, the
steam in je c tio n
was discontinued.
Temperature o f the autoclave was
maintained w ith in ± 2°C o f the chosen operating value by venting vapor
to cool o r adding steam to heat.
When the predetermined time had elapsed the autoclave was vented
to the cold tra p , being ca re fu l not to a llo w any uncondensed vapors to
escape the
autoclave
tra p .
and
This procedure allowed
its
contents.
Average
fo r ra p id
heat-up
and
co o lin g o f the
cool-down
times
observed were 141 ± 65 seconds and 75 ± 15 seconds, re s p e c tiv e ly .
Once the autoclave had been p ro p e rly vented i t could be opened and
the assembly o f packets removed.
f i ca tio n te s ts ,
the o th e r fo u r packets were opened and the contents
q u a n tita tiv e ly removed.
copious
Four packets were saved fo r d e lig n i-
The autohydrolyzed substrate was washed w ith
amounts o f deionized w ater,
w ater, and then allowed to a ir d ry .
suctioned to
remove the excess
When the sub strate was a ir dry i t
was cha racte rized as described e a r lie r .
However, the ethanol-benzene
e x tr a c tiv e s , te s t was not run.
The amount o f liq u id
in the autoclave, cold tra p , and Parr bomb
was measured to tra ce the liq u id 's movement during the a u to h yd ro lysis.
A ir
d rie d
te s tin g .
m a teria l
was
sealed
in
a sample
b o ttle
fo r
subsequent
26
Delig n if ic a tio n
The
next
step
in
the
pretreatm ent
process
was
an
a lc o h o lic
e x tra c tio n to remove lig n in th a t was rendered solu ble by the autohy­
d ro ly s is .
A 50:50 volu m etric s o lu tio n o f 95% ethanol
water was used.
and deionized
The fo u r remaining packets from the a u toh ydro lysis were reassem­
bled in to t h e ir holder and placed in the autoclave.
The cold tra p was
reconnected to the system and as before placed in an ic e bath.
Six
hundred m il l i t e r s o f the 50:50 ethanol-w ater s o lu tio n were added to the
autoclave, the autoclave was heated to ISO0C1 and the a g ita to r adjusted
to 300 rpm.
Temperature was c o n tro lle d by the methods used during the
a u to h y d ro ly s is .
The autohydrolyzed substrate was l e f t in the alcohol
m ixture fo r one hour.
At the end o f the hour the autoclave was vented
to the cold tra p , the autoclave was opened, and the contents removed.
The sub strate was q u a n tita tiv e ly washed from the pouches w ith deionized
w ater, vacuum f ilt e r e d to remove the excess w ater, and then a ir d rie d .
Once the sub strate was a ir dry i t was cha racterized using the proce­
dures p re v io u s ly described.
mined.
Again, the e x tr a c tiv e s were not d e te r­
The a i r d rie d sample was then sealed in a sample b o ttle fo r fu tu re
experiments.
B all M illin g
Chromedia,
one o f the
substrates
used,
was b a ll
attem pt to produce an amorphous c e llu lo s ic m a te ria l.
m ille d
in
an
The b a ll m illin g
was c a rrie d out w ith both wet and dry su b stra te .
The i n i t i a l
Chromedia su b stra te ,
the dry b a ll
m ille d Chromedia,
and the wet m ille d Chromedia were analyzed to determine the re la tiv e
amounts o f the various c e llu lo s e types present.
were performed on a ll three m a te ria ls .
Acid h y d ro ly s is te s ts
Since Chromedia is e s s e n tia lly
pure c e llu lo s e , lig n in determ inations were unnecessary.
27
Dry B all M illin g
Steel
b a ll
bearings o f various sizes
(1/4 inch diameter to
inch diam eter) were used to m ill the dry Chromedia.
1/2
The b a ll bearings
were loaded in to a p o rce la in g rin d in g j a r and Chromedia was added to
fill
the void spaces between the b a lls .
The to ta l
the j a r was approxim ately one t h ir d o f i t s volume.
and placed on a tum bler ro ta tin g
m inute.
space occupied in
The j a r was sealed
between 30 and 40 re v o lu tio n s per
The temperature was approxim ately 20°C.
Total m illin g time
was 96 hours w ith p e rio d ic down times to scrape the sub strate from the
corners o f the j a r .
The dry b a ll- m ille d substrate was stored in Nalgene sample ja r s .
Wet B all M illin g
Wet b a ll m illin g was c a rrie d out using p o rce la in c y lin d e rs (20 mm
by 20 mm) in the same j a r used in the dry m illin g o p e ra tio n .
Chromedia
was mixed w ith deionized water to form a s lu r r y .
The s lu r r y was vacuum
f ilt e r e d
in
to
remove excess w ater,
then
placed
the
ja r
w ith
the
c y lin d e rs .
Chromedia s lu r r y was added to ju s t cover the porcelain
c y lin d e rs .
The j a r was f i l l e d , as before to about o n e -th ird cap acity.
The j a r was ro ta te d w ith the same v e lo c ity and the same length o f time
as the dry m illin g o p e ra tio n .
The m illin g operation was again stopped
p e r io d ic a lly to scrape the m a terial from the corners o f the j a r .
Once
the 96-hour period had passed the sub strate s lu rr y was washed in to a
fla s k and c e n trifu g e d .
The water was decanted o f f and the Chromedia
tra n s fe rre d to glass sample ja r s so i t could be stored wet fo r fu tu re
a n a ly s is .
Acid and Enzymatic H ydrolysis
P retreated substrates were tested to asce rtain the r e la tiv e ease
o f h y d ro ly s is among e x tra c t-fre e ,
autohydrolyzed, and autohydrolyzed.r
a lc o h o lic
The
e xtracte d
m a te ria ls .
substances
several d iffe r e n t means during the hydrolyses.
were
hydrolyzed
by
One o f the methods was
a two step acid h y d ro ly s is using e ith e r s u lfu r ic o r h y d ro c h lo ric a cid .
The o th e r methods were various enzymatic hydrolyses using e ith e r s in g le
28
enzymes o r mixed enzymes.
Acid H ydrolysis
A v a ria tio n o f ASTM D 1106-56 Standard Test Method f o r Lignin in
Wood [31] was used f o r the acid hydrolyses.
This te s t uses a two stage
acid h y d ro ly s is in which the co n d itio n s are strong enough to hydrolyze
a ll
of
the
behind.
polysaccharrides
S e ve rity
fra c tio n
of
the
of
the
leaving
co n d itio n s
c e llu lo s e
would
the
lig n in
and
were lessened
hydrolyze.
ash
fra c tio n s
so th a t
only
These co n d itio n s
a
were
a rriv e d a t using a t r i a l and e rro r experimental procedure.
C onditions were sought th a t would re a d ily hydrolyze lig n in - fr e e
su b stra te , but would not hydrolyze e x tra c t-fre e sub strate to any great
e x te n t.
These
co n d itio n s
were
not
fu lly
achieved
(a t
le a s t
w ith
the
c o n d itio n s th a t were attem pted), so co n d itio n s were chosen where the
experimental work-up o f the sample was e a s ie s t.
co n d itio n s
increased
in
s e v e rity ,
c o llo id a l
These suspensions were very d i f f i c u l t
work-up d i f f i c u l t .
to
As the acid hyd ro lysis
suspensions would form.
filte r ,
thus making sample
The co n d itio n s chosen f o r s u lfu r ic acid were:
1.
100:1 weight r a tio
acid s o lu tio n
2.
30°C fo r one hour
3.
D ilu te to 0.7 N acid strength
4.
SO0C f o r two hours
Acid
hydrolyses
were
o f actual
run
in
acid to sub strate using 16 N
a 250 ml
approxim ately 300 m illig ra m o f s u b s tra te .
h y d ro ly s is ,
sub strate
th a t
fla s k
w ith
When 2 hours had lapsed fo r
had not hydrolyzed was tra n s fe rre d
tared Gooch c ru c ib le w ith a coarse f r i t t e d
m a teria l
ErTenmeyer
d is c .
to
a
The non-hydrolyzed
was washed w ith copious amounts o f deionized water and the
c ru c ib le placed in a dryin g oven w ith the temperature between IOS0C and
IlO 0C.
The c ru c ib le was kept in the oven ove rnig ht before weighing.
The exte nt o f h y d ro ly s is was reported as the m o is tu re -fre e weight Toss
o f the sample.
29
Enzymatic Hydr o ly s is
Three methods o f
h y d ro ly s is
c e llu la s e
used
enzymatic
a c e llu la s e
and a c e llo b ia s e ,
h y d ro ly s is
enzyme,
were
another
and the
th ir d
used.
used
One method
a m ixture
of
a
used a m ixture o f three
enzymes, a c e llu la s e , a c e llo b ia s e , and a hemic e llu la s e .
The enzyme s o lu tio n s were made by mixing the desired amounts and
types o f enzymes w ith a 0.1 M c it r a t e b u ffe r s o lu tio n .
B u ffe r s o lu ­
tio n s o f the desired pH, 4 .8 , were mixed from 0.1 M stock s o lu tio n s o f
c i t r i c acid and sodium c it r a t e .
To ob tain a s o lu tio n th a t buffered to
a pH o f 4 .8 , 46 volume p a rts o f c i t r i c acid s o lu tio n were mixed w ith 54
volume p a rts o f sodium c it r a t e s o lu tio n .
About 100 m illig ra m s o f the substrate were placed in a 200 x 25 mm
te s t tube and wetted w ith one m i l l i t e r o f the c it r a t e b u ffe r .
w e ttin g was complete,
fo u r m illit e r s
were added to the su b stra te .
water bath
and s tir r e d
s t i r r e r and s t i r
ba r.
fo r
Once the
o f the desired enzyme s o lu tio n
The te s t tube was submerged in a SO0C
the
time
of
h y d ro ly s is
v ia
a magnetic
At the end o f the desired time p e riod ,
the
contents o f the tube was tra n s fe rre d to a 100 x 13 mm te s t tube and
c e n trifu g e d .
The
c le a r
supernatant
liq u id
was
frozen
fo r
fu tu re
reducing sugar a n a ly s is .
Reducing sugar was analyzed using a method described by M ille r
[3 2 ].
This te s t used a reagent re fe rre d to as M ille r 's reagent.
The
reagent was made from the fo llo w in g re c ip e :
77.75%
Deionized water
20%
Rochelle s a lt (Potassium sodium ta r tr a te )
1%
3 ,5 - d in it r o s a lic y I ic acid
1%
Sodium hydroxide
0.2%
Phenol
0.05%
Sodium S u lfite
For the
M ille r method,
one m i l l i t e r
o f te s t
sample and three
m il l i t e r s o f M ille r 's reagent were placed in a 100 x 13 mm te s t tube.
The te s t
tube was then placed
in
a b o ilin g water bath fo r fifte e n
minutes to develop the c o lo r f o r an o p tic a l d e n sity te s t .
A blank was
30
generated using two m illit e r s o f the b u ffe r s o lu tio n and s ix m illit e r s
o f the M ille r 's
reagent.
The o p tic a l d e n sity o f the re s u ltin g s o lu ­
tio n s was found using a Varian DMS 90 U V -V isible Double Beam Spectro­
photometer th a t was in te rfa c e d w ith an Apple I I
Plus computer.
wavelength was set a t 575 nm and the s l i t w idth a t 4 nm.
photometer
was
c a lib ra te d
using
standard
s o lu tio n s
The
The spectro­
of
D-glucose;
th e re fo re , the concentration u n its obtained were m illig ra m o f apparent
glucose per m illit e r s
sugars.
s o lu tio n
and not absolute amounts o f reducing
The f in a l u n its reported were m illig ra m apparent glucose per
gram o f dry sub strate o r m illig ra m o f apparent glucose per gram o f
to ta l c e llu lo s e in the sample.
The
M ille r 's
reagent
would
not
accu rate ly
in d ic a te
a
sugar
concentration higher than one mg/ml o f s o lu tio n .
Due to th is fa c t, the
spectrophotometer was
glucose
about 0.35 mg/ml.
s o lu tio n
u su a lly
c a lib ra te d
w ith
s o lu tio n s
of
Although a t times a less concentrated c a lib ra tio n
had to be employed, because a less concentrated hyd ro lysis
s o lu tio n needed to be evaluated.
L in e a rity o f the re la tio n s h ip between
li g h t absorbance and sugar concentration existed only in a narrow range
around the c a lib r a tio n co n ce n tra tio n .
Therefore, samples to be tested
were d ilu te d (w ith b u ffe r s o lu tio n s ) and evaluated u n til t h e ir concen­
tr a tio n s were close to the c a lib r a tio n con centratio n.
The enzyme preparations themselves contained sugar before contact
w ith any su b stra te .
This sugar le v e l was accounted f o r by preparing a
standard enzyme s o lu tio n subjected to SO0C fo r the standard h yd rolysis
time o f 6 hours.
The amount o f apparent glucose was then determined
and subtracted from subsequent values o f hydrolyzed samples.
value was the apparent glucose achieved from h y d ro ly s is alone.
The fin a l
31
RESULTS AND DISCUSSION
A utohydrolysis o f Substrates
The f i r s t goal o f th is study was to evaluate h y d ro ly s is character­
is t ic s
o f three sub stra te s.
The three substrates were b a rle y straw,
lodge pole pin e, and Douglas f i r .
Degree o f lig n in removal a fte r an
au to h yd ro lysis and ethanol-w ater e x tra c tio n was determined.
C ellulose
conversion amounts v ia an acid h y d ro ly s is and an enzymatic hyd ro lysis
were also determined.
A utohydrolysis co n d itio n s
were 205 °C and 10 minutes.
used f o r th is
work
These c o n d itio n s were those found to be
optimum f o r the d e li g n i f i ca tio n o f wheat straw during previous work at
th is la b o ra to ry [2 4 ].
Table 2 contains re s u lts o f the substrate c h a ra c te riz a tio n s .
Wheat
straw composition (generated by Nakaoka [2 4 ]) was included fo r comparison
purposes.
The ash percentages reported were those from te s ts performed
before the ethanol-benzene e x tra c tio n o f the su b stra te s.
Subsequent
ashings o f ethanol-benzene extracted substrates revealed th a t some ma­
t e r ia l th a t te ste d as ash was removed during the e x tra c tio n procedure.
Table 3 shows the ash percentages a fte r the ethanol-benzene e x tra c tio n .
Al I subsequent mass balance c a lc u la tio n s were made using the Table 3
ash re s u lts .
Table 2.
Weight Percent Composition o f Substrates
(M o istu re -fre e b a s is ).
Substrate
C ellulose
Hemi c e ll Ulose
Lignin
Ash
B a rley
straw
43.6
15.6
23.9
7.7
9.2
Wheat
straw
39.3
14.1
28.9
8.3
9.4
Douglas
fir
50.8
21.2
26.1
0.2
1.7
48.3
21.3
24.6
0.4
5.4
Lodge pole
pine
.
E x tra c tib le
32
Table 3.
Weight Percent Ash o f Ethanol-Benzene Extracted Substrates.
Substrate
Barley straw
Wheat straw
Douglas f i r
Lodge pole pine
The desired
hydrolyze
most
r e s u lt
of
the
of
the
Ash
4.2
5.8
0.1
0.2
autoh ydro lysis
hemic e llu lo s e
and
solu ble in an ethanol-w ater s o lv e n t.
pretreatm ent was to
render
the
lig n in
h ig h ly
The c e llu lo s e residue from the
au to h yd ro lysis ( i f the above goals were met) should be in a form th a t
allow s easy h yd ro lysis to glucose.
The theory o f an a u to h yd ro ly s is , as discussed be fore , is th a t an
acid
environment
groups.
re s u lts
from decomposition o f hemic e llu lo s e
acetyl
The acid co n d itio n s catalyze the h y d ro ly s is o f hemic e llu lo s e
and some c e llu lo s e in the su b stra te .
This h y d ro ly s is produces water
solu ble products th a t are e a s ily removed from the lig n o c e llu lo s e m a trix
during the a u to h yd ro lysis.
A utohydrolysis
co n d itio n s
hemic e llu lo s e - lig n in
fa s t.
During
bonds.
can
also
break
lig n in - lig n in
and
These re action s are thought to be very
slower re a c tio n s ,
th a t
fo llo w
these
carbon-carbon bonds are formed between lig n in
fa s t
re a ctio n s,
monomers.
The f i r s t
re a ctio n s produce products th a t are solu ble in solvents th a t u su a lly
w ill
not d isso lve
Once the
carbon-carbon
becomes h ig h ly
se rie s
lig n in ,
in s o lu b le
such as water o r ethanol-w ater s o lu tio n s .
bonds
in
are
formed,
the
the above solvents
re a ctio n mechanism, t i m e-at-temperature is
lig n in s o lu b ilit y .
at-tem perature
repolymerized
[3 3 ].
lig n in
Due to
th is
very im portant fo r
Wayman and Lora observed th is a u toh ydro lysis t i me­
e ffe c t
on residual
lig n in
in
aspen woodmeal
using a
dioxane-w ater solve nt (see Figure 8) [3 0 ].
Barley Straw
The a u toh ydro lysis and a u to h y d ro ly s is /e x tra c tio n o f ba rle y straw
produced re s u lts s im ila r to those observed w ith wheat straw [2 4 ].
33
195° C
5 10 -175’ C
autohydrolysis time at temperature
(minutes)
Figure 8 .
E ffe c ts o f A utohydrolysis and Dioxane E xtraction on Aspen
Woodmeal Lignin [3 0 ].
34
Since these two substrates are c lo s e ly re la te d species, th is
tency o f re s u lts would be expected.
consis­
Table 4 presents a comparison o f
au to h yd ro lysis re s u lts fo r wheat and b a rle y straw.
Douglas F ir and Lodge Pole Pine
Table
5
is
a
summary
of
autoh ydro lysis
e x tra c tio n runs on the Douglas f i r
and
a u to h y d ro ly s is /
and lodge pole p in e .
The o v e ra ll
weight losses from the autoh ydro lysis and the a u to h y d ro ly s is /e x tra c tio n
runs
fo r
the
components
two woods were w ith in
such
as
c e llu lo s e ,
2% o f
each o th e r.
hemic e llu lo s e ,
and
lig n in
Removal
were
of
also
reasonably close f o r these two su b strates.
Table 5 shows th a t removal o f lodge pole hemi c e llu lo s e from the
lig n o c e llu lo s e
This
m a trix continued during the ethanol-w ater e x tra c tio n .
continued
Douglas f i r .
d is s o lu tio n
of
hemic e llu lo s e
was not
observed w ith
Lig nin analyses seem to in d ic a te th a t more lig n in was
removed from Douglas f i r du ring the a u to h y d ro ly s is /e x tra c tio n than from
lodge pole during the pretreatm ent.
But an inconsistency in the lodge
pole lig n in data makes th is conclusion suspect.
C a lcu la tio n s in d ica te d
th a t more lig n in was removed from the lodge pole during the autohydrol­
y s is than was removed during the a u to h y d ro ly s is /e x tra c tio n , which was
not p o s s ib le .
The e rra n t data was probably due to s u b je c tiv e judgments
th a t have to be made during the lig n in determ ination procedure.
Due to
th is apparent inconsistency and the time involved in repeating the run,
fu tu re work w ith wood was performed using Douglas f i r as the sub strate.
Approxim ately tw ice as much lig n in
was removed from the straws
than the woods (on weight percent basis) during the pretreatm ents.
percent
s im ila r .
of
hemic e llu lo s e
ba rley
straw
and woods were
Given the in tim a te re la tio n s h ip o f these two c o n s titu e n ts in
the lig n o c e llu lo s e m a trix ,
lig n in
removed from
The
may have
tog eth er these two fa c ts suggest th a t the
repolymerized
before
its
d is s o lu tio n
could
occur.
Lig nin re polym e rizatio n would in d ic a te th a t e ith e r the tim e-at-tem peratu re was too long o r the temperature too high.
Table 4.
Comparison o f A utohydrolysis Experiments fo r Barley Straw and Wheat Straw.
%
Substrate
Weight
Loss
% Lignin
Removed
% Carbohydrate
Removed
% C ellulose
Removed
% Hemicellulose
Removed
Autohydrolyzed
Barley Strawa
43.9
66.2
37.6
20.6
85.1
Autohydrolyzed/
Extracted Barley
Straw3
46.7
75.7
38.0
19.8
88.5
Autohydrolyzed
Wheat Strawb [24]
42.8
59.3
37.4
no
analysis
no
analysis
Autohydrolzed/
Extracted Wheat
Strawb [24]
48.6
78.8
39.0
no
analysis
no
analysis
a - m o istu re -fre e basis
b - a ir dry basis
Table 5.
Summary o f A utohydrolysis Experiments on Wood Substrates (Moisture-Free Basis).
% Weight
Loss
% Lignin
Removed
Autohydrolyzed
Douglas F ir
35.2
28.2
Autohydrolyzed/
Extracted
Douglas
F ir
37.1
Autohydrolyzed
Lodge Pole
Pine
Autohydrolyzed/
Extracted Lodge
Pole Pine
Substrate
% Carbohydrate
Removed
% C ellulose
Removed
% Hemicellulose
Removed
37.8
16.1
89.8
34.6
38.0
16.8
88.7
34.8
29.4
36.8
18.5
78.3
36.1
27.2
39.3
17.2
89.3
37
Acid H ydrolysis Experiments
Barley Straw
The low percentages o f lig n in
and hemic e llu lo s e in ba rley straw
a ft e r pretreatm ent should have re s u lte d in a substrate whose c e llu lo s e
was h ig h ly su sce p tib le to h y d ro ly s is .
When p retre ate d b a rle y straw was
subjected to the s u lfu r ic acid h y d ro ly s is co n d itio n s less than 10% o f
the
remaining carbohydrate was hydrolyzed to water solu ble product.
Table 6 shows th a t weight losses due to
acid hydrolyses were very
s im ila r fo r both wheat and b a rle y straw .
Carbohydrate weight losses
from the o v e ra ll process (acid h y d ro ly s is plus various pretreatm ents)
were s im ila r fo r both p re tre a te d and non-pretreated straws although the
la tte r
are
in d ic a te
(s u rp ris in g ly )
the
existence
of
somewhat
h ig her.
a carbohydrate
hydrolyzed by an a c id ic environment.
seem to have l i t t l e
e a s ily hydrolyzed.
These re s u lts
fra c tio n
th a t
seem to
is
e a s ily
Pretreatments as performed here
e ffe c t on the carbohydrate fra c tio n
th a t is not
(Another possible reason fo r the low weight losses
in p re tre a te d m a te ria ls w i l l be discussed la te r in th is w ork.)
Douglas F ir and Lodge Pole Pine
Table 7 contains the weight loss re s u lts from the acid hyd ro lysis
o f the woods.
woods reveal
The o ve ra ll-p ro ce ss weight loss c a lc u la tio n s fo r the
an in te re s tin g r e s u lt.
Carbohydrate w eight losses were
about 70% higher fo r p retre ate d substrates in comparison w ith those o f
non-pretreated e x tra c t-fre e woods.
This suggests th a t pretreatm ents
have a g re a te r e ffe c t on wood c e llu lo s e than on straw c e llu lo s e .
though the data
in d ic a te d
th a t
an au tohydrolysis
Even
enhances the acid
h y d ro ly s is
o f wood c e llu lo s e ,
the h y d ro ly s is y ie ld s were s t i l l
less
than 50%.
Therefore, another serie s o f experiments was performed to
t r y to exp la in the low c e llu lo s e conversions.
A p o ssible reason f o r low h yd ro!yza biI i t y
was the
high amounts o f lig n in
m a trix a ft e r pretreatm ents.
s till
o f the wood c e llu lo s e
present in
the
lig n o c e llu lo s e
On a r e la tiv e b a sis, the weight percent
Table 6.
Carbohydrate Conversion Results o f Acid H ydrolysis on Straw Substrates
(Weight %).
Substrate
Total
Carbohydrate Converted
During Acid H ydrolysis
Total
Carbohydrate Converted
During Overall Process
(Pretreatment + H ydrolysis)
E xtract-Free
Barley StravA
48.0
—
Autohydrolyzed
Barley StravA
8.4
42.9
Autohydrolyzed/
Extracted Barley
Straw3
5.8
41.6
E xtract-Free
Wheat Strawb [24]
54.9
Autohydrolyzed
Wheat Strawb [ 24]
11.2
43.5
Autohydrolyzed/
Extracted Wheat
Strawb [24]
5.4
39.6
a - Moisture-Free Basis
b - A ir Dry Basis
—
Table 7.
Carbohydrate Conversion Results o f H2 SO4 H ydrolysis o f Wood Substrates
(M oisture-Free Basis; % Weight Loss).
Substrate
Total
Carbohydrate Converted
During Acid H ydrolysis
Total
Carbohydrate Converted
During Overall Process
(Pretreatment + H ydrolysis)
E xtract-Free
Douglas F ir
24.3
Autohydrolyzed
Douglas F ir
4.5
40.6
Autohydrolyzed/
Extracted
Douglas F ir
3.6
40.2
E xtract-Free
Lodge Pole Pine
24.3
Autohydrolyzed
Lodge Pole Pine
5.3
40.2
Autohydrolyzed/
Extracted
Lodge Pole Pine
4.6
42.1
—
—
40
lig n in in the pre tre a te d woods was higher than th a t found in the nonpre tre a te d woods.
Wayman and Lora's re s u lts w ith aspen suggested th a t dioxane-water
so lve n t may be more e ffe c tiv e
water s o lv e n t.
w ith
f o r d e li g n i f i ca tio n than the ethanol-
Therefore, the ethanol-w ater e x tra c tio n was replaced
a 9:1 dioxane-water e x tra c tio n
tio n s .
f o r p o st-a u to h yd ro lysis
e x tra c ­
See Table 8 fo r a comparison o f the before and a ft e r e x tra c tio n
lig n in percentages and the percent lig n in removed fo r the two solve nts.
Although more lig n in was
fin a l
removed by the dioxane-water e x tra c tio n , the
lig n in percent in the residue was higher than th a t found in the
ethanol-w ater e xtracte d sample.
This r e s u lt was a ttr ib u ta b le to the
fa c t th a t the a u toh ydro lysis was not as e ffe c tiv e removing lig n in as
th a t
observed
during
autohydrolyzed f i r
yzed
samples
Total
a previous
Douglas
fir
run.
Therefore,
the
samples contained more lig n in than the autohydrol-
used during
the
amounts o f carbohydrates
ethanol-w ater
e x tra c tio n
experiments.
removed during the pretreatm ents and
acid hydrolyses were about the same fo r both runs (See Tables 7 and 9 ).
This in d ic a te s th a t the small increase in lig n in content between th is
run and the previous run had l i t t l e
conversion.
Table 8.
Comparison o f Douglas F ir Lig nin E x t r a c t ib ilit y by Two
Solvents (M oisture-Free Basis; Weight %).
E xtra ctio n Solvent
To
e ffe c t on the to ta l carbohydrate
% Lignin Before
E xtra ctio n
% Lignin A fte r
E xtra ctio n
% Lignin
Removed
Ethanol-Water
29.5
27.6
9.0
Dioxane-Water
34.7
31.6
12.1
a sce rta in
the
extent
of
the
e ffe c t
of
lig n in
on an acid
h y d ro ly s is , two hydrolyses were performed using an e x tra c t-fre e and a
d e lig n ifle d e x tra c t-fre e sample o f Douglas f i r .
The d e lig n ifie d sample
was prepared using the c h lo rin e gas bleaching procedure o f the Jig n in
d e term inatio n.
The
carbohydrate
conversions
fo r
the
e x tra c t-fre e
sample and the lig n in - fr e e sample were 33.4% and 27.7%, re s p e c tiv e ly .
Table 9.
Carbohydrate Conversion Results o f Autohydrolyzed Dioxane-Water
Extracted Douglas F ir (M oisture-Free Basis; % Weight Loss).
Pretreatment
Total
Carbohydrate Converted
During Acid H ydrolysis
Total
Carbohydrate Converted
During Overall Process
(Pretreatment + H ydrolysis)
Autohydrolyzed
4.1
44.6
Autohydrolyzed/
Extracted
4.9
44.4
42
The comparison o f these two re s u lts seems to in d ic a te th a t lig n in does
not
have
a detrim e ntal
e ffe c t
on
c e llu lo s e
h y d ro ly s is .
However,
Saddler e t a l . have shown th a t as lig n in is removed, enzymatic conver­
sion
of
c e llu lo s e
increases
[3 4 ].
If
an acid
h y d ro ly s is
can be
compared to an enzymatic h y d ro ly s is , the lower conversion o f a d e lig n ifie d sample suggests a problem w ith the acid h y d ro ly s is te s t.
C e llu lo se analyses were performed on the residues from the acid
hydrolyses
of
the
two
Douglas
fir
samples ju s t
discussed.
These
analyses were done to a sce rtain the e ffe c ts o f acid catalyzed h yd ro ly­
s is on the various c e llu lo s e types.
the
various
Table 10 contains a comparison o f
carbohydrate amounts before and a fte r
the
h y d ro ly s is .
Acid h y d ro ly s is o f the d e lig n ifie d sample re s u lte d in the conversion o f
19% o f
the
fr a c tio n .
c e llu lo s e
In
fra c tio n
and
the e x tra c t-fre e
only
48% o f
sample a ll
the
hemic e llu lo s e
o f the hemic e llu lo s e was
hydrolyzed and on ly 6% o f the c e llu lo s e fra c tio n was hydrolyzed.
The
re s u lts
fo r
The
removal
o f lig n in
c e llu lo s e
these
experiments
on c e llu lo s e
were as expected.
increased access to c e llu lo s e
conversion.
The fa c t
th a t
a ll
re s u ltin g
o f the
in
higher
hemi cel lu lo s e
was
hydrolyzed in the e x tra c t-fre e f i r and only 48% was hydrolyzed in the
d e lig n ifie d
reverse,
fir
higher
was not expected.
hemic e llu lo s e
The expected re s u lt would be the
conversion
fo r
d e lig n ifie d
samples.
Lig n in and hemic e llu lo s e are bonded tog eth er in untreated" Tignocellu ­
lose .
The removal o f a ll the lig n in may cause changes in the lig n o -
c e llu lo s e m a trix and the chemical and physical nature o f the hemic e llu ­
lo se .
These changes in hemic e llu lo s e may render i t less susceptible to
h y d ro ly s is .
Table 10.
C ellulo se A nalysis A fte r Acid H ydrolysis
(B asis: Ig E xtract-Free Wood, M oisture-F ree).
C ellulo se Type
a - C ellulo se
/? - C ellulo se
7 - C ellulo se
E xtract-Free
Douglas F ir
Before A fte r
0.525
0.321
0
0.173
0.216
0
Deli g n i f i ed-E xtract-F ree
Douglas F ir
'
Before A fte r
0.525
0.302
0
0.121
0.216
0.113
43
F u rthe r acid hydrolyses were performed s u b s titu tin g h yd ro ch lo ric
acid fo r the s u lfu r ic acid o f previous te s ts .
fo r these hydrolyses were:
H ydrolysis con dition s
SN HClr 3 hours, and 80°C.
The purpose o f
these experiments was to examine the e ffe c ts o f HClr ra th e r than H2SO4 ,
and a more severe h yd ro lysis environment.
The substrates hydrolyzed
were e x tra c t-fre e Douglas f i r , d e lig n ifie d Douglas f i r , and autohydrolyzed
eth an ol-w ate r extracte d Douglas f i r .
these hydrolyses.
Table 11 contains the re s u lts o f
The HCl h yd ro ly s is o f e x tra c t-fre e f i r re su lte d in a
carbohydrate conversion about 25% higher than the 33.4% conversion ob­
served fo r the previous H2SO4 h y d ro ly s is .
The h y d ro ly s is conversion o f
the autohydrolyzed/extracted substrate w ith h yd ro c h lo ric acid was essen­
tia lly
unchanged from th a t observed when s u lfu r ic acid was used.
When
HCl was used fo r the h y d ro ly s is , the o v e ra ll process carbohydrate con­
versions o f e x tra c t-fre e f i r and p re tre a te d f i r were e s s e n tia lly equal,
u n lik e th a t observed fo r the H2SO4 .
w ith the straw s,
This suggests, lik e th a t observed
a c e rta in c e llu lo s e
hydrolyzed and th is
fra c tio n
fra c tio n
e x is ts th a t is
is not increased by pretreatm ent.
e a s ily
Like
th a t observed w ith H2SO4 , a lower c e llu lo s e conversion was observed fo r
d e lig n ifie d wood in comparison to e x tra c t-fre e wood fo r HCl hydrolyses.
Wheat Straw
F urther hydrolyses were performed w ith h y d ro c h lo ric a c id , but wheat
straw was used instead o f Douglas f i r .
The substrate change was made
to a sce rta in the e ffe c ts o f an HCl h y d ro ly s is on a straw su b strate.
C onditions f o r these hydrolyses were as fo llo w s :
one hour, 3 N, 80 °C fo r two hours.
12.4N HCl, 30 °C fo r
These c o n d itio n s were s im ila r to
x
those used f o r the s u lfu r ic acid hydrolyses, except the number o f hydrogen
ions in s o lu tio n was «50% higher fo r the f i r s t hour and «200% higher
fo r the la s t two hours.
The re s u lts o f these h y d ro ly s is co n d itio n s fo r
e x tra c t-fre e wheat, autohydrolyzed wheat, and autohydrolyzed/extracted
wheat are presented in Table 12.
The e x tra c t-fre e wheat and the auto­
hydrolyzed wheat showed a 30% increase o f conversion f o r the whole pro­
cesses (pretreatm ent and acid h y d ro ly s is ) over th a t obtained w ith H2SO4 .
Table 11.
Carbohydrate Conversions o f Douglas F ir Substrates by HCl
(M oisture-Free Basis; % Weight Loss).
Substrate
Total
Carbohydrate Converted
During Acid H ydrolysis
E xtract-Free
Douglas F ir
41.1
Lignin-Free
Douglas F ir
34.4
Autohydrolyzed/
Extracted
Douglas F ir
8.4
Total
Carbohydrate Converted
During Overall Process
(Pretreatment + H ydrolysis)
43.2
Table 12.
Carbohydrate Conversions o f Wheat Straw Substrates by HCl
(M oisture-Free Basis; % Weight Loss).
Total
Carbohydrate Converted
During Acid H ydrolysis
Total
Carbohydrate Converted
During Overall Process
(Pretreatment + H ydrolysis)
Extract-Free
Wheat Straw
72.4
—
Lignin-Free
Wheat Straw
31.0
56.5
Autohydrolyzed/
Extracted
Wheat Straw
32.2
58.8
Substrate
46
C e llu lo se conversion fo r the autohydrolyzed/extracted
sample was 23%
higher fo r the HCl h y d ro ly s is .
Chromedia
The observation th a t a fte r three hours o f acid h y d ro ly s is
less
than 50% o f the amorphous carbohydrate in autohydrolyzed Douglas f i r
was hydrolyzed by H2SO4 and only about 60% by HCl
seemed u n lik e ly .
S im ila r re s u lts were also observed w ith o th e r sub strates tested
Table 13).
(see
Therefore, a serie s o f experiments was run to te s t the acid
h y d ro ly s is o f a pure c e llu lo s e substrate w ith a high fr a c tio n o f amorphous
c e llu lo s e .
To produce the h ig h ly amorphous c e llu lo s e a pure c e llu lo s e sub strate,
Chromedia, was b a ll m ille d .
Both dry and wet m illin g s were performed.
B all m illin g the dry Chromedia fo r 96 hours doubled i t s amorphous c e l l ­
ulose con ten t, w h ile wet m illin g reduced the amorphous c e llu lo s e fra c tio n
to less than 1% o f the to ta l c e llu lo s e .
This was an unexpected r e s u lt.
However, researchers a t American Viscose Corporation observed th a t by
w e ttin g an amorphous b a ll m ille d c e llu lo s e , the c r y s t a l li n i t y could be
increased to the degree o f c r y s t a l li n i t y found in the non-ball m ille d
su b strate [3 5 ].
The r e c r y s ta lliz a tio n o f wet c e llu lo s e may explain the
decrease in the amorphous nature o f the wet b a ll m ille d samples.
Because
o f the apparent r e c r y s ta lliz a tio n o f wet m ille d Chromedia, the dry m ille d
sample was chosen fo r acid h yd ro ly s is comparison w ith non-mi I led Chromedia.
Table 14 contains a summary o f the r e la tiv e amounts o f the c a r­
bohydrate fra c tio n s found in the three samples (n o n -m ille d , wet m ille d ,
and dry m ille d Chromedia ).
The acid h y d ro ly s is (w ith 5 N HCl, 80 °C fo r 3 hours) o f non-mi lie d
and d ry -m ille d Chromedia produced the re s u lts presented in Table 15.
I t is evident th a t doubling the amorphous nature o f the substrate had
an e q u iva le n t e ffe c t on the c e llu lo s e conversion.
s till
The conversion was
very low f o r samples th a t contained approxim ately 40% amorphous
c e llu lo s e .
These low conversions o f amorphous c e llu lo s e support con­
clu sio n s concerning the inadequacies o f the acid h y d ro ly s is te s ts .
Table 13.
Comparison o f Amorphous C ellulose Content versus Amount o f
C ellulose Converted.
Substrate
Acid
Grams o f Amorphous
C ellulose
Present
Total Grams o f
Carbohydrate
Hydrolyzed
Autohydrolyzed
Barley Straw
H2SO4
0.125
0.064
Autohydrolyzed/
Extracted
Barley Straw
H2SO4
0.106
0.046
Autohydrolyzed
Douglas F ir
H2SO4
0.080
0.031
Autohydrolyzed/
Extracted
Douglas F ir
H2SO4
0.104
0.026
Autohydrolyzed/
Extracted
Douglas F ir
HCl
0.104
0.060
Autohydrolyzed
Lodge Pole Pine
H2SO4
0.131
0.038
Autohydrolyzed/
Extracted
Lodge Pole Pine
H2SO4
0.107
0.032
48
Table 14.
Table 15.
C ellulo se Analyses on M ille d and Non-Milled Chromedia
(Weight %).
M illin g
Alpha
C ellulose
Beta
C ellulose
Gamma
C ellulo se
None
81.2
18.4
0.4
Dry
62.6
36.7
0.7
Wet
90.0
0.4
9.6
Results o f Acid H ydrolysis on Chromedia
(M oisture-Free B a s is ).
C ellulo se Converted
(Weight %)
Mi l l i n g
C ellulo se
were
higher
None
4.1
Dry
7.3
conversions observed during acid catalyzed
fo r
a ll
non-pretreated
observed fo r p re tre a te d su b strates.
substrates
versus
hydrolyses
conversions
These experimental re s u lts suggest
th a t the ease o f c e llu lo s e h y ro ly s is is not increased by the p re tre a t­
ments performed during th is
tio n s
presented
conversion
could
in
the
be
in v e s tig a tio n .
lit e r a t u r e
increased
by
have
While several
suggested
pretreatm ents
th a t
s im ila r
in v e s tig a ­
c e llu lo s e
to
those
performed during th is study [2 5 ,3 6 ], these c ita tio n s and the experimen­
ta l re s u lts from th is la b o ra to ry in d ic a te th a t acid h y d ro ly s is may not
be
in d ic a tiv e
of
the
e ffe c t
of
autoh ydro lysis
as
a pretreatm ent.
Therefore, a se rie s o f experiments was performed using enzyme catalyzed
hydrolyses
conversion.
to
a sce rta in
the
e ffe c ts
o f a u toh ydro lysis
on c e llu lo s e
F urther discussions on the low acid h y d ro ly s is re s u lts
are presented in the next se ctio n .
Enzymatic H ydrolysis Experiments
/
As a fin a l
d ro ly s is
se rie s o f experiments to te s t the e ffe c ts o f autohy­
on rendering
a substrate more conducive to
h y d ro ly s is ,
an
enzymatic h y d ro ly s is was performed.
The sub strate used f o r the enzymatic hydrolyses was an o p tim a lly
autohydrolyzed and ethanol-w ater e xtracte d wheat straw .
The sample was
l e f t wet a ft e r the e x tra c tio n to avoid adverse e ffe c ts o f drying on
enzymatic
hydrolyses
observed
at
Colorado
State
U n iv e rs ity
w ith
lo b lo lly pine (although subsequent experiments showed th a t drying had
no detrim e ntal e ffe c t on wheat straw conversion) [3 6 ].
fo r th is
h y d ro ly s is were as fo llo w s :
fo u r m i llit e r s
The condition s
o f enzyme s o lu ­
tio n , one m i l l i t e r o f c it r a t e b u ffe r s o lu tio n , a t SO0C fo r s ix hours.
The enzyme s o lu tio n consisted o f 3.6 gm o f a c e llu la s e enzyme dissolved
in c it r a t e b u ffe r to make 100 ml o f s o lu tio n .
o f an i n i t i a l
h yd ro lysis
Reducing sugar analysis
liq u o r revealed 561 m illig ra m s o f apparent
glucose per gram o f dry su b stra te , which was a conversion o f about 74%
o f the c e llu lo s e in the sample.
sample ( a ir d rie d )
S u lfu r ic acid h y d ro ly s is o f a s im ila r
re su lte d in only a 5.4 % conversion o f c e llu lo s e ,
and a 12.4 N h y d ro ly s is w ith h y d ro c h lo ric acid y ie ld e d a 32.2% c e llu ­
lose conversion.
Comparing the enzyme and acid hydrolyses in d ica te s
th a t an acid h y d ro ly s is te s t was a poor measurement o f the e ffe c tiv e ­
ness o f a u toh ydro lysis as a pretreatm ent.
A po ssib le reason f o r the low conversions obtained w ith acid is
th a t enzymes are very s p e c ific in the way th a t they a tta c k c e llu lo s e .
A
complete
c e llu la s e
enzyme
a c tu a lly
consists
of
three
separate
enzymes: an endoglucanase, an exoglucanase, and a c e ll obi ase.
glucanohydrolase,
an
endoglucanase,
randomly
hydrolyzes
1,4 -
c e llu lo s e
in s id e the m olecular chain, which re s u lts in a ra p id reduction in the
degree o f p o lym e riza tio n .
The exoglucanase, /? - l,4 - c e llu lo b iohydrolase,
a tta cks c e llu lo s e polymers from the reducing end producing c e llo b io s e ,
a dimer o f glucose which is water s o lu b le .
Exoglucanase can a tta ck the
ends found in the n a tive c e llu lo s e o r an end produced by a endoglu­
canase.
The la s t type o f enzyme, /?-glucosidase
(ce T lo b ia se ), hydro­
lyzes c e llo b io s e and o th e r water solu ble glucose oligomers to glucose.
50
Figure
9
is
h y d ro ly s is .
of
a ll
a schematic
of
th is
proposed
mechanism o f
enzymatic
Due to th is method o f c e llu lo s e a tta c k , a high percentage
in d iv id u a l
h y d ro ly tic
Acids a tta c k c e llu lo s e
re action s
in a t o t a l l y
soluble
produce
a solu ble
random fa sh ion.
h y d ro ly tic
re action s
y ie ld
products.
h y d ro ly s is
by acids
was measured by weight
Therefore,
Since
lo s s ,
product.
the
few
degree o f
products
must be
so lu b le to be measured as conversion.
Clausen
and Gaddy obtained
data
showing
a 90% conversion
of
c e llu lo s e could be obtained w ith 14 N HCl a t room temperature in a CSTR
(no time frame was given in t h e ir lite r a tu r e )
th a t 8 N HCl converted 90% o f the c e llu lo s e
using a se rie s o f CSTR1s.
[3 7 ].
They also found
in 30 minutes a t IOO0C
Therefore, another p o ssible explanation fo r
low conversions o f substrate v ia acid hydrolyses could be mass tra n s fe r
lim ita tio n s ,
m ixtu res.
re s u ltin g
from
low
shear
s t ir r in g
of
the
h y d ro lysis
Acid h y d ro ly s is m ixtures were s tir r e d on ly p e rio d ic a lly w ith
a glass s t i r r in g rod, w h ile enzymatic h y d ro ly s is m ixtures were s tir r e d
throughout the h y d ro ly s is .
Since
in itia l
enzymatic
hydrolyses
re s u lte d
in
high
c e llu lo s e
conversions, the use o f enzymes to evaluate the e ffe c ts o f autohydrolyses was adopted.
Enzyme strengths th a t gave the highest conversion in
6 hours were evaluated.
C e llu c la s t 1.5 L, a c e llu la s e supplied by Novo C orporation, was
the f i r s t enzyme whose strength was optim ized.
C e llu c la s t 1.5 L is an
enzyme prepa ratio n made from the submerged ferm entation o f a selected
s tra in
re s u lts
o f the fungus Trichoderma re e s e i.
obtained
from the
c e llu la s e charge v a rie d .
various
Figure 10 is
6 hour hydrolyses,
in
a p lo t o f
which the
I t can be seen from th is graph th a t a charge
higher than 2.8 gm o f cel lu la s e prepa ratio n per gram o f dry substrate
would not produce much improvement in c e llu lo s e conversion.
Therefore,
th is concentration o f c e llu la s e was chosen fo r fu tu re experiments.
o p tim a lly
autohydrolyzed
wheat
straw
was
used
fo r
th is
series
Wet
of
experiments.
Experiments eva lu a tin g the e ffe c ts o f h y d ro ly s is time on conver­
sion using C e llu c la s t 1.5 L were also performed (see Figure 11).
51
P — I
, 4—
gJ
L
i-icran^JLucanoliyclr-oI a
C e I l u l o a
e e l JLulo
C
c e p t I b l e
P ~ I „4 —
glucan
f$— I , 4—
glucan —
glucanobyd r oIae
glucoe
glueoeld
Figure 9.
A Proposed Mechanism fo r an Enzymatic H ydrolysis [5 ].
mg apparrent glucose/g dry fiber
600 - -
500 - -
400 - -
300 - -
200-
-
100 - -
g cellulase/g dry fiber
Figure 10.
E ffects o f C ellulase A c tiv ity fo r a
6
hour H ydrolysis.
800
L.
7 0 0 -600 - 500 - 400 - 300 - -
200-
-
100 --j
0.0
4.0
8.0
12.0
16.0
Time (hours)
Figure 11.
Reaction Time E ffects on a C elluiase H ydrolysis.
20.0
24.0
54
Increasing
h y d ro ly s is
conversion
by
re s u lte d
in
from
Lengthening
28% more conversion
h y d ro ly s is .
h y d ro ly s is
19%.
time
6 to
12 hours
the
h y d ro ly s is
than
increased
time
was observed
to
at
c e llu lo s e
24
hours
6 hours
of
Even though higher conversions were obtained w ith longer
tim es,
p ra c tic a l
experimental
considerations
h y d ro ly s is tim e o f 6 hours fo r sub strate e va lu a tio n .
used f o r these
wheat straw .
experiments
was again wet,
C e llu c la s t 1.5 L contains l i t t l e
o p tim a lly
d ic ta te d
a
The substrate
autohydrolyzed
c e llobiase a c t iv it y .
Therefore,
the m a jo rity o f the h yd ro lysis products would be c e llob io se and not
glucose.
Since in a reducing sugar te s t, a c e llo b io s e molecule would
appear as on ly
in d ic a te d .
one molecule,
a low h y d ro ly tic
conversion would be
To a lle v ia te th is problem a second enzyme, /?-glucosidase,
was added to the h yd ro lysis m ixtures.
supplied by Novo C orporation,
Cellob ia se 250 L ) .
It
its
This enzyme prepa ratio n was also
tra de name being Novozym 188 (o r
was produced by submerged ferm entation o f a
selected s tra in o f the fungus A s p e rg illu s n ig e r.
The c e ll o b iase preparation was added to the optim ized c e llu la s e
s o lu tio n s
biase.
in
varying amounts to ob tain the optimum amount o f c e llo ­
Figure 12 is a graph o f the re s u lts from varyin g ce llo b ia s e
amounts.
Charges o f c e llo b ia s e
higher than 0.28 gm o f
p repa ratio n per gram o f dry substrate had l i t t l e
conversion.
Therefore,
subsequent hydrolyses.
ce llo b ia se
e ffe c t on c e llu lo s e
the 0.28 gm amount was chosen fo r charging
Wet o p tim a lly
autohydrolyzed/extracted wheat
straw was the substrate used fo r these experiments.
Time stud ies were also performed w ith the mixed enzyme s o lu tio n .
As the time increased from 6 hours to 12 hours, c e llu lo s e conversion
increased
14%.
A h yd ro lysis
time
of
24 hours
d id
not
re s u lt
in
increased conversion over th a t observed during the 12 hour h yd rolysis
(see Figure
13).
The substrate
used fo r these experiments was an
o p tim a lly autohydrolyzed and ethanol-w ater extracted wheat straw .
Using the optimum concentrations determined by the above e x p e ri­
ments, p re tre a te d and non-pretreated e x tra c t-fre e samples o f wheat were
evaluated fo r c e llu lo s e conversion.
The p retre ate d samples were wet
mg apparrent glucose/g dry fiber
9 0 0 -800 - 700 - 600 - 500 H
300200
-
■
100 - -
g cellobiase/g dry fiber
Figure 12.
E ffects o f Cellobiase A c tiv ity on a 6 hour H ydrolysis.
d) 11OO - -
900 - 800 - 7 0 0 -600 - 500 - 4 0 0 --
Time (hours)
Figure 13.
Reaction Time Effects on a Cellobiase-Cellulase Hydrolysis.
57
autohydrolyzed wheat,
wet autohydrolyzed and ethanol-w ater extracted
wheat, dry autohydrolyzed wheat, and dry autohydrolyzed and ethanolwater e xtracte d wheat.
tio n s .
Table 16 contains the re s u lts o f these evalua­
Comparison o f the e x tra c t-fre e wheat c e llu lo s e conversion w ith
those o f the p re tre a te d
samples demonstrates th a t the pretreatm ents
were very e ffe c tiv e in increasing the c e llu lo s e conversion.
Also, the
high conversions obtained w ith substrates th a t had not been extracted
to remove lig n in suggests th a t e x tra c tio n was an unnecessary p re tre a t­
ment step.
It
is also evident from the data th a t d ryin g had l i t t l e
e ffe c t on the enzymatic h yd ro ly s is o f wheat straw .
Colorado S tate U n iv e rs ity ,
Murphy e t a l . , at
also had good conversions o f c e llu lo s e in
autohydrolyzed wheat w ith dry samples [3 8 ].
They d id no evaluations on
the e ffe c ts th a t dryin g o f samples had on enzymatic h y d ro ly s is .
Table 16.
Results o f Enzymatic Hydrolyses on Wheat Straw.
Substrate______
% C ellulose Hydrolyzed
Dried E xtract-F ree
12.8
Dried E xtract-F ree
Hem icellulase
Added to Enzyme
S olutio n
17.4
Dried Autohydrolyzed
94.5
Dried Autohydrolyzed/
Extracted
.89.4
Wet Autohydrolyzed
96.5
I
Wet Autohydrolyzed/
Extracted
An experiment
hem icellula se,
98.0
was performed
was added to
the
in
which
h y d ro ly s is
a t h ir d
enzyme type,
m ixtu re.
Although
a
the
researchers a t Colorado State found th a t the Novo c e llu la s e enzymes had
hem icellulase
a c t iv it y ,
more
hem icellulase
s ig n ific a n t hem icellulase a c t iv it y [3 6 ].
p repa ratio n was Gamanase 1.5 L.
was
added
to
assure
a
The trade name o f th is enzyme
Although the Novo Corporation suggests
58
a dosage o f 0.0005 gm o f Gamanase 1.5 L per gram o f dry s u b stra te , 0.25
gm o f enzyme per gram o f dry substrate was used fo r th is experiment to
assure an excess o f enzyme fo r the h y d ro ly s is .
The purpose o f th is
experiment was to determine i f an enzyme could remove the hemic e llu lo s e
from
a
non-pretreated
c e llu lo s e .
ment.
substrate
and
thus
increase
access
to
the
H ydrolysis time was increased to 12 hours fo r th is e x p e ri­
A carbohydrate conversion o f 17.4% in d ic a te s th a t the use o f the
th ir d enzyme did not a tta in the e ffe c ts rendered by an autohydrolysis
pretreatm ent.
Murphy e t a l .
suggested th a t
f o r a process to
be economically
fe a s ib le ,
the enzyme charge ra te could be no more than 10 lU/gm o f
su b strate
[3 6 ].
(An IU is
an in te rn a tio n a l
accepted u n it o f measure fo r enzyme s tre n g th s .
enzyme u n it and is
the
The u n it is defined as
the release o f I pmol o f glucose per u n it.)
The fin a l
two-enzyme
s o lu tio n used in th is work was evaluated fo r enzyme stre ngth using the
Mandels e t a l . procedure and was found to contain 0.94 lU/ml o f enzyme
s o lu tio n [3 8 ].
This tra n s la te s in to about 38 lU/gm o f s u b stra te , which
is high fo r a commercial process.
An enzyme
s o lu tio n
(using
the
same Novo enzyme preparations)
prepared by Bertran and Dale had a stre ngth o f 0.55 lU/ml
[3 9 ].
This
s o lu tio n contained 3 gm o f C e llu c la s t 1.5 L and 1.5 gm Novozym 188 per
100 ml o f s o lu tio n .
The enzyme used fo r the fin a l evaluations by th is
la b o ra to ry contained 7.2 gm o f C e llu c la s t 1.5 L and 0.72 gm o f Novozym
188 per 100 ml o f s o lu tio n w ith a stre ngth o f 0.94 lU /m l.
Comparing
the strengths o f these two enzyme s o lu tio n s suggests th a t most o f the
enzyme stre ngth was the re s u lt o f the c e llu la se and not the c e ll o b iase.
The steep i n i t i a l slope o f a p lo t o f a c t iv it y versus c e ll o b iase charge
supports the conclusion th a t the dominant v a ria b le determ ining enzyme
stre n g th is c e llu la s e charge (see fig u re 12).
Reducing the amount o f
c e llu la s e by 75% (to «0.7 gm cellulase/gm dry fib e r ) would re s u lt in an
enzyme stre ngth o f about 10 lU/gm o f s u b stra te .
Figure
10 in d ic a te s
th a t
the
decreased
reduce the 6 hour conversion by about 30%.
Data presented in
enzyme stre ngth
The
would only
c e llu la s e time studies
in d ic a te th a t incre asing h yd ro ly s is time to 24 hours (a reasonable time
59
period fo r a commercial process) could increase the conversion 20% over
th a t observed in 6 hours (see fig u re 11).
Based on these conclusions,
adequate conversions o f c e llu lo s e could be obtained w ith reduced enzyme
charges by in cre asing the h yd ro ly s is tim e.
Therefore,
a g ric u ltu ra l
an o v e ra ll
residues
process
follow ed
by
using a u toh ydro lysis
enzymatic
h y d ro ly s is
to
p re tre a t
and
yeast
ferm entation could be a fe a s ib le route to obtain useful liq u id hydro­
carbons.
A great
deal
of
fu r th e r
work
is
needed to
confirm
the
economic p o te n tia l o f such a process, but the basic technology looks
q u ite prom ising.
60
CONCLUSIONS
1.
When enzymes were used to catalyze the h y d ro ly s is o f pretreated
wheat straw su b stra te ,
higher conversions were observed versus
those f o r non-pretreated straw .
However, s im ila r re s u lts were
observed w ith both acid and enzyme catalyzed hydrolyses fo r
d e lig n ifle d and non-delig n ifie d substrates whether o r not the
substrates were p retre ate d before d e li g n i f i c a tio n .
This in d i­
cates th a t among the e ffe c ts o f pretreatm ent on the lig n o c e llulose m a trix , changes in c e llu lo s e morphology have a g reate r
e ffe c t in incre asing c e llu lo s e conversion than does a decreased
lig n in con ten t.
2.
Low conversions o f c e llu lo s e observed during acid catalyzed
hydrolyses versus conversions observed fo r mixed enzyme hydro­
ly s is
re su lte d from d iffe re n c e s in th e ir re sp e ctive re action
mechanisms.
c e llu lo s e
Enzyme systems a tta c k s p e c ific
molecule w ith
lo c a tio n s on a
a high percentage o f these attacks
producing solu ble re a ctio n products.
W hile,
acid c a ta ly s ts
randomly a tta c k c e llu lo s e bonds producing few so lu b le products
per in d iv id u a l re a c tio n .
c e llu lo s e
Thus, using the w eight loss from a
residue to measure extent o f re a c tio n , y ie ld s
low
conversions when acids are used to catalyze the re a c tio n .
3.
S im ila r to ta l c e llu lo s e conversions observed fo r acid hyd roly­
s is only versus acid h y d ro ly s is plus a u to h yd ro lysis suggests
the existence o f a carbohydrate fra c tio n th a t is e a s ily hydro­
lyzed by acid c a ta ly s ts .
Conversion o f th is fra c tio n o f c e l l ­
ulose does not seem to be increased by pretreatm ent.
4.
Straw lig n in
was rendered more e x tr a c t!ble
by pretreatm ent
than wood lig n in fo r the same autoh ydro lysis c o n d itio n s .
Ti me­
at-tem perature e ffe c ts may have caused wood lig n in to re p o ly ­
m erize, thus reducing i t s s o lu b ilit y in ethanol-w ater s o lv e n t.
61
SUGGESTIONS FOR FUTURE RESEARCH
1.
I f the use o f acid h yd ro ly s is to determine the e ffe c ts o f c e l l ­
ulose pretreatm ents is to be continued as an adjunct to enzyme
hydrolyses, the fo llo w in g suggestions should be adopted.
The
hydrolyses should be s tir r e d continuously and v ig o ro u s ly during
the experiments, and the reducing sugar a n alysis method should
be adopted to determine the degree o f c e llu lo s e conversion in
a ll cases.
2.
The tim e -at-tem p era ture c o n d itio n s used fo r three
substrates
(b a rle y straw , lodge pole pin e, and Douglas
were those
fir )
found to be optimal fo r the removal o f lig n in from wheat straw .
These co n d itio n s may not be optim al fo r the o th e r substrates
o r the optimal co n d itio n s f o r c e llu lo s e conversion.
Therefore,
experiments should be performed to determine the optimal con­
d itio n s fo r c e llu lo s e conversion fo r each su b s tra te .
3.
The liq u o rs from a u toh ydro lysis experiments should be analyzed
to determine i f
sugars produced by au to-catalyzed hyd ro lysis
re action s are in ta c t in the liq u o r o r i f they are destructed
to furfuraTs..
4.
Wood substrates o f in te re s t should be tested to determine i f
t h e ir enzymatic h yd ro ly s is ra te is increased by pretreatm ent
as observed fo r wheat straw .
62
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63
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Worthy, W.
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Nakaoka, R. K.
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Murphy, V. G., Linden, J. C., M oreira, A. R. and Lenz, T. G.
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Wiley and
/
\
29.
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in P u lp ," TAPPI (1974).
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Lora, J. H. and Wayman, M.
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ASTM Standard Test Method D1106 (1977).
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M ille r , G. L.
A n a l y t .
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F o r m a t i o n ,
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"L ig n in
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Table 17.
Results o f the Acid H ydrolysis Development Experiments
Substrate
Acid
Concentration
(°c)
Time
(hr)
Temperature
%
Hydrolyzed
ION
40
1.0
4.2
Autohydrolyzed
Wheat Straw
ION
40
1.0
3.7
Autohydrolyzed
Wheat Straw
12N
40
1.0
3.4
Delig n in fie d
Wheat Straw
ION
40
1.0
1.9
D e lig n in fie d
Wheat Straw
14N
70
3.2
19.0
Autohydrolyzed
Wheat Straw
14N
70
3.5
12.5
D e lig n in fie d
Autohydrolyzed
Wheat Straw
18N
70
3.0
80.2
D e lig n in fie d
Autohydrolyzed
Wheat Straw
18N
80
3.0
89.0
E xtract-Free
Wheat Straw
18N
80
3.0
56.2
APPENDIX
E xtract-Free
Wheat Straw
TABLE 17.
Results o f the Acid H ydrolysis Development Experiments (continued)
Substrate
Acid
Concentration
Temperature
(°c)
Time
(hr)
Hydrolyzed
%
Delig n in fie d
Wheat Straw
18N
80
3.0
98.7
E xtract-Free
Wheat Straw
18N
0.7N
«20
80
3.0
4.0
62.8
E xtract-Free
Wheat Straw
18N
0.7N
«20
80
1.5
4.0
60.6
Extract-Free
Wheat Straw
18N
0.7N
«20
80
1.0
2.0
59.2
D e lig n in fie d
Autohydrolyzed
Wheat Straw
18N
0.7N
«20
80
1.0
2.0
23.8
E xtract-Free
Wheat Straw
16N
0.7N
25
80
1.0
2.0
48.9
Extract-Free
Wheat Straw
18N
0.7N
25
80
0.5
2.0
56.0
Autohydrolyzed/
Extracted
Wheat Straw
18N
0.7N
25
80
0.5
2.0
15.1
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