Seasonal changes in lignin and cellulose content of Montana range grasses
by Leonard F Gieseker, Jr
A THESIS Submitted to the Graduate Committee in partial fulfillment of the requirements for the
degree of Master of Science in Chemistry
Montana State University
© Copyright by Leonard F Gieseker, Jr (1941)
Abstract:
Lignin in Gallatin Valley grasses increased with advancing season, beginning at about five per cent in
May, and rising to a high of eighteen per cent for Bromus inermis at the end of August, contrasted with
fourteen per cent for Bromus marginatus. The wheatgrasses ranged in between, with Agro-pyron
pauciflorum remaining lower in lignin than crested wheatgrass during the first half of the season.
Cellulose content closely paralleled lignin content. Grasses at Judith Basin followed a similar course,
but matured about a month earlier. y
IO
SEASONAL CHANGES IN LIGNIN AND CELLULOSE CONTENT
OF MONTANA RANGE GRASSES
L. F. QIBSEKEE, JR .
A THESIS
S u b m itted t o th e G ra d u ate Committee
in
p a r t i a l f u l f i l l m e n t o f th e re q u ire m e n ts
f o r th e d e g re e o f
M a s te r o f S o len o e i n C h em istry
at
M ontana S ta te C o lle g e
A pprovedI
I n Charge o f M ajo r Work
C haizm an, E xam ining Committee
C h airm an , G ra d u ate Committee
Bozeman, M ontana
J u n e , 1941
%'
. . .
-
1-
TABLE OF CONTENTS
Page
I.
ABSTRACT.................................................................................................
2
II.
INTRODUCTION...........................................................................................
3
III.
REVIEW OF LITERATURE.........................................................................
5
A.
H ia to ry o f F e e d L A n a ly see .....................
B.
L ig n in ...........................................................
C.
C e llu lo s e ......................................................................................25
5
12
IV .
STATEMENT OF PROBLEM......................................................................... 38
V.
EXPERIMENTAL WORK............................................................................... 39
V I.
EXPERIMENTAL RESULTS........................................................................... 46
V II.
DISCUSSION..........................................
59
V III.
SUMMARY....................................................................................................... 63
IX .
ACKNOWLEDGMENT...................................................................................... 64
X. BIBLIOGRAPHY........................................................................
65
136884
ABSTRACT
L ig n in i n C a l l a t i n V a lle y g r a s s e s in c r e a s e d w ith a d v a n c in g s e a s o n , b e ­
g in n in g a t a b o u t f i v e p e r c e n t i n May, and r i s i n g t o a h ig h o f e ig h te e n p e r
c e n t f o r Bromus ln e rm is a t th e end o f A u g u st, c o n tr a s te d w ith f o u r te e n p e r
c e n t f o r Bromus m a r g in a tu s .
The w h e a tg ra s a e s ra n g e d i n b e tw ee n , w ith A g ro -
p y ro n p s u o iflo ru m re m a in in g lo w e r i n l i g n i n th a n c r e s t e d w h e a tg ra s s d u r in g
th e f i r s t h a l f o f th e s e a s o n .
c o n te n t.
C e llu lo s e c o n te n t c l o s e l y p a r a l l e l e d l i g n i n
G ra s s e s a t J u d i t h B asin fo llo w e d a s i m i l a r c o u r s e , b u t m atu red
a b o u t a month e a r l i e r
-3 -
INTRODDOTIC®
Q ra se e a a re w ith o u t do u h t th e moat im p o rta n t p l a n t s f o r m a n 's w e lf a r e
o f a l l p l a n t s grown i n th e w o rld , b e c a u se he l i v e s c h i e f l y upon g r a s s e s and
th e a n im a ls who depend on them f o r fo o d .
Range la n d i s v a s t l y im p o r ta n t
f o r th e p r o d u c tio n o f th e s e a n im a ls , and in th e U n ite d S t a t e s a lo n e i t r e p ­
r e s e n t s a p p ro x im a te ly f o r t y p e r c e n t o f th e c o u n t r y 's t o t a l a r e a .
W ith o u t
g r a s s l a n d s , man would have t o r e s t r i c t h i e home and a c t i v i t i e s t o an amaz­
i n g l y s m a ll p o r t i o n o f th e la n d he now i n h a b i t s on t h i s e a r t h , and human c i ­
v i l i z a t i o n w ould be s i m i l a r l y l i m i t e d .
Wheat and c o m a re o n ly two o f th e many g r a s s e s t h a t a re o f g r e a t im ­
p o rta n c e i n th e U n ite d S t a t e s .
H ow ever, w ith o u t ev en t h e s e two o u r e n t i r e
p a t t e r n o f l i f e w ould have t o be c h an g e d , b ecau se th e y p la y en o rm o u sly im­
p o r t a n t p a r t s i n o u r p r e s e n t- d a y l i f e .
M ontana i s composed p r im a r i ly o f ra n g e la n d , a s th e amount u n d e r c u l t i ­
v a t i o n i s i n s i g n i f i c a n t a s compared w ith th e t o t a l a c re a g e u sed f o r g r a z in g
p u rp o se s.
I f th e p e o p le o f M ontana a re t o p r o s p e r , i t i s n e c e s s a ry f o r them
t o have a t t h e i r d i s p o s a l a l l p o s s ib le in f o r m a tio n r e g a r d in g M ontana ra n g e
g r a s s e d , s in c e th e s e g r a s s e s a re o f v i t a l im p o rtan c e t o them .
One way o f o b ta in in g a g r e a t e r u n d e r s ta n d in g o f g r a s s e s i s by a chem i­
c a l s tu d y .
By d e te r m in in g th e v a r io u s ch em ica l c o n s t i t u e n t s o f g r a s s e s i t
i s p o s s ib le t o probe i n t o t h e i r i n n e r s t r u c t u r e s , and many tim e s v a r i a t i o n s
t h e r e a re a p p a r e n t w hich a re n o t v i s i b l e t o th e e y e .
More p a r t i c u l a r l y , an
a n a l y s i s o f t h e i r l i g n i n and c e l l u l o s e c o n te n ts w i l l o f f e r s p e c i f i c in fo rm a ­
t i o n r e g a r d in g t h e i r n u t r i t i v e
v a lu e .
From a n u t r i t i o n a l p o in t o f v iew a
-4 -
knowledge o f th e s e a s o n a l changes o f th e lig n in and c e l l u l o s e c o n te n ts o f
Montana range g r a s s e s sh ou ld he a d e f i n i t e a id , as w ith t h i s knowledge i t
i s p o s s ib le t o compare th e s h i f t i n g s e a s o n a l fe e d v a lu e s o f th e g r a s s e s
s t u d ie d .
Such a com parison sh ou ld n a t u r a lly lea d t o a w i s e r u t i l i z a t i o n o f
Monteina range la n d , a n i i t i s hoped th a t t h i s w i l l e v e n t u a lly be the c a s e .
-
5*
REVIEW OF LITERATURE
A.
H is to r y o f Feed A n alyses
S h o r tly a f t e r th e b eg in n in g o f th e n in e te e n th o en tu ry Davy 2 j / l l / v a l ­
ued fe e d in g s t u f f s a cco rd in g to th e amount o f e x tr a c t t h a t was removed by
d ig e s t io n w ith h o t w a te r .
In 1809 Thaer 9 1 / in tro d u ced h i s system o f hay
e q u iv a le n t s , u s in g meadow hay as th e sta n d a rd .
He e stim a te d th a t 90 pounds
o f dry h ay, or c lo v e r , or v e tc h , or a l f a l f a , or s a in f o in , o r 200 pounds o f
p o t a t o e s , or 266 pounds o f c a r r o t s , or 350 pounds o f ru tab agas w ith t o p s , or
460 pounds o f b e e ts w ith to p s , or 525 pounds o f r a d is h e s or 600 pounds o f
w h ite cabbage were eq u a l to 100 pounds o f meadow hay in fe e d in g v a lu e .
T hese
hay v a lu e s were an advance over th e o ld e r methods o f v a lu a t io n , but th e f a i l ­
ure t o c o n s id e r th e p r o te in f r a c t io n o f th e feed le d to d isc o rd a n t r e s u l t s .
O ther o b se rv e r s ob tain ed d if f e r e n t hay v a lu e s , and so a mass o f c o n tr a d ic ­
to r y t a b le s began to accum ulate in th e l i t e r a t u r e .
B o u e sin g a u lt, %] in
1 8 4 4 , proposed th e v a lu a tio n o f c a t t l e fe e d s upon th e b a s is o f t h e i r n it r o ­
gen c o n te n t as compared w ith ord in a ry hay o f 1 .3 4 $ n itr o g e n c o n te n t.
I t was th e g r e a t s e r v ic e o f Henneberg to break away e n t i r e l y from th e
o ld hay e q u iv a le n t c o n cep tio n and t o base th e v a lu a tio n o f fe e d in g s t u f f s on
th e p ercen tage o f th e th r e e major n u t r i t i v e c o n s titu e n ts *
and c a rb o h y d ra tes.
p r o t e in s , f a t s
Crude f i b e r , assumed t o be i n d i g e s t i b l e , was determ ined
by a m o d ific a tio n o f th e o ld E in h o f p roced u re, which c o n s is t e d o f rem oving
th e more e a s ily -h y d r o ly z e d c e l l u l o s e c o n s t it u e n t s w ith s u c c e s s iv e p o r tio n s
o f b o il in g d il u t e a c id and a l k a l i .
The sum o f th e p e r ce n ta g es o f m o is tu r e ,
—6 —
c ru d e f a t , cru d e p r o t e i n (N
x
6 .2 5 )» a sh and orude f i b e r s u b tr a c te d from IQO
gave th e p e rc e n ta g e o f n i t r o g e n - f r e e e x t r a c t .
The c ru x o f th e w hole H - f r e e
e x t r a c t problem i s in th e n a tu r e o f th e s u b s ta n c e s rem oved by th e b o i l i n g
a c id and b o i l i n g a l k a l i tr e a tm e n t i n th e d e te r m in a tio n o f orude f i b e r .
Q u e s tio n s o f te rm in o lo g y b o th e re d H en n eh e rg , 4 j / a s he s t a t e d t h a t th e su b ­
s ta n c e c a l l e d wood f i b e r i s e v i d e n t l y n o t alw ays th e same s u b s ta n c e , s in c e
th e p ro d u c t o b ta in e d from th e same f e e d in g s t u f f v a r i e s w ith d i f f e r e n t p ro ­
c e s s e s o f m e c h a n ic a l com m inution.
For t h i s re a so n he c o n s id e re d i t d e s i r ­
a b le t o g iv e th e r e s id u e o b ta in e d b y th e tr e a tm e n t w ith a c id and a l k a l i a
l e s s d e f i n i t e d e s ig n a tio n th e n wood f i b e r ( i . e . , c e l l u l o s e ) .
T h is i s e s p e ­
c i a l l y n e c e s s a r y s in c e th e r e s id u e s o b ta in e d from d i f f e r e n t s u b s ta n c e s by
e x a c t l y th e same p r o c e s s have a v e ry d i f f e r e n t c o m p o sitio n ( c lo v e r hay r e s i ­
d u e , f o r e x am p le, i s q u i t e d i f f e r e n t from t h a t o f w heat s tra w o r from t h a t
o f th e f e c e s o f an a n im a l fe d c lo v e r h a y , e t c . ) , and a re e s s e n t i a l l y u n lik e
th e p u re wood f i b e r o r c e l l u l o s e o f th e c h e m is t.
He f e l t i t n e c e s s a r y ,
t h e r e f o r e , t o p ro p o se f o r th e r e s i d u e i n q u e s tio n th e name "orude f i b e r . "
F o r a s i m i l a r re a s o n th e names " N -fre e e x t r a c t " and "o ru d e p r o te in " were de­
v is e d .
The o ld H enneberg method o f fe e d a n a l y s i s h a s been u s e d s in c e 1866 w ith
v e ry few c h a n g e s .
I t i s s t i l l th e s ta n d a r d method o f fe e d a n a l y s i s .
H enneberg sy stem o f a n a l y s i s i s a s f o llo w s :
1.
Orude f a t ( e t h e r e x t r a c t ) .
2.
Crude f i b e r ( e x t r a c t i o n s , d i l u t e a l k a l i and a c i d s ) .
3.
A sh.
The
4.
P r o t e i n (N x 6 .2 5 ) •
5*
N itr o g e n - f r e e e x t r a c t (b y d i f f e r e n c e ) .
A f te r a s tu d y o f th e above sy stem o f a n a ly s is one w i l l f in d t h a t n o t
one o f th e above e x i s t i n g s o - c a l l e d f r a c t i o n s i s a p u re compound, b u t e a c h
i s a m ix tu r e .
I.
E th e r E x t r a c t I j /
True f a t s ( t r i g l y c e r i d e s ) and f a t t y a c id s w hich a re u t i l i z e d by a n im a ls
a s s o u r c e s o f e n e rg y and o f th e e s s e n t i a l f a t t y a c i d s .
P h o s p h o lip id s , s t e r o l s , waxes and r e s i n s , and p la n t p ig m e n ts.
T h is
g ro u p may make up a s h ig h a s f o r t y p e r c e n t o f th e e t h e r e x t r a c t , and w ith
th e e x c e p tio n o f p h o s p h o lip id s and c a r o t e n e , a p p e a r t o be o f l i t t l e u se t o
th e a n im a l.
F a t - s o l u b l e v ita m in s Ai ( c a r o t e n e ) . D.E and K.
E s s e n t i a l d i e t a r y con­
s titu e n ts .
2.
Crude P r o t e i n (N
x
6 .2 5 )
The K je ld a h l m ethod f o r d e te r m in in g crude p r o t e i n i n fe e d s i s r e a l l y
o n ly a m ethod f o r d e te r m in in g t o t a l n i t r o g e n .
S in c e many p u r i f i e d p r o t e i n s
h ave been fo und t o c o n ta in 16$ n i t r o g e n , 100 d iv id e d by t h i s f ig u r e g iv e s
6 . 2 5 , w hich i s th e cu sto m ary f a c t o r u s e d f o r e x p r e s s in g r e s u l t s a s cru d e
p ro te in .
The r e q u ire m e n ts o f an an im al a r e f o r c e r t a i n amino a c id s w hich
a re c o n s t i t u e n t s o f p r o t e i n s .
Of more th a n t h i r t y amino a c id s w hich have
b een i d e n t i f i e d so f a r , a p p ro x im a te ly t e n have been shown by Rose 6 0 / t o be
e s s e n t i a l f o r norm al g row th and m a in te n a n c e o f r a t s .
T h a t t h i s re q u ire m e n t
o f th e r a t oannot be tak en to o l i t e r a l l y fo r o th er B p e c ie a o f anim als wae
r e c e n t ly shown by A lm q u ist, who found th a t g ly o in e i s an e s s e n t i a l amino
a o id f o r o h io k s. 2 / P r e v io u s t o t h i s tim e g ly o in e has alw ays been con­
s id e r e d t o be a n o n - e s s e n t ia l amino a o id .
R egarding rum inants (th e typ e o f anim als w ith which we are c o n c er n e d ),
th er e i s a s e r io u s q u e s tio n as t o w hether th e amino a o id con ten t o f th e fe e d
( i n o th er w ords, th e q u a lit y o f th e p r o te in ) i s o f v e r y g r e a t s ig n if i o a n o e .
T h is i s because th e s e h erb ivorou s anim als depend la r g e l y fo r t h e ir n u t r ie n t s
upon th e a c tio n o f m icroorganism s which n o t o n ly break p r o te in s down in t o
amino a c id s , but are a ls o a b le t o s y n th e s iz e e s s e n t i a l amino a c id s which are
th en used by th e an im al.
o a ttle .
An example o f t h i s i s th e u t i l i z a t i o n o f u rea fo r
Urea i s a sim ple o rg a n ic n itr o g e n compound which i s not known t o
have any food v a lu e i t s e l f , but which oan be used by m icroorganism s in th e
rumen in s y n t h e s iz in g e s s e n t i a l amino a c id s .
For t h i s reason i t seems prob­
a b le th a t an e x te n s iv e su rvey o f th e amino aoid co n ten t o f fe e d s fo r rumi­
n an ts would be o f dubious v a lu e , and th e t o t a l n itr o g e n d eterm in a tio n i s
probably s a t i s f a c t o r y f o r most p u rp o ses.
3.
Ash
T h is in c lu d e s a l l m in eral elem en ts e x ce p t th a t p o r tio n which e sc a p e s
d u rin g i g n i t i o n , p a r t ic u la r ly s u lf u r , c h lo r in e and io d i n e .
The im portance o f m ineral e lem en ts in th e n u t r it io n o f anim als has been
known fo r a lo n g tim e .
However, u n t i l r ec e n t y e a r s th e study o f m in e r a ls in
t h i s r e la t io n h as been lim ite d t o a few o f the major e le m e n ts , s p e c i f i c a l l y
c a lciu m , phosphorus, sodium , p o ta ssiu m , magnesium and c h lo r in e .
These are
-9 -
" m ajo r" e le m e n ts o n ly i n th e se n se t h a t th e y a re r e q u ir e d in r e l a t i v e l y
l a r g e q u a n t i t i e s by th e a n im a l.
Of no l e s s im p o rtan c e a re th e s o - o a l l e d
"m in o r" e le m e n ts , whioh a re r e q u ir e d i n s m a lle r q u a n t i t i e s bu t a re none th e
le s s e s s e n tia l.
I o d i n e , f o r i n s t a n c e , n e e d s be p r e s e n t i n th e p l a n t o n ly in
q u a n t i t i e s m easured in p a r t s p e r b i l l i o n , and y e t a d e f ic ie n c y o f io d in e i n
p l a n t m a t e r i a l u se d a s fe e d c a u s e s p ro fo u n d changes in th e a n im a ls th u s de­
p r iv e d o f t h i s e le m e n t.
S p e c i f i c know ledge o f th e n u t r i t i o n a l s i g n if ic a n c e o f m in e ra ls i s due
p r i m a r i l y t o r e s e a r c h w ith in th e l a s t q u a r t e r o f a c e n tu r y .
I t i s now known
t h a t i n a d d i t i o n t o t h e "m ajo r" e le m e n ts m entioned a b o v e , i o d i n e , i r o n , s u l ­
f u r , m anganese, c o p p e r, z in c and c o b a lt a re e s s e n t i a l e le m e n ts i n n u t r i t i o n .
I t i s p ro b a b le t h a t f u r t h e r work w i l l add more m in e r a ls t o th e l i s t , p o s­
s i b l y i n c lu d in g s i l i c o n , f l u o r i n e , b o ro n , b ro m in e, alum inum , a r s e n i c o r
n ic k e l.
A ll o f th e s e a re found r e g u l a r l y i n th e a n im al body.
4 . Crude F ib e r
( E x t r a c t i o n w ith d i l u t e a c id and a l k a l i )
C e llu lo s e 6^ / i s a m ajo r o o n s titu d e n t o f th e crude f i b e r f r a c t i o n .
The
c e l l u l o s e i n p l a n t c e l l w a lls i s p r a c t i c a l l y i n d i g e s t i b l e by c a r n iv o r a and
d i g e s t i b l e to a w id e ly v a r ia b le e x te n t by om nivora and h e r b iv o r a .
C e llu lo s e
u t i l i z a t i o n by any an im al i s d e p e n d e n t on in te r m e d ia r y a c ti o n by b a c t e r i a o f
th e d i g e s t i v e t r a c t f o r i t s d e g r a d a tio n t o g lu c o s e .
C e llu lo s e i s an im p o r­
t a n t so u rc e o f e n e rg y t o h e r b iv o r o u s a n im a ls .
O nly p a r t o f th e l i g n i n i s fo u n d i n th e cru d e f i b e r f r a c t i o n .
m a in d e r i s found i n th e n i t r o g e n - f r e e e x t r a c t .
The r e ­
L ig n in i s i n d i g e s t i b l e i t -
-I O s e l f and a ls o i n h i b i t s th e d ig e s t io n o f c e l l u l o s e .
I t was o r ig in a lly a s­
sumed th a t th e crude f i b e r f r a c t io n was in d i g e s t i b l e and th e n it r o g e n - fr e e
e x tr a ct d ig e s t ib le .
One can see from th e above th a t t h i s i s d e c id e Iy n ot th e c a s e .
A ls o ,
th e f a c t th a t th e crude f i b e r c o n ta in s v a r ia b le amounts o f lig n in makes any
assu m ptions drawn from th e crude f ib e r co n te n t o f p la n t m a te r ia ls o f l i t t l e
v a lu e •
5 . N itr o g e n -fr e e E x tra ct
(O btained by d if f e r e n c e )
The f a c t th a t t h i s f r a c t io n i s o b ta in ed by d if f e r e n c e i s o n ly one o f
th e o b j e c tio n s t o i t s u s e .
I t i s c u sto m a r ily assumed th a t n it r o g e n - fr e e e x ­
t r a c t i s a measure o f th e d i g e s t i b l e e n e r g y -su p p ly in g m a te r ia l o f f e e d .
The
n it r o g e n - f r e e e x t r a c t c o n ta in s most o f th e l i g n i n , which i s approxim ately
98 per cen t i n d i g e s t i b l e by a n im a ls.
To make m a tters even w orse, a high l i g ­
n in co n ten t 1 1 /7 4 / may preven t th e d ig e s t io n o f th e e a e i l y - d i g e s t i b l e por­
t io n s o f th e p la n ts by in o r u s tin g them and th us k eep in g them from coming
in t o c o n ta c t w ith i n t e s t i n a l b a c t e r ia and d ig e s t iv e j u i c e s .
Thus i t i s pos­
s i b l e fo r them t o p ass through and remain in t a c t in th e f e c e s o f th e a n im a l.
The n it r o g e n - f r e e e x tr a c t a ls o c o n ta in s hemic e l l u l o s e , s ta r c h e s and
su g a rs which are more or l e s s e a s i l y d ig e s t e d and are e n e r g y -su p p ly in g ma­
te r ia ls .
The hemic e l l u l o s e s 74/ are w id e ly d is t r ib u t e d in th e v e g e ta b le kingdom
and n ext to c e l l u l o s e are undoubtedly th e most abundant o f the m a te r ia ls o f
p la n t o r ig in .
H e m io e llu lo se may be d e fin e d as carbohydrate su b sta n ces t h a t ,
11
in t h e i r n a tu r a l s t a t e , are in s o lu b le in b o ilin g w ater bu t are s o lu b le in
d i l u t e aqueous s o lu t io n s o f a l k a l i s .
Upon h e a tin g them w ith d ilu t e a c id s a t
atm osp h eric p r e s s u r e , hemic e l l u l o s e s a ffo r d sim ple su g a rs and fr e q u e n tly a l ­
so u r o n ic a c id s .
In co n n ectio n w ith c e r t a in ty p e s o f hem ic e ll u lo s e two
term s have r e c e n t ly come in t o u s e ; nam ely, "polyuronides" and " o e llu lo s a n a ."
The term "polyuronides" i s formed by th e c o n ju g a tio n o f c e r ta in sugar a c id s
(g lu c u r o n ic and g a la o tu r o n io a c id s ) w ith s u g a r s.
While most o f th e hem ic e l ­
l u l o s e s are p o ly u r o n id e s , i t must be em phasized th a t not a l l p o ly u ro n id e s
are hem ic e l l u l o s e s , fo r p e c t i n s , gums and m u cilages a ls o c o n ta in u r o n ic
a c id s .
The term " o e llu lo sa n s " r e f e r s t o th o se hemic e l l u l o s e s th a t are u s u a lly
f r e e o f u r o n ic a c id and are in t im a t e ly a s s o c ia te d w ith c e l l u l o s e .
An e x c e lle n t review o f the p r e se n t knowledge o f hem ic e l l u l o s e s i s found
in Norman's book, B io ch em istry o f C e llu lo s e , P o ly u r o n id e s , L ignin e t c .
From th e above d is c u s s io n o f th e Henneberg system o f fe e d a n a ly s is one
would assume th a t some o th e r method o f a n a ly s is would be much b e t t e r .
i s in d eed th e c a s e .
T h is
A com plete a n a ly s is o f fe e d s in which one would a n a ly ze
fo r th e v a r io u s carb ohydrates ( f o r exam p le, p e n to sa n s, s t a r c h e s , hemic e l l u ­
l o s e , e t c . ) would be v ery lo n g and te d io u s and could o n ly be performed in a
few o a s e s .
were need ed .
I t would a ls o be im p r a c tic a b le where a la r g e number o f a n a ly s e s
Taking t h i s in t o c o n s id e r a tio n , a system o f a n a ly s is which
overcom es th e f a u l t s o f th e crude f ib e r method and y e t i s sim ple enough f o r
r o u tin e u se has been p resen ted by Norman.
cedure I
He su g g e ste d th e fo llo w in g pro­
121.
S th er e x t r a c t .
2.
Crude p r o te in (H x 6 .2 5 ) •
3.
C e llu lo s e .
4.
L ig n in .
5.
Ash.
6.
E a s ily - d ig e s t e d carb ohydrates (b y d i f f e r e n c e ) .
T his system d is c a r d s a f r a c t io n a t io n which i s w h o lly a r b itr a r y and
w ith o u t b i o l o g i c a l s ig n if ic a n c e in fa v o r o f a s e p a r a tio n o f the fe e d in t o
groups which can be t r u ly e v a lu a te d from a n u t r it iv e s ta n d p o in t.
The d e te r m in a tio n o f p la n t c e l l u l o s e , fo r in s t a n c e , o f f e r s th e advan­
ta g e th a t i t i s more s p e c i f i c than th e crude f ib e r f r a c t io n .
I t i s known
th a t c e l l u l o s e per se i s alm ost c o m p le te ly d ig e s t ib l e in th e rum inant.
The d e te r m in a tio n o f lig n in i s a d e f i n i t e s te p forw ard , sin c e l i g n i n
i t s e l f i s a t l e a s t 98$ i n d i g e s t i b l e and a ls o d e f i n i t e l y r e ta r d s th e d i g e s t i ­
b i l i t y o f o th e r c o n s t it u e n t s .
B.
L ig n in
The fu n c tio n o f li g n i n in th e p la n t may be t o g iv e s tr e n g th and r i g i d ­
i t y to th e c e l l w a ll.
U n lig n if ie d m a te r ia l u s u a lly i s s o f t and c o n ta in s
a h igh p ercen tage o f w a ter.
m a t e r ia l.
I t a ls o breaks much more e a s i l y than l i g n i f i e d
On th e o th er hand, a h igh p ercen tage o f l i g n i n w ill o fte n make
th e woody m a te r ia l b r i t t l e .
t e r i a l a c t io n .
L ig n in i s ex trem ely r e s i s t a n t to decay and bac­
Wood b u ried at l e a s t 5 0 0 ,0 0 0 y ea rs has been found and on
a n a ly s is much o f th e l ig n in was in an a p p a ren tly u n a lte r e d s t a t e .
-13'
The l i g n i n in anim al m etabolism i s v e ry s i g n i f i c a n t , s in c e i t i s e x ­
tr e m ely r e s i s t a n t t o d ig e s t io n by enzym es.
When i t i s fe d to dogs 2 1 / th ere
i s a p a r t ia l breakdown o f th e l i g n i n , a s ev id en ced by an in c r e a s e in th e
amount o f b e n z o ic a c id e lim in a te d as h ip p u rio a c id and a ls o a l o s s in m etho x y l c o n te n t o f th e l ig n in from th e f e c e s .
I t i s p o s tu la te d th a t such break­
down o f l ig n in as does occur i s p robab ly not due t o b a c t e r ia l a c tio n but
th a t i t ta k e s p la c e in th e stom ach, p o s s ib ly through some enzyme p r e se n t in
th e g a s t r i c j u i c e .
In th e human body th e r e i s no e v id e n c e at p re se n t th a t
any o f th e li g n i n in g e s te d can be u t i l i z e d .
L ign in i s th e in o r u s tln g p art o f p la n t and wood t i s s u e which i s charac­
t e r iz e d by i t s p h e n o lic c h a r a c t e r is t ic s and by i t s m ethoxyl c o n te n t, and i s
th a t p o r tio n o f p la n t t i s s u e reco v ered by th e 72 per cen t s u lf u r ic a c id or
fuming h y d r o c h lo r ic a c id method f o r d eterm in a tio n o f l i g n i n .
Recent work
2 2 /5 S /6 ^ / h as fu r th e r su b d ivid ed th e in o r u s tln g or fib e r -b o n d in g m a te r ia ls
in wood in t o two f r a c t io n s , the lig n in -b o n d in g m a te r ia l and the n o n - lig n in ­
bonding m a t e r ia l.
T h is i s o f g r e a t i n t e r e s t , sin c e th e more g e n e r a l d e f i n i ­
t i o n o f li g n i n must be r e v is e d t o take t h i s in t o c o n s id e r a tio n .
S in ce no
c le a r -o u t d e f i n i t i o n o f lig n in has been o ff e r e d , t h i s attem pt o f fu r th e r
s u b d iv is io n o f p la n t - in c r u s t in g m a te r ia ls may le a d to c o n fu sio n in te r m in o l­
ogy.
E xperim ental work has in d ic a te d f a i r l y d e f i n i t e l y th a t both l i g n i n and
an oth er m a te r ia l are concerned in th e bonding to g e th e r o f wood f i b e r s .
The
n o n -lig n in bonding m a te r ia l has been examined in h o lo c e llu lo s e s e c t io n s o f
wood; i t has been found t h a t trea tm en t w ith d ilu t e s o lu t io n s o f sodium hy­
d roxid e w i l l remove i t and so perm it m a cera tio n .
The l i g n i n i s removed by
—14—
c h l o r i n a t i o n o f th e wood sam ples and w ash in g w ith w a te r .
Both tie l i g n i n
and n o n - l i g n i n "bonding m a t e r i a l m ust he removed b e fo re f i b e r s w i l l s e p a r a t e ;
th e rem oval o f e i t h e r i s n o t s u f f i c i e n t t o cause su ch s e p a r a t i o n .
The n a ­
t u r e o f t h i s n o n - li g n in b o n d in g m a t e r i a l h a s n o t been d e f i n i t e l y worked o u t
b u t may c o n s i s t o f hem ic e l l u l o s e c o n s i s t i n g o f x y la n , u r o n io a c id and p ro b ­
a b ly g lu o o s a n , t o g e t h e r w ith o th e r com ponents whose e x a c t n a tu r e i s n o t known.
S o hulze 84/ i s g e n e r a l l y c r e d i t e d w ith th e i n t r o d u c t i o n o f th e term
" l i g n i n " i n t o p l a n t c h e m is tr y , 7j / a s i t was e a r l y re c o g n iz e d t h a t t h e r e was
p r e s e n t i n p l a n t s a woody " l i g n i f le d " p o r t i o n .
By t r e a t i n g wood w ith n i t r i c a c id and p o ta ssiu m h y d ro x id e Payne 7 0 / ob­
t a i n e d a more o r l e s s pu re c e l l u l o s e and p o in te d o u t t h a t some s u b s ta n c e had
b een removed w hich was r i c h e r in carb o n th a n c e l l u l o s e .
T h is s u b s ta n c e h a s
b een named l i g n i n .
L ig n in 6 ^ / i s a s u b s ta n c e o r m ix tu re o f s u b s ta n c e s w hich composes th e
more r e s i s t a n t o r woody p o r ti o n s o f p l a n t t i s s u e .
I t s h o u ld be p o in te d o u t
c l e a r l y t h a t l i g n i n i s a v a g u e , c ru d e , g e n e r a l te rm and c o v e rs th e woody
p o r t i o n o f p l a n t s much a s th e word " p r o t e i n " c o v e rs th e l i v i n g n itr o g e n o u s
p o rtio n of liv in g p la n ts .
The c h e m is try o f l i g n i n to d a y i s in th e s t a t e
t h a t th e c h e m is tr y o f p r o t e i n s was i n tw e n ty y e a r s a g o .
T hus, i n th e d e te r m in a tio n o f l i g n i n when one i n v e s t i g a t o r r e p o r t s a
p e rc e n ta g e o f l i g n i n i n a sam ple and a n o th e r , by a d i f f e r e n t method o f a n a ly ­
s i s , r e p o r t s a d i f f e r e n t p e rc e n ta g e i n th e same sam p le, no one a t p r e s e n t
CEin
sa y w hich one i s c o r r e c t .
To overcome t h i s d i f f i c u l t y one ch o o ses a
m ethod o f a n a l y s i s o f l i g n i n t h a t b e s t s u i t s h i s n e e d s .
I n th e d e te r m in a -
-1 5 -
t l o n o f l i g n i n in fe e d one ohooaee a method th a t h a st r e p r e s e n ts th a t por­
t i o n o f th e p la n t th a t i s in d ig e s t i b le by an im als, as determ ined by d ig e s ­
t io n t r i a l s .
With t h i s in mind a b r i e f d is c u s s io n o f th e chem istry o f l i g ­
n in w i l l be p r e se n ted h e r e .
The l i g n i n p r e se n t in woody m a te r ia l may be c h e m ic a lly combined w ith
c a rb o h y d r a te s.
There are two v iew s h e ld a t p r e se n t as t o th e lin k a g e o f
l i g n i n w ith th e carbohydrate m a te r ia l; one i s th a t th e r e i s an e s t e r - l i k e
26/ 100/ u n ion betw een an a c id ic group in the lig n in and an hydroxyl o f th e
ca rb o h y d ra te, w h ile th e o th er view 51/ 38/ 62/ i s th a t an e t h e r - lik e lin k a g e
e x i s t s betw een li g n i n and the ca rb o h y d ra te.
Due to the f a c t th a t l ig n in i s
more d i f f i c u l t to sep a ra te in some p la n ts than in o th e r s , i t i s p o s s ib le
th a t e i t h e r one or both lin k a g e s are p r e se n t in v a r io u s p la n ts .
There has been c o n sid e r a b le s p e c u la tio n as to the p recu rso rs o f l i g n i n .
Some o f the probably p recu rso rs are*
c e l l u l o s e , 36/ s o lu b le ca rb o h y d ra tes,
p e n t o s e s , m ethyl p e n t o s e s ,5 ^ /5 ^ /5 ^ /7 8 /7 2 /8 5 / hexosea and p e c t in .24/
Some o f
th e e a r ly in v e s t ig a t o r s o f lig n in even went so fa r as t o s t a t e th a t li g n i n
o f p h e n o l!0 c h a r a c te r was not p r e se n t in p la n ts and wood, but formed as a
r e s u lt o f th e a c tio n o f chem ical r e a g e n ts on th e ca rb o h y d ra tes.
T h is view ­
p o in t has been d e f i n i t e l y d is c a r d e d ;6%/ how ever, i t has been r eco g n ized t h a t
v a r io u s methods f o r th e d eterm in a tio n o f lig n in 64/ may form in s o lu b le con­
d e n sa tio n produ cts from the carbohydrate a o f p la n t s .
These carbohydrate con­
d e n s a tio n p rod u cts seldom amount t o as much as 10 per cen t o f the t o t a l l i g ­
n in determ in ed .
L ign in
shows a number o f c o lo r r e a c t io n s .7 6 /
N e a rly one hundred y e a r s
-1 6 -
a g o , Runge 8 j / found t h a t p in e o i l , when t r e a t e d w ith p h e n o l and h y d r o c h lo r ic
a c i d , assum ed a g r e e n is h - b lu e c o l o r a t i o n , and when t r e a t e d w ith a n i l i n e - s u l ­
f a t e , a y e llo w c o lo r was p ro d u c e d .
b een em ployed.
B cth o rg a n ic and in o r g a n ic r e a g e n ts have
Among th e fo rm e r, " p h lo r o g lu o in o l r e a g e n t" o r " W ie s e n e r1s r e ­
a g e n t," w hich g iv e s a r e d d i s h - v i o l e t c o l o r a t i o n w ith d i g n i f i e d m a t e r i a l , h a s
b een w id e ly u s e d i n t e s t i n g f o r l i g n i n .
C o lo rs i n g e n e r a l are p ro d u ced when
l i g n i f i e d t i s s u e s a re t r e a t e d w ith p h e n o ls and w ith a m in e s.
The c o l o r r e a c ­
t i o n s g iv e n by l i g n i f i e d m a te r ia l w ith a ro m a tic compounds a re p ro b a b ly due to
an a ld e h y d ic s u b s ta n c e w hich i s c l o s e l y a s s o c ia te d w ith th e l i g n i n co m p lex .
I n u s in g c o lo r t e s t s f o r l i g n i n one m ust r e a l i z e th e y may be m is le a d in g .
A c r i t i c a l s u rv e y sh o u ld p re c ed e th e u se o f any c o lo r t e s t f o r l i g n i n t o he
s u re t h a t i t i s s p e c i f i c f o r l i g n i n and n o t due i n p a r t t o o th e r c o n s t i t u e n t s
o f th e p l a n t .
A c co rd in g t o H a r r i s , 4 ^ / l i g n i n can he i s o l a t e d o n ly w ith th e a p p l i c a ­
tio n o f h y d ro ly tic re a c tio n s .
Even th e s o - c a l l e d " n a t iv e lig n in " ( K la e o n ) ,
th e a lc o h o l - s o l u b le l i g n i n o b ta in e d from wood w ith o u t th e u s e o f added a c i d ,
was found t o he a r e s u l t o f th e a c t i o n o f h y d r o l y t i c enzym es.
The q u a n t i t a t i v e e s ti m a ti o n o f l i g n i n i s d iv id e d i n t o two c l a s s e s »
th e
d i r e c t and th e i n d i r e c t m ethods.
I n d i r e c t M ethods
S ch u lze 84/ o x id iz e d l i g n i f i e d p l a n t m a te r ia l w ith n i t r i c a c id and po­
ta s s iu m c h lo r a te and assumed th e l o s s i n w e ig h t r e p r e s e n te d l i g n i n .
T h is
m ethod i s o n ly o f h i s t o r i c a l i n t e r e s t , a s th e r e s u l t s o b ta in e d a re t o o ttig h ;
-1 7 -
n o t o n ly i s th e l i g n i n c o m p le te ly o x id iz e d , b u t a l s o th e hem ic e l l u l o s e s and
p e rh a p s some o f th e c e l l u l o s e i s a ls o d e s tr o y e d .
B en e d ik t and Bam berger 6 /
d e te rm in e d th e number o f m ethoxyl g ro u p s i n wood sam p les and computed th e
p e rc e n ta g e o f l i g n i n i n th e sam p le.
T h is m ethod i s b a se d on th e a ssu m p tio n
t h a t in l i g n i f i e d m a t e r i a l l i g n i n i s th e o n ly s u b s ta n c e c o n ta in in g m ethoxyl
g ro u p s and th e m ethoxyl c o n te n t i n l i g n i n from v a r io u s s o u rc e s i s th e sam e.
C ro s s , Bevan and B rig g s 20 /b a s e d t h e i r method on th e r e a c t i o n betw een l i g n i n
and p h lo r o g l u o in o l.
Due t o th e c o m b in a tio n o f p h lo r o g lu c in o l w ith th e f u r ­
f u r a l - y i e l d i n g g ro u p s , t h i s m ethod g iv e s to o h ig h a r e s u l t .
The m ethod o f
S e id e l 8 6 / i s b a se d on th e f a c t t h a t when n i t r i c a c id i s added t o l i g n i n , ox­
id e s o f n itr o g e n a re l i b e r a t e d , and may be d e te rm in e d by t i t r a t i o n w ith p e r­
m an g an ate,
The method o f W aentig and Q ie r is o h i s b a se d on th e f a c t t h a t l i g ­
n in ta k e s up a c o n s id e r a b le q u a n t i t y o f c h l o r i n e .
By d e te r m in in g th e amount
o f c h lo r in e a d so rb e d th e p e rc e n ta g e o f l i g n i n i s c a l c u l a t e d .
M ehta 6 2 /ta k e s
a d v an ta g e o f th e f a c t t h a t a s o l u t i o n o f p h o s p h o tu n g s tio and phosphom olybdic
a c id s i n p h o s p h o ric a c id i s a v e ry s e n s i t i v e re a g e n t f o r d e t e c t i n g m inute
am ounts o f a ro m a tic s u b s ta n c e s c o n ta in in g h y d ro x y l g ro u p s .
E i o h le r 2Jg/ r e ­
g a rd s f lu o r e s c e n c e a s a v e ry good m ethod o f e s ti m a ti o n o f l i g n i f i c a t i o n , and
he re g a r d s i t a s a more s e n s i t i v e method th a n c o lo r r e a c t i o n s .
D ir e c t M ethods
The d i r e c t m ethods f o r q u a n t i t a t i v e e s ti m a ti o n o f l i g n i n a re d iv id e d
i n t o two c l a s s e s , th o s e w hich depend upon d i s s o l v i n g c e l l u l o s e and o t h e r
c a rb o h y d ra te s o u t and le a v in g th e l i g n i n a s an in s o lu b le r e s i d u e , and th o s e
f
—18—
w hich d is s o lv e out th e l i g n i n and th u s sep a ra te i t from the c e ll u lo s e and
o th e r carbohydrate m a t e r ia l.
The s u l f u r i c a c id method 2 8 /5 ^ /6 8 /5 2 / I s most commonly used to d a y .
However, th e r e are s e v e r a l o b j e c tio n s t o i t .
I t i s based on th e f a c t t h a t
c o ld 72 p er cen t s u l f u r i c a c id w i l l h yd rolyze c e l l u l o s e .
U su a lly t h i s i s
preced ed by an e x t r a c t io n w ith w ater t o remove w a te r - and a lc o h o l- s o lu b le
p o r tio n s o f th e woody m a te r ia l.
I f one adds a form aldehyde s o lu t io n t o th e
72 per cen t s u l f u r i c a c id , the tim e req u ired fo r th e h y d r o ly s is o f th e carbo­
h y d ra tes i s c o n s id e r a b ly r ed u c e d .8 2 /
The o b je c tio n s to t h i s method are th e
p o s s i b i l i t y o f in com p lete h y d r o ly s is o f th e ca rb o h y d ra tes, the form ation o f
con d en sa tio n p rod u cts o f th e carb oh yd rates and con tam in atin g the l i g n i n r e s ­
id u e w ith n itr o g e n o u s com plexes.
Norman and Jenlcins
p o in t out th a t f r u c t o s e , x y lo s e , a r a b in o se , and
p o ly s a c c h a r id e s g iv e a s o l i d r e sid u e on sta n d in g in 72 per cent s u l f u r i c
a c id and, t h e r e f o r e , in t e r f e r e w ith th e lig n in d e te r m in a tio n by t h i s method.
The tim e o f treatm en t w ith 72 per cen t s u lf u r ic a c id can be r e g u la te d so
th a t t h i s e r r o r can be reduced t o th e p o in t th a t i t i s n o t a s e r io u s f a c t o r .
The fuming h y d r o c h lo r ic a c id method 9 2 /8 1 /8 /. method i s s im ila r to th e
72 per cen t s u l f u r i c a c id method e x ce p t th a t fuming h y d r o c h lo r ic a c id i s
u sed .
T his method i s s u b je c t to two e r r o r s *7^/ o n e, incom p lete h y d r o ly s is
o f p r o te in s and tw o, form ation o f r e v e r s io n produ cts o f hum us-like m a te r ia l
when th e h yd rolyzed product i s d ilu t e d w ith w ater b efo re f i l t r a t i o n .
The l ig n in in a liq u e f ie d m a te r ia l can be removed by h e a tin g w ith a lk a ­
l i under p r e s s u r e , and a c i d i f y i n g . 6 2 /
T h is method g iv e s v a lu e s th a t are to o
•19'
low owing t o th e p a r t i a l d e g r a d a tio n o f th e l i g n i n by sodium h y d ro x id e s o lu ­
t i o n h e a te d t o a c o m p a ra tiv e ly h ig h te m p e r a tu r e .
P o r i n d u s t r i a l p u rp o s e s d e l i g n i f i c a ti o n i s b ro u g h t a b o u t by h e a ti n g
wood w ith a c id s u l f i t e s u n d e r p r e s s u r e , p ro d u c in g s o lu b le l i g n i n s u l f o n i c
a c i d , w hich i s s o ld c o m m e rc ia lly a s th e b a s i c c a lc iu m s a l t .
W hatever m ethod i s u s e d , th e f in e ly - g r o u n d p l a n t m a t e r i a l m ust f i r s t be
f r e e d from v a r io u s " e x t r a c t i v e s " su ch a s f a t t y s u b s ta n c e s , r e s i n s and v o la ­
tile
o ils .
-th e r and e t h a n o l , o r a c e to n e and e t h a n o l , o r a 1 :2 a lc o h o l- b e n ­
zene s o l u t i o n a r e u s e d f o r t h i s e x t r a c t i o n .
t r a c t i o n w ith h o t o r o o ld w a te r .
T h is may be fo llo w e d by an e x ­
I n some o ases a p r e lim in a r y tr e a tm e n t w ith
5 p e r c e n t sodium h y d ro x id e i s s u g g e s te d t o remove th e gummy s u b s ta n c e s , b u t
i n u s in g t h i s th e r e i s alw ays th e d a n g e r o f lo s in g some o f th e l i g n i n .
It
i s n o t n e c e s s a r y i n th e d e te r m in a tio n o f l i g n i n i n g r a s s e s .
T here i s a s y e t no method by w hich l i g n i n oan be i s o l a t e d u n c h an g e d .
As a r e s u lt o f a c id tr e a tm e n t,58/ l i g n i n may p o ly m er iz e, r e s i n i f y , co n d en se,
or c a r b o n iz e .
W hatever m ethod i s em ployed, a l i g n i n p r e p a r a t io n i s o b ta in e d which i s
no lo n g e r i d e n t i c a l w ith th e n a t u r a l l i g n i n .
A ls o , i t s h o u ld be p o in te d o u t
t h a t th e l i g n i n o b ta in e d by d i f f e r e n t m ethods o f a n a l y s i s may n o t be s i m i l a r
i n c o m p o s itio n .
H i l p e r t 4 8 / p o in ts o u t t h a t when one c o n s id e r s th e l i g n i n
i s o l a t e d by th e two d i f f e r e n t m ethods t o he i d e n t i c a l , one i s o v e r s te p p in g
th e l i m i t s o f t o le r a n c e i n c o m p o sitio n f u r t h e r th a n i s cu sto m ary i n chem­
is try .
Even sh o u ld two l i g n i n s a g re e i n th e m ethoxyl c o n te n t , one oan s t i l l
n o t draw c o n c lu s io n s a s to i d e n t i t y i n c o m p o s itio n .
-2 0 -
L ig n in oan be i s o l a t e d from wood by h e a ti n g w ith a lc o h o l and h y d ro c h lo ­
r i c a c i d . 3 2 /3 ^ /4 2 /4 8 /
I n p la c e o f e th a n o l o th e r h y d ro x y l compounds oan be
u s e d , su ch a s b u ty l and amyl a l c o h o l s , e th y le n e g l y c o l , th e m onomethyl e th e r
o f e th y le n e g l y c o l , and g ly c e r o l
^Cmonoohlo r o h y d rin .
I n a l l o a se s a p ro ­
d u c t i s o b ta in e d c o n ta in in g th e a l k y l o r a r y l g ro u p i n c o m b in a tio n w ith th e
lig n in .
I t h a s been s u g g e s te d t h a t a p ro d u c t i n th e n a tu r e o f an a c e t a l i s
fo rm ed , b u t t h i s h a s n o t been d e f i n i t e l y p ro v e d .
L ig n in oan a ls o be e x t r a c t e d from wood w ith p h e n o ls . Ig!/$ 2 /95/
T hese
combine c h e m ic a lly w ith th e l i g n i n , y i e l d i n g s o - c a l l e d "p h en o l l i g n i n . "
r e a c t i o n p ro d u c t i s f i l t e r e d , and th e f i l t r a t e
e x c e s s p h e n o l.
The
steam d i s t i l l e d t o remove th e
The l i g n i n re m a in s i n th e d i s t i l l i n g f l a s k .
L i g n i f i e d m a t e r i a l such a s wood and s tr a w , when h e a te d s e v e r a l h o u rs
w ith a c e t y l b ro m id e , d i s s o lv e s c o m p le te ly .
Yfhen t h i s s o l u t i o n i s p o u re d in
i c e w a te r , a p r e c i p i t a t e o f bromo l i g n i n s e p a r a te s o u t .
A r a t h e r pure l i g n i n m a t e r i a l h a s b een p re p a re d by e x t r a c t i n g w ith d i a z o ti z e d s u l f a n i l i o a c id in th e p re s e n c e o f sodium h y d ro x id e s o l u t i o n .
The
d is s o l v e d l i g n i n was p r e c i p i t a t e d w ith /^ n a p h th y la m in e h y d ro c h lo rid e o r
w ith
^>-brom oaniline h y d r o c h lo r id e .
The o b je c tio n a b le f e a t u r e o f t h i s m ethod
i s th e f a c t t h a t p a r t o f th e c e l l u l o s e i s a ls o d is s o lv e d by th e d ia z o b e n z e n e s u l f o n i c a c id t r e a t m e n t .
The c o n s t i t u e n t g ro u p s o f l i g n i n have been c a r e f u l l y s tu d ie d bv many
i n v e s t i g a t o r s . 41/ 72/ 71/ 3 0 / 3 1 / 3 2 /
The p re s e n c e o f m eth o x y l g roups ( - OCH^)
h a s been d e f i n i t e l y e s t a b l i s h e d .
S in c e th e m ethoxyl g ro u p s a re n o t r e a d i l y
s p l i t o u t , th e y a re p ro b a b ly a tta c h e d a s an e t h e r lin k a g e r a t h e r th a n an
-2 1
e a t e r lin k a g e .
arom atic n u c l e i .
A ls o , i t i s thought th a t th e m ethoxyl group® are a tta c h e d to
The r a te o f rem oval o f m ethoxyl groups in lig n in w ith h y -
d r io d io a o id warn found t o correspond c l o s e l y to th a t o f v a n i l l i n .
Ho d e f i n i t e co n ten t o f m ethoxyl groups in li g n i n h a s been a s c e r t a in e d ,
a s i t v a r ie s w ith th e source o f th e l i g n i f i e d m a te r ia l and a ls o w ith th e
method employed fo r th e i s o l a t i o n o f th e l i g n i n .
The presen ce o f hyd roxyl groups in li g n i n i s in d ic a te d by th e f a c t th a t
i t can be a o e ty la te d
and a lk y la t e d .
A lc o h o lic g ro u p s, a s w e ll as p h e n o lic
groups are p r e s e n t .
Wood and o th e r l i g n i f i e d m a t e r ia ls , when d i s t i l l e d w ith d il u t e m in era l
a c id s , g iv e form io and a c e t i c a c id s .
T h is in d ic a t e s th a t lig n in may c o n ta in
a c e t y l and form yl g ro u p s, alth ou gh t h e i r p resen ce in l i g n i n has not been de­
f i n i t e l y e s t a b lis h e d .
There i s a ls o ev id e n c e th a t carb onyl groups are p r e se n t in l i g n i n .
As
l i g n i n p r e p a r a tio n s reduce P e h lin g ’ s s o l u t i o n , a ld eh y d io groups may be p r e s ­
e n t.
But i t must be remembered th a t o th e r su b sta n ce s b e s id e s a ld eh yd es r e ­
duce F e h lin g 1B s o lu t io n .
I n d e f in it e co n d en sa tio n p rod u cts are o b ta in ed w ith
ph en ylh yd razin e and a em i-o a rb a zid e.
I t may be th a t th e form aldehyde a r i s e s
from th e m ethylene d io x id e group ( O-OHg-O) p resen t in th e lig n in m o le c u le ,
a s compounds c o n ta in in g t h i s group are known to y i e l d form aldehyde when th e y
are d i s t i l l e d w ith h y d r o c h lo r ic a o id .
The form ation o f r a th e r u n sta b le lig n o s u lf o n io a o id s by the in t e r a c t io n
o f l i g n i f i e d m a te r ia l w ith s u lfu r o u s a c id and b i s u l f i t e e may be due to an
e t h y le n io bond JJjj/ in l i g n i n .
Data on th e h a lo g e n a tio n o f l i g n i n
are r a th e r in c o n c lu s iv e a s su b -
•22
B t i t u t i o n a s w e ll a s a d d itio n o f brom ine no do u b t ta k e s p l a c e .
L ig n in oan be a o e t y l a t e d 4 2 / w ith a o e t i o a n h y d rid e s and p y r id in e o r a o e t y l c h lo rid e .
I t a p p e a r s , how ever, t h a t i s o l a t e d l i g n i n m ust be so a l t e r e d
by th e h y d r o c h lo r ic a c id s e p a r a t i o n m ethod t h a t i t s a c t i v i t y tow ard a c e t y l a ­
t i o n i s d im in is h e d .
A b e n so y l d e r i v a t i v e o f a l k a l i l i g n i n h a s been p re ­
p a re d by t r e a t i n g th e l i g n i n w ith b e n z o y l c h lo r id e and p y r i d i n e .
R e s u lts o f
th e a n a l y s i s o f th e b e n z o la te d p ro d u c t f o r carb o n and h y d ro g en i n d i c a t e t h e
p re s e n c e o f f o u r b e n z o y l g ro u p s .
L ig n in oan be r e a d i l y a l k y l a t e d 8 8 /8 6 /3 ^ / by th e u s u a l a l k y l a t i n g r e ­
a g e n ts .
One m ethod i s t o su sp en d l i g n i n i n 10 p e r c e n t sodium h y d ro x id e
s o l u t i o n and a d d d im e th y l s u l f a t e , k e e p in g th e te m p e ra tu re h t 60° C.
The
m e th y la te d l i g n i n i n some o a se s e x h i b i t s d ouble r e f r a c t i o n .
By c h l o r i n a t i o n C ro ss and Sevan 1 8 / o b ta in e d a s u b s ta n c e w hich th e y
r e p r e s e n te d by th e fo rm u la C^gE^gCl^Og.
l i n e s o l u t i o n s and i n g l a c i a l a o e t i o
T h is s u b s ta n c e d i s s o l v e s i n a lk a ­
a c id and a lc o h o l .
By a d r a s t i c c h l o r i '
n a t i o n u s in g antim ony p e n ta o h lo r id e c o n ta in in g a s m a ll amount o f i o d i n e , p e r o h lo ro e th a n e and h e x a o h lo ro h e n z e n e were o b ta in e d .
L ig n in w i l l a l s o a b s o r b
b ro m in e , w hioh i n d i c a t e s i t may have one o r s e v e r a l e t h y l e n i o b o n d s.
when l i g n i n i s
A ls o ,
b ro m ln a te d , a l o s s o f m etboxyI ta k e s p la c e t o th e e x t e n t o f
30 p e r c e n t o f th e o r i g i n a l m ethoxyl
c o n te n t.
S in ce a l l th e m ethoxyl g ro u p s
o o u ld n o t he removed hy th e b r o o i n a t i o n , i t i s p ro b a b ly t h a t a l l th e m eth­
o x y l g ro u p s i n l i g n i n a re n o t s i m i l a r l y com bined.
L ig n in oan be n i t r a t e d S ^ / v e ry e a s i l y .
An am orphous n i t r o s u b s ta n c e
can be o b ta in e d by n i t r a t i n g l i g n i n w ith 5 N n i t r i c a c i d .
T h is i n d i c a t e s
-2 3 -
t h a t l i g n i n h aa a p h e n o l! o s t r u c t u r e .
When wood and s i m i l a r l i g n i f i e d m a t e r i a l s a re h e a te d w ith a s o l u t i o n o f
s u l f u r o u s a o id and a o id s u l f i t e s , a s i n th e s u l f i t e p r o c e s s f o r p u lp in g wood,
th e l i g n i n g o e s i n t o s o l u t i o n , le a v i n g th e c e l l u l o s e i n more o r l e s s p u re
s ta te .
The s o l u t i o n c o n ta in in g th e l i g n i n ( o r w a ste s u l f i t e l i g n i n from
p u lp m i l l s ) h a s been th e s u b je c t o f num erous i n v e s t i g a t i o n s .
T h ere i e a t
p r e s e n t a d is a g re e m e n t among I n v e s t i g a t o r e as t o th e meohaniem o f th e r e a c ­
t i o n betw een l i g n i n and s u lf u r o u s a o id and h i s u l f i t e s .
When l i g n i n i s s u b je c te d t o ev en m ild o x id a tio n , co m p lete d i s r u p t i o n o f
th e m o lecu le ta k e s p la c e and sim p le d e g r a d a tio n p ro d u c ts a re o b ta in e d .
Among th e o x id a tio n p ro d u c ts 7 8 /9 2 /4 0 /5 6 /5 2 / ^ / fo u n d w ere fo rm ic and o x a l i c
a o i d ; a ls o s u o o in io a o id , m alo n io a c i d s , and a d lp io a o id .
I n some c a s e a o f
o x id a tio n s m a ll q u a n t i t i e s o f th e a ro m a tic a c id s have b een o b ta in e d , su ch aa
b e n z o ic , p h t h a l i o , i s o p h t h a l i o , t r i m e l l i t i o , h e m im e llltio , p r e h n i t i o , p y ro m e l l i t i o , benzene p e n ta o a r b o x y lio , and m e l l i t i o a c i d s .
A re c e n t p a te n t in ­
d i c a t e s t h a t v a n i l l i n i s a r e s u l t o f o x id a tio n o f l i g n i n u n d e r c e r t a i n con­
d itio n s .
No d e f i n i t e compounds have b een i s o l a t e d by th e r e d u c tio n o f l i g n i n
w hich a re o f muoh a id i n d e te r m in in g th e s t r u c t u r e o f l i g n i n .
L ig n in h a s b een d e m e th o x y la te d by h e a ti n g w ith d i l u t e a c id s u n d e r p r e s ­
su re .
There was e v id e n c e a ls o t h a t th e p ro d u c t h e re h ad u n d erg o n e c o n s i d e r ­
a b le i n t e r n a l c h an g e , a s many o f i t s r e a c t i o n s had ch an g ed .
L ig n in i s o l a t e d
from c o rn cobs h a s been shown t o be e n t i r e l y f r e e from f u r f u r » l - y i e l d i n g sub­
s ta n c e s .
H y d ro ly s is o f l i g n i n w ith h y d r o c h lo r ic a o id g iv e s no r e d u c in g su ­
•24-
g a r b u t th e fo rm a tio n o f a h y d razo n e m e ltin g a t 155° 0 . i n d i c a t e s th e p r e s ­
en ce o f a ra h in o s e #
The f u e i n o f l i g n i n 2 2 / w ith a l k a l i y i e l d s i n n e a r l y a l l o a s e s p r o t o c a te o h u lo a c i d , c a t e c h o l , o x a lic and o t h e r sim p le a l i p h a t i c a c id s .
a c id h a s a l s o been r e p o r te d a s one o f th e d e g r a d a tio n p r o d u c ts .
V a n illic
In a d d itio n ,
t h e r e i s alw ay s form ed a d a r k , am orphous, h u m in -lik e d e g r a d a tio n p ro d u c t
c a lle d lig n io a c id .
The S tr u c t u r e o f L ig n in
I t i s im p o s s ib le t o g iv e a s t r u c t u r a l fo rm u la f o r l i g n i n , s in c e l i g n i n
h a s n e v e r been i s o l a t e d i n th e p u re s t a t e .
The l i g n i n o b ta in e d by one m eth­
od o f s e p a r a t i o n d i f f e r s from th e l i g n i n o b ta in e d by any o th e r m ethod.
N e a rly a l l r e c e n t w o rk e rs a g re e t h a t o o n if e r y l a lc o h o l o r o o n if e r y l a ld e h y d e
o r some c l o s e l y - r e l a t e d compound i s th e e s s e n t i a l g ro u p in g in th e l i g n i n .
Of th e many fo rm u la s p r e s e n te d f o r l i g n i n , o n ly two w i l l be p r e s e n te d h e r e .
K u rs c h n e r*s fo rm u la f o r lig n in *
He
^
O
—{
— C = CH-^CHaOH
^C--CHa OH
I
HC — f
y-------O
C = CH-CHa OH
K u ra o h n er s u g g e s ts 5 8 / t h a t l i g n i n i s a polym er o f th e g ly c o s id e o o n i f e r i n .
H ow ever, s in c e no
n - h e x y lio d id e was i s o l a t e d from th e r e d u c tio n p r o d u c t s , i t
seem s u n l i k e l y K u rs c h n e r1B fo rm u la w i l l h o ld .
-2 5 -
F r e u d e n ler g 1B l i g n i n i
He s u g g e s ts tw elv e m o lecu les o f o a f f e io a lc o h o l condense w ith them­
s e l v e s t o form primary lig n in h avin g th e form ula c i2 0 3 144°37
"the s t r u c ­
tu re i n d i c a t e d .3 2 / Freudenberg n o te s th a t in t h i s form ula th e lin k a g e s are
d e p ic te d as ta k in g p la ce in th e para p o s i t i o n , alth ou gh a part o f th e u n i t s
may be lin k e d in th e meta p o s it io n .
P h i l l i p s 7 2 / s u g g e s ts th a t th e funda­
m ental u n it s are g u a ia o o l and n -p r o p y l-g u a ia o o l, where th e hydrogen in th e
n -p r o p y l s id e chain i s s u b s tit u te d by a lc o h o l hydroxyl g ro u p s.
C e llu lo s e
C e llu lo s e D is c u s s io n
In th e ca se o f most p la n t m a t e r ia ls in w hich c e l l u l o s e p la y s a s t r u c ­
t u r a l r o l e , th e c e l l u l o s e does n o t occu r in a pure or e a s i l y - p u r i f i e d co n d i­
t i o n , but i s found in th e most in tim a te a s s o c ia t io n w ith o th er c e l l - w a l l
c o n s t it u e n t s .
c u lty .
S ep a ra tio n ahd i s o l a t i o n i s a m atter o f c o n sid e r a b le d i f f i ­
T his le d in th e p a st to th e id e a o f compound c e l l u l o s e s , as lig n o —
c e l l u l o s e , p e o t o o e llu lo s e , o u t o o e ll u lo s e , e t c .
However, th e modern con cep t
o f th e s t e r i o s tr u c tu r e o f the c e l l u l o s e m olecule r u le s out the p o s s i b i l i t y
o f c e l l u l o s e a c tin g as a s tr u c tu r a l c o n s titu e n t w h ile in chem ical combina­
t i o n w ith any o th er la r g e m o le c u le .
C e llu lo s e d e r iv a t iv e s , when th e y do ex ­
i s t , appear to lo s e the p h y s ic a l p r o p e r tie s and s t r u c t u r a l str e n g th which
c h a r a c te r iz e c e l l u l o s e in i t s s k e l e t a l r o l e , and assume th e p h y s ic a l pro­
p e r t ie s o f a m u cila g e.
Hence th e th e o r y th a t compound c e l l u l o s e s occu r in
th e p la n t s k e le to n i s u n te n a b le .
—2 6 —
No s in g le tr e a tm e n t i s known w h ic h , w ith o u t a t t a c k i n g th e c e l l u l o s e ,
w i l l remove th e e n c r u s t i n g m a t e r i a l s , c h i e f l y l i g n i n and hem ic e l l u l o s e .
The
"best m ethod a t p r e s e n t i s a l t e r n a t e l y e x p o s in g m a te r ia l t o c h lo r in e and e x ­
t r a c t i o n w ith h o t sodium s u l f i t e s o l u t i o n .
The r e s id u e th u s f r e e d o f l i g n i n
and hem ic e l l u l o s e i s ta k e n a s th e o e l l u l o s i o a g g re g a te o f th e m a t e r i a l .
C o tto n c e l l u l o s e i s , i n f a c t , p u re g lu c o s e p o ly s a c c h a r id e , and c a n n o t
he re g a rd e d a s a t y p i c a l p la n t c e l l u l o s e .
Norman u s e s th e a n a lo g y o f ju d g ­
in g m in e ra l o r e s a g a in s t a v e in o f n a tiv e m e ta l.
P la n t " c e l l u l o s e " i s a
m ix tu r e .
By tr e a tm e n t o f c e l l u l o s e i n th e c o ld w ith 1 7 .5 p e r c e n t NaOH, a r e s i ­
d u e , term ed «<c e l l u l o s e , i s o b ta in e d w h ic h , a lth o u g h n o t a b s o lu t e ly f r e e
from a s s o c i a t e d p o ly s a c c h a r id e s , i s ta k e n a s r e p r e s e n t i n g tr u e c e l l u l o s e f o r
in d u s t r i a l p u rp o se s.
The s tr o n g a l k a l i n e e x t r a c t i s a c i d i f i e d and th e p r e c i ­
p i t a t e w hich i s form ed i s te r m e d /'’c e l l u l o s e .
s o l u t i o n i s te rm e d
The re m a in in g c e l l u l o s e i n
jrc e llu lo s e .
C e llu lo s a n im p lie s a hex o san o r p e n to s a n o c c u r r in g in te rm ix e d w ith th e
t r u e c e l l u l o s e i n th e n a t u r a l o e l l u l o s i o a g g r e g a te .
The a s s o c ia te d p o ly s a c ­
c h a r id e foun d i n m ost s t r u c t u r a l c e l l u l o s e i s x y la n .
T hus, when one sp e a k s o f n a t u r a l c e l l u l o s e , one means p la n t c e l l u l o s e
o r (tr u e c e llu lo s e + o e llu lo s a n ).
B e a rin g i n mind th e above d e f i n i t i o n o f n a t u r a l c e l l u l o s e , any m ethod
o f d e te r m in in g c e l l u l o s e must y i e l d a r e s id u e c o n ta in in g a l l th e o e l l u l o s a n s
s in c e t h i s f r a c t i o n i s t o be re g a rd e d a s an i n t e g r a l p a r t o f th e o e l l u l o s i o
fram ew ork
-2 7 -
The n a t u r a l e e l l u l o e e a o f p l a n t m a t e r i a l s a re much l e a s r e s i s t a n t t o
th e a c t i o n o f a c id s and a l k a l i s th a n i s c o tto n c e l l u l o s e .
T h is i s p ro b a b ly
due t o th e i n f u s i o n o f x y la n c h a in s o f s h o r t e r le n g th th a n th e p u re c e l l u ­
lo s e c h a in s , w hich a re more e a s i l y h y d ro ly z e d th a n th e p u re c e l l u l o s e .
Norman s t a t e s i
"There i s a p o s s i b i l i t y which has n o t been co n sid e re d
by workers on c e l l u l o s e ; nam ely, th a t th e r e may be mixed ch ain s o f x y la n an
c e l l u l o s e , o r , more a c c u r a t e ly , o f x y lo s e and g lu c o s e .
The p resence o f a
few such mixed ch a in s m ight account fo r th e extreme s t a b i l i t y o f a sm a ll pro­
p o r tio n o f th e x y la n , which r e s i s t s e x t r a c t io n s by lo n g b o il in g w ith 10 per
cen t sodium h y d r o x id e .”
I t i s su g g e ste d th a t th e ch a in s are not a ju m ble, but th a t a sh o rt
ch ain o f x y lo s e i s connected t o a chain o f g lu c o se u n i t e .
In o th e r w ords,
i t i s u n lik e ly th a t s e v e r a l g lu c o se m o le c u le s , th en a x y lo s e m o le c u le , th en
s e v e r a l g lu c o s e m o le c u le s , are hooked to g e th e r a t random.
B a c t e r io lo g ic a l s t u d ie s em ploying n a tu r a l c e l l u l o s e s in p la ce o f th e
h ig h ly - p u r if ie d o e l l u l o s i o s u b s tr a te s c u sto m a r ily u sed m ight y ie ld r e s u l t s
o f c o n sid e r a b le i n t e r e s t .
I t seems l i k e l y th a t th e a b i l i t y o f b a c t e r ia t o
u se c e l l u l o s e in th e p resen ce o f o th e r a v a ila b le carb ohydrates w i l l be more
common than g e n e r a lly supp osed.
An an a lo g y here would be th e case o f an a n i­
mal w hich, i f fe d e n t i r e l y on s t a r c h , would d ie , but w hich would l i v e and
p rosp er and be a b le to u t i l i z e s ta r c h i f i t were fe d to th e e x te n t o f $0 per
cen t o f h is d ie t alo n g w ith o th er n e c e s s a r y in g r e d ie n t s .
The u se o f pure
c e l l u l o s e p robab ly has le d many b a c t e r i o l o g i s t s t o erron eou s c o n c lu s io n s
when a ttem p tin g to determ ine i f c e l l u l o s e i s a tta ck ed by c e r ta in o rg a n ism s.
-
28 -
The Chem ioal C o n s t i t u t i o n o f C e llu lo s e
Anaelme Payne 7 0 / i s u s u a l l y c r e d i t e d w ith th e f i r s t i s o l a t i o n o f c e l l u ­
l o s e i n 1838.
I n 1895 T o lle n s 46/ p ro p o se d a fo rm u la f o r c e l l u l o s e t h a t was
g e n e r a l l y a c c e p te d u n t i l a d v a n c in g r e s e a r c h p o in te d o u t c e r t a i n p r o p e r t i e s
o f c e l l u l o s e t h a t th e T o lle n s fo rm u la c o u ld n o t e x p l a i n .
At th e tim e t h a t T o lle n s s u g g e s te d h i s fo rm u la f o r c e l l u l o s e th e f o llo w ­
in g f a c t s were known c o n c e rn in g c e l l u l o s e 1
I.
The p ro d u c t o f h y d r o ly s is o f c e l l u l o s e i s g lu c o s e .
2.
A o e to ly s ie y i e l d s a t r i - o r d is a o o h a r id e .
3.
T here a re p ro b a b ly n o t more th a n th r e e h y d ro x y ls which r e a c t a s a l ­
c o h o lic g ro u p s .
4.
T here i s l i t t l e
i n d i c a t i o n o f f r e e a ld eh y d e g ro u p .
T o lle n s th o u g h t, t h u s , t h a t c e l l u l o s e was a lo n g c h a in o f g lu c o s e anhy­
d r i d e s lin k e d t o g e t h e r th ro u g h g lu c o s e a n h y d rid e u n i t e .
Ha. H H H
111
1
I-I O
I
i n
C - C - C - C - C-O
i
I
H
I
I
l
l
l
O
H
O
H
O
H
O
H
I-LH
11
c - c - e - c - c —c
l
I
O
H
H
O
O
O
H
H
'
l
l
11
H
1 /
H H H O O
I
I
I
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C-C-C— c
I
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l
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1
1
1
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x
C -C - C-C-C— O
H a H
H I
C -C I
/
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-2 9 -
The amorphous nature o f c e l l u l o s e , I t s i n s o l u b i l i t y and o th e r proper­
t i e s have p la ced i t in a c la s s o f su b sta n ce s o f a high m o lecu la r w e ig h t.
T h is form ula shown i s o n ly a sm a ll p o r tio n o f th e t o t a l m o lecu le o f c e l l u ­
lo s e .
T o lle n s had a d e f i n i t e id e a as t o how th e g lu c o se an h yd rid es are lin k e d
t o g e t h e r , as shown by h i s form u la.
S in ce the tim e o f T o lle n s ’ proposed form ula th ere has accum ulated in
th e li t e r a t u r e a la r g e number o f s tr u c t u r a l form ulas fo r th e c e l l u l o s e mole­
c u le .
At p r e se n t one can p robab ly sa y th a t th e b e st one i s v e r y s im ila r t o
T o l l e n s 1 o r ig in a l form ula e x c e p t th a t th e amylene o x id e , g lu c o s e , i s p r e s e n t ,
and th a t lin k a g e occu rs a t th e 1 -4 p o s i t i o n s .
CH j l OH
The D egradation o f C e llu lo s e by B io lo g ic a l P r o c e s s e s
C e llu lo s e , s in c e i t i s p r e se n t in many p la n ts in a c o n sid e r a b le per­
c e n ta g e , p la y s a v ery im portant part in many b io l o g ic a l a c t i v i t i e s .
Nature
has p rovided f o r th e d e s tr u c tio n o f c e l l u l o s e by the a c t i v i t i e s o f m icroor­
ganism s; i . e . , fu n g i and b a c t e r ia .
By o o n t r o ll lin g th e o o h d tio n s o f b io lo ­
g i c a l d e s tr u c tio n (f e r m e n ta t io n ), v a lu a b le produ cts such a s a lc o h o ls , a c id s ,
e t c . may be o b ta in e d .
T h is form s th e b a s is o f im portant branches o f in d u s­
t r i a l u t i l i z a t i o n o f c e l l u l o s e w a stes
—3 0 -
Ferment at io n o f c e ll u lo s e e v e n tu a lly r e s u l t s in a number o f g a s e s , de­
pending upon th e ty p e s o f m icroorganism s u se d .
Four k in d s o f fe r m en ta tio n
oan be d i f f e r e n t i a t e d .
1.
Methane fe r m e n ta tio n , brought about by th e a c tio n o f sewage bac­
t e r i a , produces c h i e f l y m ethane, w ith sm a lle r amounts o f carbon d io x id e and
th e low er f a t t y a c id s (from form ic to b u t y r ic ) .
2.
Hydrogen fe r m e n ta tio n , produced by Bacterium fe r m e n ta tio n is , y i e l d s
m ain ly hydrogen, w ith sm a lle r amounts o f carbon d io x id e and th e low er f a t t y
a c id s m entioned in ( l ) .
3.
M ethane-hydrogen fe r m e n ta tio n , produced by v a r io u s th e r m o p h ilic
b a c t e r ia , such as are capable o f a s s im ila t in g n itr o g e n from th e a ir or o th er
n itr o g e n s o u r c e s , y ie ld s n itr o g e n and carbon d io x id e .
4.
N itr o g e n fe r m e n ta tio n , produced by d e n it r i f y in g b a c t e r ia , such a s
are capable o f a s s im ila t in g n itr o g e n from the a i r or o th er n itr o g e n s o u r c e s ,
y i e l d s n itr o g e n and carbon d io x id e .
As to th e form ation o f m ethane, i t i s assumed by Symons and B usw ell and
o th e r s th a t p r im a r ily carbon d io x id e and hydrogen are lib e r a t e d , and th e s e
th en r e a c t t o form methane and w a te r , w ith o r g a n ic a c id s a s in t e r m e d ia t e s .
Although w ith o u t q u estio n o r g a n ic a c id s are in te r m e d ia te s i t seems h ig h ly
improbable th a t carbon d io x id e and hydrogen, once lib e r a t e d , would a g a in r e ­
a c t to form m ethane.
The p r o c e ss o f ferm en ta tio n i s preceded by h y d r o ly s is through h y d ro ly ­
t i c enzymes.
I t has been p o s s ib le t o d ir e c t t h i s p r o c e ss so th a t e i t h e r c e l —
lo b io s e or g lu c o s e p r e v a ils by a llo w in g e i t h e r o f th e enzym es, o e llo h ia s e or
31-
o e I lu la e e , to a o t.
T his may he aooom pliahed by a d ju s tin g tem perature
c o n d itio n s t o th e optimum and by a r r e s tin g th e p r o c e s s o f fe r m en ta tio n by
means o f a n t i s e p t i c s .
In view o f t h i s , th e assum ption may be made th a t the
b i o l o g i c a l p r o c e s s p a s s e s through th e same s ta g e s a s c e l l u l o s e su b je c te d t o
h y d r o ly s is by a ohem ioal p r o c e ss in th e la b o r a to r y .
I t appears l i k e l y th a t
th e b a c t e r ia a ls o e f f e c t a gradual sh o r te n in g o f th e c e l l u l o s e c h a in , and
th a t w ith r e f in e d methods o lig o s a c c h a r id e s and even f r a c t io n s o f h ig h er mo­
le c u l a r w e ig h ts m ight he i s o l a t e d .
S tep s in t h i s d ir e c t io n may be seen in
th e work which le d to th e r e c o g n it io n th a t o lea v a g e o f th e lo n g e r ch ain s
(from o e llu lo s e down to th e w a te r -s o lu b le d e x tr in e ) i s a p p a re n tly e f f e c t e d
e x c l u s i v e l y by o e llu la e e whereas th e o lig o s a c c h a r id e s undergo s c i s s i o n
o h i e f l y under the a c tio n o f o e llo b ia s e .
M -glyoop o ly a a o o h a ra ee" and "
Thus one may d is t in g u is h between
-g lu o o b lig o s a o o h a r a s e ."
However, nc c o n n ectio n i s apparent betw een o b se r v a tio n s o f t h i s kind
and th e f a c t th a t some b a c te r ia cea se t o a ot a f t e r th ey have sh o rten ed th e
c e l l u l o s e ch ain to a c e r ta in e x t e n t .
I t would appear th a t accu m ulation o f
d e g r a d a tio n produ cts a t t h i s s ta g e i n h i b i t s fu r th e r growth o f b a c t e r ia .
The number o f is o la t e d and w e ll-d e fin e d b a c te r ia capable o f c e ll u lo s e
d e s t r u c t io n has g r e a t ly in c r e a s e d d u rin g r e c e n t y e a r s , and v e r y in t e r e s t in g
r e s u l t s o f e x te n s iv e r ese a r c h work, e s p e c i a l l y in the f i e l d o f s o i l m icro­
b io lo g y , are a v a ila b le .
A lle n and C arlson l / found th a t w ild r a t s could n o t d ig e s t c e l l u l o s e ,
and th a t in a l l o a se s th e w ild r a ts d ie d o f s t a r v a tio n or e n t e r i t i s .
Enzyme
p r e p a r a tio n s from th e anim al d ig e s t iv e t r a c t had no d ig e s t in g e f f e c t on c e l -
-3 2 -
lu l o s e from oel o t e x .
The c a rn iv o ro u s anim als oannot d ig e s t a p p re c ia b le q u a n t it ie s o f c e l l u ­
lo s e .
The rum inants a r e , how ever, a b le t o u t i l i z e a la r g e p o r tio n o f th e
c e llu lo s e .
Jacq u es 5 1 / found th a t P leo tr id iu m o e llu lo y tio u m i s o l a t e d from
th e rumen o f c a t t l e ferm en ts c e l l u l o s e w ith the fo rm a tio n o f f a t t y a c id s*
The t o t a l q u a n tity o f f a t t y a c id formed r e p re sen ted 80 p er cent o f the c e l ­
lu lo s e .
Woodman and Stew art 4 2 / in v e s t ig a t e d the a c tio n o f therm op hylio c e l l u ­
l o s e - s p l i t t i n g b a c te r ia on the f i b e r o f a number o f t y p i c a l fe e d in g s t u f f s .
They s ta te d th a t a sm a ll amount o f lig n o o e llu lo e e was probably r e s p o n s ib le
f o r a d i s t i n c t lo w e rin g o f d i g e s t i b i l i t y , caused not e n t i r e l y by th e p r e s­
ence o f l i g n o o e l l u l o e e , but a ls o by th e manner o f i t s d e p o s itio n w ith in the
c e l l w a lls .
A ls o , th e y dem onstrated th a t during th e growth o f p e r e n n ia l rye
g r a s s th e in c r e a s e in l i g n i n co n te n t was a s s o c ia te d w ith reduced d i g e s t i ­
b i l i t y , a c o n c lu s io n which i s in harmony w ith o th e r fe e d in g t r i a l s .
L a te r
i n v e s t ig a t i o n h as s u b s ta n tia te d t h e i r f in d in g s , w ith th e e x c e p tio n o f t h e i r
u se o f th e term " lig n o o e llu lo e e ."
I t has n e t been proved f a r i l y d e f i n i t e l y
th a t lig n o o e llu lo e e as a compound does n o t e x i s t , and t h a t the d e crea se in
d i g e s t i b i l i t y o f th e c e ll u lo s e i s due in main t o th e manner o f d e p o s it io n o f
l i g n i n in th e c e l l .
P ry a n ish n ik o v 7%/ found th e d i g e s t i b i l i t y o f roughage may he in flu e n c e d
by th e fo llo w in g fa c to r s *
the p resen ce o f l i g n i n , th e co n ten t o f s i l i c o n in
th e c e l l w a l l s , th e d if f e r e n t m ic e lla r c o n d itio n s o f c e l l u l o s e and th e modi­
f i c a t i o n s in th e c h a r a c te r o f th e c lo s e c o n n e c tio n s betw een c e ll u lo s e and
33-
in o ru e ta n ta .
Wakaman 9 j / s t a t e s t h a t when l i g n i n i s removed from p l a n t m a t e r i a l s th e
r a t e o f c e l l u l o s e d e c o m p o sitio n i n th e ru m in an t in c r e a s e s h u t th e p re s e n c e
o f o n ly 8 p e r c e n t l i g n i n was foun d t o he s u f f i c i e n t t o re d u c e th e r a t e o f
c e l l u l o s e d e c o m p o s itio n by 50 p e r c e n t.
A r e d u c tio n o f th e l i g n i n c o n te n t
t o 1 .5 p e r c e n t e n a b le s th e c e l l u l o s e t o decompose a s r e a d i l y a s i n th e p u re
fo rm .
He c o n s id e r s th e d e p r e s s in g e f f e c t o f l i g n i n on c e l l u l o s e d ecom posi­
t i o n t o he due t o th e m anner o f c o m b in a tio n w ith c e l l u l o s e , which i s p ro ­
t e c t e d from r a p i d a t t a c k by s a p r o p h y tic m ic ro o rg a n is m s .
Cram pton h a s made e x te n s iv e s t u d i e s on th e e f f e c t o f l i g n i n on d i g e s t i ­
b ility of g ra sse s.
X jJ
He s t a t e s t h a t th e n u t r i t i v e v a lu e o f
h e rb a g e may change a p p r e c ia b ly i n a te n - d a y p e rio d i n th e summer.
I n mid­
summer th e a v a i l a b i l i t y
o f th e c a r b o h y d ra te f r a c t i o n o f th e h erb ag e i s ma­
t e r i a l l y re d u c e d .
i s due t o th e amount and k in d o f d e p o s it io n o f th e
T h is
l i g n i n , w hich a f f e c t s th e d i g e s t i b i l i t y and n u t r i t i v e v a lu e s o f h e r b a g e .90/
Cram pton a l s o s u g g e s ts th e u se o f r a b b i t s a s a s u b s t i t u t e f o r s t e e r s i n d i ­
g e s t i b i l i t y t r i a l s , a s t h e r e i s a h ig h c o r r e l a t i o n b etw een th e two s p e c ie s
i n c ase o f d i g e s t i b i l i t y o f cru d e p r o t e i n and l i g n i n .
I t i s o f i n t e r e s t t o n o te t h a t p e p s in , r e n n in , c a t a l a s e and o t h e r e n ­
zymes can he rem oved from s o l u t i o n s by a s in g le f i l t r a t i o n th ro u g h c o t t o n ,
o r t o a c e r t a i n e x t e n t by f i l t e r p a p e r , due to a d s o r p tio n o f enzymes by c e l ­
l u l o s e . 44/
F o r f u r t h e r in f o r m a tio n on c e l l u l o s e d i g e s t i o n th e f o llo w in g a r ­
t i c l e s a re r e f e r r e d to *
9 8 /9 9 /lO O /lO /.
I t h a s b een found t h a t in t e r m i t e s t h a t oause d e s t r u c t i o n o f wood th e
■34-
o e l l u l o a e i s d e s tr o y e d by b a o t e r i a w ith in th e i n t e s t i n e s o f th e t e r m i t e s . ^ /
The D e g ra d a tio n o f C e llu lo s e by Aoida
H y d ro ly s is 3 2 / w ith m in e ra l a c id s y i e l d s g lu c o s e t o a l e s s e x te n t th a n
d o e s h y d r o l y s i s w ith o r g a n ic a c i d s .
I f th e h y d r o ly s is i s n o t co m p lete s
number o f in te r m e d ia te p ro d u c ts r e s u l t .
o f th e c e l l u l o s e m o le c u le .
They a re composed o f b ro k e n c h a in s
T h is i s p ro b a b ly c au se d by t h e f a c t t h a t th e
c e l l u l o s e m o le c u le i s so la r g e t h a t h y d r o ly s is m ust s t a r t on th e s u r f a c e and
p e n e t r a t e in w a rd .
T h u s, i f one w ere to s to p th e h y d r o ly s is b e fo re i t was
co m p lete one would f i n d a m ix tu re o f compounds w ith th e fo rm u la (^5® i2°6^X e
The "X" i n t h i s fo rm u la would v a r y from one to th e num ber r e p r e s e n t i n g th e
t r u e c e l l u l o s e m o le c u le .
J u s t how many g lu c o s e u n i t s from a c e l l u l o s e m ole­
c u le can n o t be a c c u r a t e l y d e te rm in e d , and i t i s v e ry p ro b a b le t h a t th e num­
b e r v a rie s in c e llu lo s e s o f d if f e r e n t p la n ts .
The in te r m e d ia te p ro d u c ts o f th e d e g r a d a tio n o f c e l l u l o s e can b e s t be
o b ta in e d by h e a t i n g w ith d i l u t e a c i d s .
By h e a tin g c e l l u l o s e f i b e r w ith d i ­
l u t e h y d r o c h lo r ic a c id a p ro d u c t i s o b ta in e d w hich re s e m b le s o x y c e llu lo s e i n
a p p e a ra n c e and b e h a v io r .
Thus th e c e l l u l o s e em erges from th e tr e a tm e n t a s a
w eakened f i b e r w hich may e a s i l y he ru b b e d t o a pow der betw een th e f i n g e r s .
I t i s term ed h y d r o o e l l u l o s e .
I n th e fo rm a tio n o f h y d r o c e llu lo s e th e o n ly
change c o n c e iv a b le i s a s h o r te n in g o f lo n g c h a in s .
U nder th e h y d r o ly z in g e f ­
f e c t o f th e a c i d , a number o f oxygen b r id g e s may b re a k u p , r e s u l t i n g i n a
s a c c h a r id e h a v in g a pronounced r e d u c in g e f f e c t , due t o th e p re se n c e o f more
a ld e h y d e g ro u p s .
-3 5 -
In h e a tin g h y d r o o e Ilu lo e e w ith d i l u t e a lk a l i ( 4 - 6 p er cent sodium hy­
d r o x id e ) , th e raw h y d r o o e llu lo e e p r e p a r a tio n s may be d iv id e d in t o two com­
p o n e n ts.
The f i l t r a t e shows pronounced red u cin g power on F e h lin g 's s o lu ­
t io n ; th u s i t must c o n ta in th e low er d eg ra d a tio n p r o d u c ts.
The r e s id u e , how­
e v e r , o n ly shows a v ery s l i g h t red u cin g power, which means i t c o n ta in s th e
h ig h e r d e g r a d a tio n p r o d u c ts, and i s a ls o s o lu b le in h ig h e r a lk a l i co n c en tr a ­
t i o n s (8 per cen t sodium h y d r o x id e ).
The p u r ifie d a l k a l i - s o l u b l e h y d r o o e l-
lu l o s e has been termed " o e llu lo se A ."
I t i s p o s s ib le by u se o f m in eral a c id t o con vert 99 p er cen t o f th e
o r ig in a l c e l l u l o s e in t o a l k a l i - s o l u b l e c e l l u l o s e .
O rganic a c id s such as
fo r m ic , a c e t i c , and o x a lic may a ls o be u sed fo r c o n v e r sio n .
With c o n cen tra ted a c id s such a s s u l f u r i c , n i t r i c , h y d r o c h lo r ic , hydro­
f l u o r i c , or p h o sp h o r ic, c e ll u lo s e s w e lls c o n s id e r a b ly , becomes p e p tiz e d , and
f i n a l l y d i s s o lv e s c o m p le te ly .
U s u a lly , im m ed iately a f t e r p rep a ra tio n th e
s o lu t io n shows red u cin g power toward F e h lin g 's s o lu t io n .
Upon d i l u t io n w ith
w ater im m ed iately a f t e r d is s o lv in g and w ith thorough c o o lin g , th e g r e a t e r
p a r t o f the c e l l u l o s e may be r eg e n e r a ted in the form o f w h ite f la k e s w hich
show th e b eh a v io r and X -ray diagram o f m ercerized c e l l u l o s e .
The prepara­
t io n ob tain ed i s o fte n termed "am yloid," m erely because i t g iv e s th e same
b lu e c o lo r a t io n w ith io d in e in th e p resen ce o f t r a c e s o f s u lf u r ic a c id a s
s ta r c h (amylum) d o e s .
On sta n d in g o f th e c e l l u l o s e d is s o lv e d in a c id , d eg ra d a tio n p r o c e e d s ,
and by th e a d d itio n o f a lc o h o l a p r e c ip it a t e c h a r a c te r iz e d by i t s h ig h r e ­
d u cin g power may be o b ta in e d .
I t i s p a r t ly or e n t i r e l y s o lu b le in w ater and
-
h a e th e name " c e l l o d e x t r i n . "
36-
I t h a s p la y e d a g r e a t p a r t I n th e e n d e a v o rs t o
i s o l a t e homogeneous in te r m e d ia te s i n th e c o u rse o f th e d e g r a d a tio n o f c e l l u ­
lo s e .
I n th e l i g h t o f modern c o n c e p tio n o f th e c h em ica l c o n s t i t u t i o n o f c e l ­
l u l o s e , c e l l u l o s e d e x t r i n i s f a r from b e in g a homogeneous p r o d u c t.
I t is
r a t h e r re g a rd e d a s a m ix tu re o f more o r l e s s s h o rte n e d g lu c o s e a n h y d rid e
c h a i n s $ i n o t h e r w o rd s, a m ix tu re o f o lig o s a c c h a r id e s o f v a ry in g c h a in
l e n g t h , th e lo n g e s t l i n k o f which may co m p rise t h i r t y o r l e s s g lu c o s e anhy­
d rid e s .
S in c e th e m o le c u le s o f s m a lle r s i z e c r y s t a l l i z e , th e whole m ass ap ­
p ears c r y s ta ll in e .
C e r ta in b io s e a n h y d rid e s which a t one tim e were c la im e d
t o have been i s o l a t e d a s homogeneous d e g r a d a tio n i n t e r m e d i a t e s , a l s o a p p a r­
e n t l y r e p r e s e n t m ix tu r e s o f o lig o s a c c h a r id e s o f v a r io u s c h a in le n g th s s im i­
l a r to c e llu lo s e d e x trin e .
M ethods o f A n a ly s is o f C e llu lo s e
A lth o u g h num erous m ethods f o r th e d e te r m in a tio n o f c e l l u l o s e a p p e a r in
th e l i t e r a t u r e th e g e n e r a l p ro c e d u re i s q u i t e s i m i l a r i n a l l o f th em .
L ig ­
n i n , p e o tin and r e l a t e d compounds a r e rem oved by o x id a tio n w ith c h lo r in e g a s ,
n i t r i o a o id , h y p o c h lo r ite s o l u t i o n o r o t h e r r e a g e n ts w hich w i l l c au se s o lu ­
t i o n o f th e r e s i s t a n t com ponents o f f i b r o u s m a t e r i a l .
A weak h y d r o ly s is may
o r may n o t he em ployed, d e p en d in g on w hat p o r tio n o f th e p e n to s a n s and h e m io e l lu lo e e one w is h e s t o c o n s id e r a s b e in g a component p a r t o f th e " p la n t
c e llu lo s e ."
The C ro ss and Sevan \%J m ethod o f d e te r m in a tio n o f c e l l u l o s e was one o f
th e f i r s t m ethods o f d e te r m in a tio n o f c e l l u l o s e t o he e x t e n s i v e l y u s e d i n
-3 7 -
p la n t a n a ly s is .
I t i s th e c h l o r i n a t i o n m ethod.
I t depends upon o x id a tio n
w ith o u t h y d r o ly s is and does n o t remove th e p e n to s a n s o r hem ic e l l u l o s e .
Many
o t h e r m th o d e a re a m o d if ic a tio n o f th e o r i g i n a l Oroae and Bevan m eth o d .
The K onig 5 2 / m ethod in v o lv e s d i g e s t i o n a t 137° w ith g ly c e r o l and H2 SO^.
Waksman and S te v e n s % / remove th e l i g n i n from th e o r g a n ic m a tte r w ith a c id
sodium s u l f i t e s o l u t i o n , fo llo w e d by e x t r a c t i o n o f th e c e l l u l o s e w ith ammnni a c a l c o p p er s o l u t i o n and i t s p r e c i p i t a t i o n w ith h y d r o c h lo r ic a c i d .
O st and
W ilk e n in g 6 8 / u s e h y d r o ly s is o f th e c e l l u l o s e - c o n t a i n i n g m a t e r i a l , p r e v io u s ly
f r e e d from s u g a r s , s t a r c h e s and hernlc e l l u l o s e w ith c o n c e n tr a te d a c i d s , th e n
c o m p le tin g th e h y d r o ly s is t o g lu c o s e by b o i l i n g w ith th e d i l u t e a c i d .
The
c e l l u l o s e c o n te n t i s c a l c u l a t e d from th e amount o f r e d u c in g s u g a r fo rm ed .
The Norman and J e n k in s 66/m ethod i s b a se d on th e a ss u m p tio n t h a t th e o e l l u lo s a n s o r p o ly s a c c h a r id e m a te r ia l a s s o c i a t e d w ith "p u re c e l l u l o s e " m ust r e ­
m ain i n t a c t i n any s a t i s f a c t o r y m ethod o f c e l l u l o s e e s t i m a t i o n .
The m ethod
p ro p o se d f o r s tr a w s and woods in v o lv e s f i r s t two tr e a tm e n ts w ith n e u t r a l hy­
p o c h l o r i t e Eind th e n t h r e e o r more w ith a c id h y p o c h l o r i t e , each fo llo w e d by
b o i l i n g w ith sodium s u l f i t e .
W illia m s and O lm stead 9 6 / and o th e r s f i r s t f r e e
th e f i b e r a s c o m p le te ly a s p o s s ib le from th e s t a r c h , p r o t e i n and f a t by en ­
zym ic d i g e s t i o n .
R e ce n tly th e monoethanolamine method o f R eid , N elso n and Aronovsky 8 o /
h as a t t r a c t e d th e a t t e n t io n o f in v e s t i g a t o r s .
I t i s a r a p id method and con­
s i s t s o f r e f lu x in g two hours w ith monoethanolamine which decomposes th e ma­
jo r p o r tio n o f th e l ig n in in p la n t m a te r ia l and removes th e l e s s - r e s i s t a n t
p e n to sa n s, but has l i t t l e e f f e c t on th e
t e r i a l or in th e i s o l a t e d form.
^ c e l l u l o s e , e i t h e r in p la n t ma­
■38-
STATEMEWT OF PROBLEM
The problem f o r t h i s t h e s i s c o n s is te d
o f th e d e te r m in a tio n o f a s h , e t h e r e x t r a c t
n i t r o g e n , l i g n i n and c e l l u l o s e i n s e v e r a l
ra n g e g r a s s s p e c i e s , and i n a d d i t i o n , a
s tu d y o f th e m ethods o f a n a l y s i s f o r l i g n i n
and c e l l u l o s e
-3 9 -
EXPKRIMENTAL work
The g r a s s sam ples were c o lle c t e d from Experiment S t a tio n p lo t s in Gal­
l a t i n and J u d ith B asin C o u n tie s.
These c o l l e c t i o n s were made a t one-w eek
i n t e r v a l s , b e g in n in g May 1 4 , 1 9 40 , and l a s t i n g u n t i l September 1 5 , 1 940.
The annual growth c y c le
o f th e s e g r a s s e s was d iv id e d in t o fo u r d e f i ­
n i t e d evelop m en tal s ta g e s and ten d to o f f e r a co n v en ien t b a s is fo r p r e s e n t­
in g ex p erim en ta l d a ta .
These s ta g e s a r e i
A.
Snow d isap p earan ce to flo w e r s t a l k s f i r s t in e v id e n c e .
B.
Flow er s t a lk s f i r s t in e v id e n c e to heads f u l l y o u t .
0.
Heads f u l l y out to flo w e r s in bloom.
D.
Flow ers in bloom t o s e e d s f u l l y r ip e .
B.
Seeds s h a t t e r in g .
L is t o f G rasses C o lle c te d and Analyzed
G a lla t in County
Agropyron o r ista tu m (L .) G aertn. , Standard c r e s te d w heatgrase
Agropyron o r ista tu m , Fairway c r e s te d w heatgrass
Agropyron pauoiflorum (Sohw ein) H it c h ., s le n d e r w heatgrass
Bromus ln erm is L e y s s ., smooth brome g r a ss
Bromus m arginatus N e e s ., mountain brome g r a ss
Elymus junoeus F is o h ., R ussian w ild -r y e
J u d ith B asin County
Agropyron o r ista tu m ,
’ andard c r e s te d w heatgrass
A gropyron o r is ta tu m , 19537, S ta n d a rd c r e s te d w h e a tg ra s s ( s e l e c t i o n )
40-
Agropyron o r ista tu m , M 24-3» Standard crea ted w heatgraae ( s e l e c t i o n )
Agropyron o r ista tu m , Fairway c r e a te d w heatgrasa
Agropyron o r ista tu m , 1350, Fairway c r e s te d w h ea tg ra ss ( s e l e c t i o n )
Elymus ju n o e u s, R ussian w ild -r y e
The g r a s s e s from G a lla t in upon c o l l e c t i o n were im m ed iately brought in ­
t o th e la b o r a to r y and p laced in an oven a t 50° G. u n t i l d ry .
They were
d r ie d as q u ic k ly as p o s s ib le to preven t any a v o id a b le change in t h e i r com­
p o s it io n .
The g r a s s e s c o lle c t e d a t J u d ith B a sin , due t o the d is ta n c e o f
th a t s t a t io n from our la b o r a to r y , were o n ly a ir -d r ie d b e fo r e s h ip p in g , and
o v e n -d rie d a f t e r a r r i v a l .
A fte r d r y in g , th e g r a s s e s were ground in a W iley m i l l u n t i l th ey p a ssed
through a o n e -h a lf m illim e te r s c r e e n .
A ll a n a ly se s were run in d u p lic a t e .
M oisture was determ ined by p la c in g a 2-gram sample f o r 5 hours in a
vacuum oven a t 100° C. and 25 mm.
T h is was used in c a lc u la t in g a l l o th e r
a n a ly s e s on an oven-d ry b a s is .
Ash was determ ined by h e a tin g a 1-gram sample fo r one hour a t 600° 0 .
in a m u ffle fu r n a c e .
T o ta l n itr o g e n determ ined by th e K jeld a h l method was m u ltip lie d by 6 .2 5
t o g iv e th e crude p r o te in c o n te n t.
Ether e x t r a c t was determ ined by e x t r a c t in g a 1-gram sample o v e r n ig h t
w ith anhydrous e th e r and w eighing th e e x tr a c te d m a t e r ia l, which was d r ie d
f o r one hour a t 100° 0 . a f t e r th e e th e r had been ev a p o ra ted o f f on a steam
p la te .
"Other carbohydrates" was determ ined by s u b tr a c tin g th e t o t a l a sh ,
e th e r e x t r a c t , p r o te in , lig n in and c e l l u l o s e from 1 0 0 .
-
41 -
L jgnln Procedure
The method used f o r the d e te r m in a tio n o f l ig n in was th a t o f Grampton
and Maynard, I j / which i s a m o d ific a tio n o f the 72 per cen t s u l f u r i c a c id
m ethod.
Weigh out an a ir -d r y 1-gram sample o f g r a ss or fe c e s and e x t r a c t
w ith e th e r f o r 16 hours by means o f a B a iley-W alk er e x t r a c t o r .
T ra n sfer the
d r ie d r e sid u e in t o a 5 0 -c o . g la s s - s to p p e r e d Erlenmeyer f la s k and add 40 oo.
o f a 2 .0 $ s o lu t io n o f p ep sin in 0 .1 N_ HCl.
D ig e st o v e rn ig h t a t 40° C.
Re­
co v er th e u n d ig e ste d r esid u e by f i l t r a t i o n through 200-m esh b o lt in g s i l k in
a Qooch c r u c ib le .
In th e a n a ly s is o f f e c e s , u se a No. 54 Whatman f i l t e r pa­
per in s te a d o f b o lt in g s i l k .
Wash s u c c e s s iv e ly w ith h o t w ater, hot a lc o h o l,
h o t b en zen e, hot a lc o h o l and e t h e r .
T ra n sfer the washed r esid u e t o a 1 0 0 -c o .
b e a k e r , and a llo w th e e th e r to e v a p o r a te .
40$ form aldehyde.
sample (2 m in u te s ).
M oisten th e r e sid u e w ith 4 o o . o f
Then add 4 oo. o f 72$ H^SO^, a llo w in g i t to p e n e tr a te the
Add 6 oo. o f co n cen tra ted HgSO^ and s t i r v ig o r o u s ly
w ith a g la s s rod to a id in s o lu t io n , which should be com plete in from 10 to
15 m in u te s.
P a r tly immerse th e beaker in cold w ater bath to preven t th e
tem perature from r i s i n g above 70° 0 .
When sample i s d is s o lv e d , s t i r in 35
o o . o f a g r a n u la tin g rea g en t c o n s is t in g o f a 1 :6 m ixture (b y volum e) o f
chloroform and g l a c i a l a c e t i c a c id , and pour th e whole in t o 500 oo. d i s ­
t i l l e d w ater in an 8 0 0 -oo. b eak er.
B o il g e n tly u n t i l th e chloroform has
been d r iv e n o f f (1 5 m in u te s ), a f t e r which the s o lu t io n should c le a r and the
l i g n i n s e t t l e in g ra n u la r form.
w ith s u c t io n .
F i l t e r (u s in g a s b e s to s ) in a Qoooh c r u c ib le
Wash in not l e s s th an 200 o o . o f 5$ HCl.
determ ine l ig n in by l o s s on i g n i t i o n .
Dry a t 110° C. and
—42—
D ieo u a sio n o f th e Crampton-Maynard Method fo r L ig n in D eterm in ation
T h is method c o n s is t s o f s o lu t io n o f the sample in co n cen tra ted HgSO^
a f t e r p retreatm en t to remove f a t s , su g a rs and p r o te in s w hich o th erw ise
would in t e r f e r e w ith th e l ig n in v a lu e s o b ta in e d .
The s p e c ia l problem in th e a n a ly s is o f forage and f e c e s l i e s in th e
rem oval o f th e p r o te in w ith ou t a sim u lta n eo u s removal o f a part o f th e l i g ­
n in .
A ccording t o p r e se n t in fo r m a tio n , l ig n in i s s o lu b le in v a ry in g d e g r ee s
in d il u t e a l k a l i (h o t or c o ld ) , b o il in g w ater and d il u t e m ineral a c id ( 1 . 25%)
a t b o il in g tem p eratu re.
been u se d .
To overcome t h i s d i f f i c u l t y , enzyme d ig e s t io n has
V arious enzymes have been u s e d .
W illiam s and O lm stead, $ 6 / work­
in g w ith human d i e t s and f e c e s , proposed th e use o f p a n o rea tin in a s o lu t io n
b u ffe r e d a t pH 8 .
The o b je c tio n t o th e u se o f p a n o re a tin i s the f a c t t h a t
l i g n i n i s s l i g h t l y s o lu b le in a p a n o re a tin s o lu tio n b u ffe r e d a t a pH o f 8;
a l s o , enzyme d ig e s t io n r e sid u e i s not c o m p le te ly s o lu b le in con cen tra ted
s u l f u r i c a c id .
U sin g t h i s method in a s t e e r d ig e s t io n t r i a l , 25 per cen t
more li g n i n was reco v ered in th e f e c e s th an was consumed!
P e p s in , on th e o th er hand, i s a c t iv e in a c id medium, and i t has not y e t
been shown th a t l ig n in i s s o lu b le in d i l u t e m ineral a c id a t tem p eratures a t
which t h i s enzyme i s a c t i v e .
The e f f e c t i v e n e s s o f p e p s in , a ccord in g to H or-
w i t t , 50/ in rem oving n itr o g e n from sp in a ch le a v e s , was about 89 p er cen t o f
th e t o t a l n it r o g e n .
I t i s assumed th a t th e p r o te in i s reduced by p e p sin d i ­
g e s t io n t o a l e v e l where i t w i l l no lo n g e r s e r io u s ly in t e r f e r e w ith th e l i g ­
n in d e te r m in a tio n .
The problem o f c o m p letely and r a p id ly d is s o lv in g th e u n d ig e sted r e s id u e
in the s tr o n g aold was s o lv e d hy Eoss and H i l l ,8 2 / who found th a t l i g n i f i e d
t i s s u e would d is s o lv e prom ptly ( 1 0 -1 5 m in u tes) in 72$ EgSO^ i f f i r s t m ois­
ten ed w ith fo r m a lin .
The use o f form aldehyde has not been a ccep ted by some
in v e s t i g a t o r s , due t o the f a c t th a t form aldehyde co n d en sa tio n produ cts may
be formed le a d in g to h igh lig n in v a lu e s .
With th e sample in c o n ta c t w ith
th e a c id fo r so sh o r t a tim e , co n d en sa tio n o f th e carb ohydrates (p e n to s e s
and h e x o se s) which might add t o th e l i g n i n value i s presum ably la r g e l y a v o id ­
ed.
A ccep tin g th e h y p o th e s is th a t li g n i n i s not u t i l i z e d by the a n im a l, th e
p r a c t ic a l u s e f u ln e s s o f any lig n in method would be in d ic a te d by a l ig n in
b alan ce t r i a l .
Crampton and Maynard found p r a c t ic a lly q u a n tit a tiv e r e c o v e r y
o f the d ie t a r y l i g n i n , 9 7 .8 $ in r a b b its and 9 9 .3 $ in s t e e r s , u s in g t h e i r
m ethod.
The most s e r io u s o b je c tio n t o th e d eterm in a tio n o f l i g n i n in fe e d
a n a ly s e s i s th a t th e p resen t methods o f l ig n in d e te r m in a tio n are so lo n g and
tim e-con su m in g.
I f a rap id method o f e s tim a tio n o f l i g n i n o f s u it a b le a c­
curacy could be found, th e d e te r m in a tio n s o f l ig n in and c e l l u l o s e would no
doubt be more g e n e r a lly a ccep ted as a replacem ent o f the Henneberg (cru d e
f i b e r ) method.
C e llu lo s e P ro c e d u re
The method used in th e d e te r m in a tio n o f c e ll u lo s e was a m o d ific a tio n
o f th e Crampton and Maynard m ethod.I j /
I t c o n s is t s o f t r e a t in g p la n t mate­
r i a l w ith a b o il in g m ixture o f n i t r i c a c id and a c e t i c a c id .
The n i t r i c a c id
•44'
o x id iz e s th e l i g n i n and r e la t e d compounda ao th a t th e y go in t o s o lu t io n .
The a c e t i c a c id h y d r o ly z e s th e l e s s - r e s i s t a n t ca rb o h y d ra tes.
A m o d ific a tio n
o f t h i s method was chosen because i t compares fa v o r a b ly w ith r e s u lt s o f
o th e r c e l l u l o s e m ethods5 in a d d itio n , i t i s a v ery ra p id method.
S in ce
th e r e i s a v a r ia t io n in th e amount and com p osition by th e d if f e r e n t methods
a method was chosen which was s u it a b le f o r use in anim al s t u d i e s , one t h a t
gave a h igh r ec o v e r y o f no n -sta r c h y h e x o sa n s.
The d e t a i l s o f th e procedure used are as fo llo w s :
dry sample in a 6-in o h Pyrex c e n tr ifu g e tu b e .
and 1 .5 00. oono. HNO^.
Add I 5 Co. o f 80$ a c e t i c a c id
P lace a w a t e r - f i l l e d B a tte r se a c r u c ib le , to p diame­
t e r 30 mm., in t o the neck o f th e c e n tr ifu g e tu b e .
f i e d r e f lu x con d en ser.
P la ce a 1-gram , a i r -
T his s e r v e s as a s im p li­
B o il g e n t ly fo r 20 m inutes on a sand b a th .
c e n tr ifu g e tu b e s w ith co ld w ater and remove B a tte r se a c r u c ib le s .
20 0 0 . o f a lc o h o l, m ixing w e ll w ith s t i r r i n g rod.
p it a t e s e t t l e s (5 m in u te s ).
Decant th e l iq u id .
Cool
Add about
C en trifu g e u n t i l p r e c i­
Wash ( i n c e n tr ifu g e tu b e )
s u c c e s s iv e ly w ith a lc o h o l (2 w a sh in g s ), h o t b en zen e, h ot a lc o h o l, e t h e r .
Dry r e sid u e in c e n tr ifu g e tube a t room tem perature u n t i l no n o t ic e a b le e t h e r
odor rem ain s, and p la ce in oven a t IOO0 C. fo r one hour.
Remove p r e c ip it a t e from th e c e n tr ifu g e tu b es and w eig h .
C a lc u la te c e l ­
lu l o s e as l o s s on i g n i t i o n .
Our e x p e r ie n c e has shown t h i s method to g iv e r ep ro d u cib le r e s u l t s .
The
o n ly o b j e c tio n i s th a t i t i s r a th e r d i f f i c u l t t o remove th e c e ll u lo s e q u a n ti­
t a t i v e l y from th e c e n tr ifu g e tu b e s .
T h is d i f f i c u l t y a r i s e s from th e f a c t
th a t th e c e l l u l o s e becomes e l e c t r i c a l l y charged and i s a tt r a c te d by th e g l a s s .
T h is may be p a r tly overcome by b ru sh in g a rubber policem an w ith a cam el’ s -
-4 5 -
h a ir b ru sh , c a u sin g th e c e l l u l o s e t o be a tt r a c te d more t o th e rubber p o lic e
man th an t o th e g l a s s .
In th e method o f d e te r m in a tio n o f c e l l u l o s e which was u sed , in which
th e c e l l u l o s e i s d is s o lv e d by a c e t i c a c id s o lu t io n c o n ta in in g n i t r i c a c i d ,
by r e f lu x in g and th en p r e c ip it a t i n g in a lc o h o l, i t i s probably th a t we are
d e a lin g here w ith c e l l u l o s e d e x tr in e as a f i n a l p ro d u ct.
RESULTS OF RANGE-GRASS ANALYSES*
62
63
65
66
67
68
69
70
Growth M o is tu re
D ate
K ind o f
G rass C o lle c te d S ta g e ( d r y b a s i s )
S td .
5 /1 6
5 .4 #
Al
In e rm is
5 .9
Al
5 /1 5
Fwy.
6 .2
5 /1 6
A1
m a rg in .
6 .4
5 /1 6
Al
5 /1 6
p a u o i.
6 .3
Al
6 .2
5 /2 2
Ju n e.
A2
ju n o .
4 /1 8
3 .7
a2
Ju n e.
5 /2 2
6
.5
a3
6 .8
Fwy.
5 /3 1
Al
S td .
5 .4
5/3 1
Bi
m a rg in .
6 .7
5 /3 1
Bi
In e rm is
6 .4
Bi
5 /3 1
p a u o i.
4 .7
5 /3 1
Al
6 .8
6 /6
ju n o .
C2
6
.1
m a rg in .
6 /1 2
Bi
6
.2
In e rm is
6 /1 2
Bi
6 /1 2
p a u o i.
5 .4
Al
6 .1
S td .
6 /1 2
Cl
Fwy.
6 /1 2
5 .4
Cl
June.
5 .6
6 /1 9
B2
ju n o .
6 /6
5 .9
B3
Fwy.
8 .2
6 /6
B3
6 /6
5 .7
19537
B3
6 /6
7 .2
1350
B3
6 /6
S td .
6 .9
C3
6
/6
7 .5
M24-3
B3
Fwy.
6 /2 6
Bi
6 .5
6 /2 6
S td .
Bi
5 .9
6 /2 6
5 .6
m a rg in .
Bi
6 /2 6
in e r m is
6 .3
Bi
f aug l .
W
%
T o ta l
Ash
1 0 . 0%
1 1 .7
1 0 .1
1 3 .6
1 0 .6
1 0 .5
9 .4
1 0 .8
9 .7
8 .7
1 0 .1
1 0 .9
9 .5
1 0 .1
1 0 .5
9 .6
8 .7
8 .6
9 .0
8 .6
8 .7
7 .8
7 .7
1 0 .0
7 .8
8 .0
8 .5
6 .9
8 .6
§ :?
7 .3
E th e r Crude P r o E x t r a c t t e i n ( 6 .2 5 )
4 .o %
2 6 .4 %
5 .0
2 5 .9
4 .5
2 5 .1
5 .3
2 5 .4
2 4 .3
5 .5
3 .0
2 4 .3
3 5 .5
3 .9
2 6 .7
3 .3
1 9 .0
4 .5
4 .3
2 2 .5
4 .4
19 .7
5 .0
2 2 .9
5 .2
2 0 .3
2 3 .8
3 .4
1 7 .0
3 .3
2
1 .8
4 .9
1
6
.2
4 .7
4 .5
2 3 .3
3 .8
1 6 .7
3 .1
1 7 .4
3 .2
1 9 .3
1
4 .6
3 .5
1 2 .0
4 .3
1 1 .2
4 .9
2 .5
1 5 .9
3 .4
1 2 .4
4 .4
1 5 .5
3 .7
1 3 .9
3 .3
1 1 .9
i:§
3 .9
1 0 .1
L ig n in C e llu ­
lo s e
5 .3 % 2 0 .2 %
2 3 .2
5 .2
5 .7
2 2 .5
2 1 .6
5 .1
2 1 .1
5 .3
2 3 .6
6 .5
1 8 .8
4 .4
6 .8
2 0 .7
7 .5
2 5 .5
7 .2
2 5 .2
6 .1
2 6 .8
6 .0
2 7 .2
2 4 .8
6 .7
8 .8
2 4 .6
2 8 .6
9 .1
2 8 .8
8 .5
2
8 .1
7 .4
2
7
.6
8 .9
9 .3
2 7 .9
1 0 .8
2 9 .2
9 .0
2 3 .2
2 6 .0
9 .4
8 .0
2 4 .5
8 .2
2 3 .6
3 0 .0
1 3 .1
9 .6
2 3 .9
1 1 .6
2 9 .1
1 3 .2
3 2 .4
1 1 .2
2 9 .1
Ii:?
9 .5
2 6 .4
O th e r Carbo­
h y d ra te
3 3 .3 %
2 9 .0
3 2 .1
2 9 .0
3 3 .2
3 2 .1
2 8 .0
31 .7
3 3 .8
3 2 .1
3 2 .9
2 8 .0
3 3 .5
2 9 .3
3 1 .5
2 6 .4
I
3 4 .9
2 7 .1
3 3 .3
3 0 .9
3 6 .6
38 .7
4 3 .5
4 2 .1
3 0 .7
4 2 .7
3 0 .9
2 9 .9
3 5.9
TABLE I
S a a p le
Number
I
2
3
4
5
6
14
20
29
30
31
32
33
34
41
42
43
44
45
52
54
55
$ :f
4 2 .8
Kind o f
frTaHH
ju n c .
Fwy.
19537
S td .
M24-3
ju n c .
Fwy.
m argin.
Inerm ls
p a n e l.
S td .
19537
M24-3
ju n c .
S td .
1350
FwjF.
ju n c .
Fwy.
S td .
p a u c l.
m argin.
Inerm is
ju n c .
Fwy.
S td .
p a n e l.
Inerm ia
m argin.
1350
S td .
Fwf.
M24-3
19537
Date
C o lle c te d
b /1 9
6 /1 9
6 /1 9
6 /1 9
6 /1 9
7 /3
7 /1 0
7 /1 0
7 /1 0
7 /1 0
7 /1 0
7 /3
7 /3
7 /3
7 /3
7 /3
7 /3
7 /1 7
7 /2 4
7 /2 4
7 /2 4
7 /2 4
7 /2 4
7 /3 0
8 /7
8 /7
8 /7
8 /7
8 /7
7 /1 7
7 /1 7
7 /1 7
7 /1 7
7 /1 7
Growth
S ta se
d3
g3
c3
C3
c3
Dg
Dl
Dl
D%
Cl
Di
D3
D3
D)
D3
d3
d3
Dg
Dl
Dl
Dl
Bi
Dl+
B2
Dl
Dl
Dl
Dl
Bi
D3
d3
d3
D3
D3
M oisture
6 .4 %
8 .1
6 .9
6 .2
8 .2
5 .0
5 .8
6 .1
6 .0
4 .8
4 .9
4 .6
4 .5
5 .6
4 .8
5 .9
5 .0
3 .6
6 .1
6 .0
6 .2
6 .1
6 .3
5 .0
4 .6
4 .6
4 .3
4 .5
4 .6
4 .8
2 .6
4 .4
4 .0
6 .4
T o ta l E ther Crude P ro- L ig n in
Ash Ph rtrao t ta ln f 6 .2 5 ) _______ _
1 1 .8 %
1 6 .0
9 .2 * 3 .2 *
1
3 .2
9
.0
2
.7
5 .4
8
.7
n
.5
1 0 .7
3 .5
1
3
.3
1
4
.1
7 .7
2 .9
1
1
.0
1
0
.0
8 .6
3 .5
1 7 .8
1 0 .6
1 1 .4
2 .7
1 1 .6
7 .8
1 2 .4
3 .7
1 0 .8
8 .2
1 0 .1
3 .3
1 0 .5
1 0 .6
4 .4
1 3 .5
9 .6
1 0 .5
7 .7
3 .5
1 2 .8
3 .8
7 .3
1 2 .5
1 2 .1
2 .8
8 .6
7 .5
3 .6
1 4 .1
3 .6
5 .1
1 1 .0
1 0 .9
3 .3
1 1 .5
7 .6
1 3 .0
7 .8
2 .9
8 .0
4 .0
1 1 .7
7 .1
4 .2
1 2 .7
8 .2
6 .3
1 8 .8
4 .0
1 0 .3
1 2 .5
7
.6
8 .8
1 2 .4
3 .5
1
3 .7
9
.1
3 .5
6 .9
1
3 .3
7 .2
5
.7
2 .9
1 3 .2
2 .6
7 .3
9 .4
1 2 .6
1 1 .6
4 .2
1 0 .4
9 .4
2 0 .4
4 .4
1 5 .1
1 1 .8
8 .4
9 .4
3 .5
1 3 .6
9 .0
3 .7
6 .7
8 .0
1 3 .3
5 .7
4 .1
7 .8
1 5 .4
2 .7
8 .7
1 1 .7
9 .6
5 .7
3 .7
1 1 .8
8 .2
3 .5
5 .5
6 .0
5 .2
1 2 .3
4 .1
3 .2
9 .2
6 .2
1 3 .7
1 1 .8
4 .8
3 .5
7 .5
3 .6
7 .6
1 2 .7
6 .5
C e llu - Other C
hvdi »teal,o a e ,
3 2 .2
2 7 .6
3 9 .8
2 9 .9
2 8 .2
3 7 .4
3 1 .0
3 0 .7
2 8 .2
3 8.7
2 8 .8
2 8 .7
3 1 .1
3 3 .4
2 9 .8
3 7 .8
3 3 .1
2 7 .9
3 2 .8
3 5 .9
3 2 .2
3 1 .4
2 8 .0
4 1 .0
3 0 .8
4 2 .8
30 .2
3 3 .1
3 7 .0
31 .7
4 0 .7
2 8 .5
3 8 .7
2 9 .9
2 6 .7
2 7 .7
3 0 .8
3 6 .9
33.1
33 .7
3 6 .6
3 4 .3
3 2 .8
34 .7
3 2 .2
2 9 .0
2 6 .1
2 4 .6
3 4 .8
3 2 .1
34 .7
3 1 .3
3 5 .8
3 3 .1
3 7 .0
2 8 .4
3 2 .2
3 7 .0
3 6 .6
3 0 .4
3 1 .6
4 0 .8
3 2 .2
3 5 .5
3 0.7
4 1 .7
39.7
2 9 .9
TABLE I ( c o n tin u e d)
Sample
'NTllmtlAI*
77
79
82
83
84
92
97
99
101
103
105
107
111
112
116
117
119
131
133
135
137
139
.141
150
153
154
155
156
157
160
162
163
I 65
167
Date
Kind o f
G rass C o lle c te d
Ju n e.
7 /1 7
19537
7 /3 1
Pwy.
7 /3 1
1350
7 /3 1
M24-3
7 /3 1
June.
7 /3 1
S td .
7 /3 1
8 /1 4
June.
8 /2 2
S td .
8 /2 2
Fvy.
m arg in . 8 /2 2
in e rm is 8 /2 2
p a n e l.
8 /2 2
8 /2 6
Ju n e.
S td .
9 /4
Pvy.
9 /4
in e rm is 9 /4
m a rg in . 9 /4
9 /4
p a u c i.
Growth M o istu re
S tag e ( d r y b a s i s )
4 .8 %
E3
7
.8
d3
3
.8
e3
4
.2
e3
4 .4
D3
5 .5
e3
4
/3
d3
4 .1
Es
4 .4
5I
Bi
4 .7
4 .4
Ei
4 .1
E1
4
.4
E1
3 .8
E2
Ei
3 .5
Bi
5 .6
Bi
4 .5
Bi
4 .7
4 .1
E1
T o ta l E th e r
Ash E x tr a c t
I 3 -8% 4 .3 I
7 -3 v 4 .1
1 0 .0
3-5
8 .6
2 .3
9 .8
3-7
4 .1
1 3.7
3 .2
7 -3
4 .0
1 4 .0
7 .5
3 .1
9 .0
2 .7
2 .8
9 .9
7 -1
2 .3
8 .1
3 .5
4 .2
1 2 .9
2 .4
5 .5
8 .6
2 .8
2 .4
8 .9
9 .2
2 .5
7 .7
2 .9
Crude P ro t e in ( 6 .2 5 )
7 .5 *
6 .9
5 -3
5 .0
8 .0
6 .3
6 .9
2 0 .8
9 .1
8 .6
6 .2
4 .6
5 .6
1 6 .6
6 .1
9 .0
6 .2
5-5
4 .3
L ig n in C e llu ­
lo s e
1 1 . 4> 31.8%
35.1
1 5 .3
33-6
13 .9
14 .1
3 2.0
1 3 .6
2 8 .5
358
11 .9
1 4 .2
33.1
2 4 .8
7 .9
1 4 .0
3 6.3
3 5 .6
13 .7
1 2 .4
34.5
1 6 .0
39-6
1 3 .7
37-3
9.5
2 9 .9
1 5 .6
4 0 .4
1 4 .1
36.9
1 7 .8
4 1 .3
38.5
1 3 .3
1 4 .0
39.7
O th er C ar­
b o h y d ra te s
3 1 .2 £
31 .3
33-7
38 .0
3 6 .4
2 8 .2
3 5 .3
28 .5
30 .0
30 .0
3 4 .2
3 0 .4
3 1 .8
2 6 .9
30 .0
2 8 .6
2 3 .4
3 1 .0
3 1 .4
Sub-1 ■ P t . E l l i s , G a l l a t i n County
Sub-2 = Campus, G a lla t in County
Sub-3 * J u d i t h B asin County
♦The c o l l e c t i o n o f g ra s s e s and some o f th e s e a n a ly s e s were perform ed b y members o f th e Chem­
i s t r y S t a f f a s a p a r t o f r e g u la r ran g e g r a s s p r o j e c t s , and a r e in c lu d e d h e re f o r th e sake o f
c o m p le te n e ss.
TABLE I (c o n tin u e d ).
Sample
Humber
170
173
175
177
179
180
182
185
193
194
195
196
197
198
205
206
207
208
209
-4 9 -
/
/ / ^ /? / / ?
/Z?7*y^
C o u r r f 1-/
/
/tfV/> user y
S-/Z* n cfay c/
4
F ig u r e I
/
50-
1S
«a/ / a
^ /jf
C o u +7
/ stCttSCf f / O^iSrrf
F ig u r e 2
+ /5
F igure 3
J
(T e / / a / o S G
< // Atf
Cf^y/SJ
•5 Tqsi c/c* r~c /
//
//
.. 4 0
/9 S 3 ?
/I -Z i-3
S z ./s
/3 S O
-J £
/
-
/
30
-^ O
~?<sSi e
'Ja/y
F ig u ra 4
Ao^ o&T
-5 3 EXPERIMENTAL RESULTS
L ig n in C ontent o f QftViatln Covinty Q rasaes
The H g n ln c o n te n t I n th e f iv e G a lla t in County g r a s s e s ( f ig u r e l ) i n ­
c re a s e d from 5 -6 p e r c e n t i n th e f i r s t c o l l e c t i o n s on May l 6 t o 13-18 p e r
c e n t on th e l a s t c o l l e c t i o n , Septem ber 4 , 1941.
I t was i n t e r e s t i n g t o n o te
t h a t th e l i g n i n c o n te n t o f th e g ra s s e s in c re a s e d more o r l e s s u n ifo rm ly u n t i l
Ju n e 26.
On th e n e x t c o l l e c t i o n , J u l y 1 0 , th e r e was a d ro p i n l i g n i n c o n te n t
o f th e f o u r g ra s s e s t h a t w ere i n s ta g e D.
Only p a u c iflo ru m , w hich was i n
s ta g e C, showed a s l i g h t in c r e a s e i n l i g n i n o v e r th e p re v io u s c o lle c tio n
d a te .
A d e c re a se i n th e H g n in p e rc e n ta g e does n o t n e c e s s a r ily mean t h a t any
l i g n i n , once form ed i n th e p l a n t , i s decomposed; i t i s more p ro b a b le t h a t
t h e p la n ts s y n th e s iz e d o th e r c o n s ti tu e n ts a g r e a t d e a l f a s t e r .
Thus, a l ­
th o u g h th e r e may have been an a c tu a l in c r e a s e i n t o t a l l i g n i n i n a p l a n t,
i f , s im u lta n e o u s ly , th e p l a n t may have s y n th e s iz e d a la r g e q u a n tity o f c a r ­
b o h y d ra te s , th e l i g n i n c o n te n t on a p e rc e n ta g e b a s is would show a d e c r e a s e .
Comparison o f th e L ignin Content o f Brcmus marginatus and
Bromur; In e m3, s
There was a c lo s e p a r a l l e l in grow th s ta g e s in ln e rm ls and m a rg in a tu s.
Both m atured e a r l y , h av in g re a ch e d s ta g e D b y t h e l a t t e r p a r t o f Ju n e, and
rem ained i n s ta g e D u n t i l e a r l y i n A ugust.
H gTvtr^ c o n te n t u n t i l s ta g e E .
e r a i s r a p id ly in c re a s e d .
T here was a c lo s e s imi l ar i t y i n
D uring t h i s p e r io d th e l i g n i n c o n te n t o f I n -
In ex m is, on Septem ber 4 , showed th e h ig h e s t l i g n i n
c o n te n t o f a l l g ra s s e s a n aly z e d .
M arginatus d id n o t show t h i s te n d en c y ,
and a t th e end o f th e grow ing sea so n had th e lo w e st l i g n i n c o n te n t o f th e
g ra s s e s p l o t t e d I n f ig u r e I .
Comparison o f Agropyron crlstatu m s e l e c t i o n s , Fairway and Standard
Fairw ay and S ta n d a rd re a ch e d th e seme grow th s ta g e s a t about th e same
tim e .
The lignin c o n te n ts o f th e s e g r a s s e s were n e a r ly th e same d u rin g
th e e a r l y grow th s ta g e s (A, B and C ).
w ere I n
s ta g e D.
S h o r tly a f t e r J u l y I , b o th g ra s s e s
Frcm t h i s tim e o n . S ta n d a rd had a c o n s id e ra b ly h ig h e r
l i g n in c o n te n t th a n F airw ay.
Fairw ay
and S ta n d a rd re a ch e d s ta g e D a b o u t
two weeks a f t e r th e brome g r a s s e s .
Agropyron pauc lfloru m
The
rem arkable f e a tu r e o f pauc lflo ru m was t h a t I t m atured l a t e i n
s e a s o n , n o t re a c h in g s ta g e D u n t i l e a r l y I n
A ugust.
th e
As m ig h t be e x p e c te d ,
th e p a u c iflo ru m had th e lo w e st l i g n i n c o n te n t o f th e g r a s s e s t e s t e d d u rin g
Ju ne and J u ly .
Elymus .Iunceus
Jun ceu s was sam pled from th e campus p l o t s , w h ile th e o th e r O e lla t ln
County g ra s s e s w ere grown a t F o rt E l l i s .
I t was n o t p o s s ib le t o compare i t
w ith th e F o r t E l l i s g r a s s e s u n le s s o th e r v a r ia b le s b e s id e s s e a s o n a l change
w ere c o n s id e re d .
I t i s o f i n t e r e s t t o n o te , how ever, t h a t t h e l ig n in con­
t e n t In c re a s e d from 4 .4 p e r c e n t on May 18 t o 1 1 .4 p e r c e n t on J u ly 3 .
a f t e r , th e r e was a s l i g h t d e c re a se i n l i g n i n
c o n te n t.
T h ere­
The re a so n f o r th e a p ­
p a r e n t d e c re a se I n p e rc e n ta g e o f l i g n i n was n o t d e te rm ine d .
-5 5 ”
C e llu lo s e In G a lla tin County Grasses
The c e llu lo s e con ten t o f th e f i v e G a lla tin county g r a sse s p lo tte d on
fig u r e 2 in crea sed from 20-24 per cen t on May l 6 t o 37-42 p er cent on Sep­
tember 4.
There was a marked tendency fo r th e c e llu lo s e t o in crea se along
w ith th e lig n in . On J u ly 10 when th e lig n in content showed a decided drop,
th e percentage o f c e llu lo s e in lnerm is and Standard lik e w is e decreased.
On
August 7 th ere was a decided drop in both lig n in and c e ll u lo s e in m arginatus.
With
th e e x ce p tio n o f paueiflorum , which had a low
lig n in and a high c e l l u ­
lo s e percentage in mid sea so n , th ere was a general tendency f o r lig n in con­
te n t t o p a r a lle l c e ll u lo s e co n te n t.
h ig h e s t in both c e ll u lo s e and lig n in .
In e n n is, a t th e end o f th e season , was
Standard, in th e l a t t e r part o f June,
was h ig h e s t in both c e llu lo s e and lig n in .
ln erm is, in th e f i r s t part o f
J u ly , had th e lo w e st c e llu lo s e and lig n in con ten ts o f any o f th e g r a s s e s .
What s ig n ific a n c e t h i s c o r r e la tio n may have i s not known ex cep t th a t th e
form ation o f lig n in c lo s e ly p a r a lle ls th e form ation o f c e ll u lo s e .
This may
have valu e in determ ining th e precursors o f lig n in .
E arly in th e season th e
c e llu lo s e con ten t o f Elymus Junceus was about
th e same as th e o th er G a lla tin County g r a s s e s .
Here again , th e c e llu lo s e
co n ten t c lo s e ly p a r a lle le d th e li g n i n , and on September 4 , had 29-9 per cen t
c e l l u l o s e , which was s e v e r a l per cen t low er than th e oth er G a lla tin County
g r a sse s grown a t Fort E l l i s .
L ignin Content o f Ju d ith B asin Grasses
S ix g r a sse s from Ju d ith B asin were analyzed; sampling began on June 6
-5 6 -
and continued a t two-week in t e r v a ls u n t i l J u ly 31*
B asin
The g r a sse s a t Ju d ith
matured e a r li e r than th o se in G a lla tin County.
At th e tim e o f th e
f i r s t c o lle c t i o n on June 6 , most p f th e g r a sse s were alread y in or p a st
s ta g e B.
I t i s t o be r e g r e tte d th a t data on th e e a r ly sta g e s o f th ese
g r a sse s cannot be p resen ted .
The lig n in con ten t o f f i v e J u d ith Basin
g r a sse s appears in fig u r e 3 .
Fairway, S e le c tio n 1350» had a d e f in it e ly
low er l i gnin con ten t than Fairway u n t i l th e l a s t sampling (J u ly 3 1 ), a t
which tim e th e lig n in con ten t o f both were about eq u al.
S e le c t io n 1350
had th e lo w e st average H gnin content during th e e n tir e sea so n , in compari­
son w ith th e o th er fou r g r a sse s s tu d ie d .
Agropyron cristatu m Standard and S e le c tio n s 19537 and M24-3
Standard had a h igh er lig n in con ten t than th e o th er s e le c t io n s when
f i r s t c o lle c t e d on June 6 .
This was probably due t o th e f a c t th a t th e
Standard samples were more mature and were already in sta g e C when f i r s t
c o lle c t e d , w h ile th e o th er s e le c t io n s were s t i l l in sta g e B.
f u l i f o th er v a r ia tio n s in lig n in co n ten t were s ig n if ic a n t .
I t i s doubt­
In f a c t , th e
e a r l i e r v alu es f o r lig n in may a l l be q u estio n ed , due t o th e f a c t th a t th e
samples from J u d ith B asin contained more or l e s s o ld grass from the previous
season .
In g e n e r a l, th e lig n in valu es o f th e J u d ith Basin g r a sse s in June were
h igh er than th o se from G a lla tin County a t th e same tim e.
mer ni I g r a sse s had very n e a r ly
Late in th e sum­
th e same lig n in con ten t ex cep t lnerm is in
G a lla tin County, which had s e v e r a l per cen t high er lig n in co n ten t than any
o th er g r a ss.
-5 7 -
C e llu lo s e Content o f J u d ith Basin Grasses
The s i m i l a r i t y o f th e c e ll u lo s e and l i g n i n c o n te n t o f th e s e g ra s s e s
t o th e c e ll u lo s e and l i g n in c o n te n t o f th e G a lla tin County g ra s s e s was p ro ­
nounced.
They were n e a r ly th e same.
S ta n d a rd had a much h ig h e r c e llu lo s e
c o n te n t when compared w ith th e o th e r g ra s s e s on June 6.
showed th e h ig h e s t c e l l u l o s e c o n te n t on J u ly 31.
S e le c tio n 19537
F airw ay , th ro u g h o u t th e
s e a s o n , bad s e v e r a l p e r c e n t more c e ll u lo s e th a n S e le c tio n 1350, as n o tic e d
i n f ig u r e 4.
In g e n e r a l, th e Ju d ith Basin g r a sse s had a s l i g h t l y low er c e llu lo s e con­
te n t than th e G a lla tin County g r a sse s.
Elymus .Iunceus - J u d ith Basin
The H g n-Ln con ten t o f junceus May 22 was 6 .8 .
I t g ra d u a lly increased
t o 1 1 .8 per cen t on June 19 and throughout th e r e s t o f th e season was n e a r ly
c o n sta n t.
At th e end o f th e season Elyraua Junceus had s e v e r a l per cen t l e s s
n g n in t-h«n th e o th er J u d ith Basin g r a s s e s .
The c e llu lo s e r o se gradually
from 2 0 .7 per cen t in th e l a t t e r p art o f May t o 35.8 per cen t eafLy in
August.
Here th e g en era l tendency f o r l ig n in to c lo s e ly p a r a lle l c e llu lo s e
co n ten t was not apparent.
Comparing Junceus from G a lla tin County
and J u d ith B a sin , th e lig n in
con ten t u n t i l June 19 was p r a c t ic a lly th e same.
T h erea fter, th e lig n in con­
t e n t in G a lla tin County Junceus decreased about 2 per c e n t, w h ile in Ju d ith
TUtairi County i t remained con stan t a t about 1 1 .5 per c e n t.
The c e llu lo s e o f
Junceus in G a lla tin County was s e v e r a l per cen t h igh er than Ju d ith B asin
-5 8 -
e a r ly In th e
J u d ith
season .
T his was reversed in m id-season, and on J u ly 31 th e
Junceus had 35*8 p er cen t c e llu lo s e --a b o u t 10 per c en t more
c e ll u lo s e than th e G a lla tin County Junceus.
-5 9 discussion
The chem ical
a n a ly s is o f variou s Montana g r a sse s has been undertaken
w ith th e hope th a t th e r e s u lt s w i l l have v a lu e in p r e d ic tin g feed in g v a lu e s.
A complete chem ical a n a ly s is o f g r a sse s a t presen t i s not
knowledge o f p la n t c o n s titu e n ts i s f a r from com plete.
p o s s ib le , as our
A lso , i f one were to
analyze one g ra ss in as much d e t a il as p resen t knowledge p erm its, i t i s
d o u b tfu l i f one could com plete such an a n a ly s is in s e v e r a l y e a rs'
tim e.
T h erefore, i t seemed d e s ir a b le t o lim it th e a n a ly s is o f g ra ss t o a few
p la n t c o n s titu e n ts th a t are o f importance in p r e d ic tin g fe e d in g v a lu es, un­
d erstan d in g, however, th a t one, by in te r p r e ta tio n o f r e s u lt s , i s not p re­
s e n tin g a complete p ic tu r e .
Attempts were made, th e r e fo r e , t o choose a
method o f a n a ly s is which would have th e g r e a te s t b io lo g ic a l s ig n ific a n c e ,
and which would be capable o f com pletion in a reasonable amount o f tim e.
For reasons given in th e in tr o d u c tio n , th e methods o f fe e d a n a ly s is
(crude
f i b e r , n itr o g e n -fr e e e x tr a c t) as presen ted by the A sso c ia tio n o f O f f ic ia l
A g r icu ltu r a l Chemists were not
used.
The a n a ly sis o f g r a sse s was confin ed
t o s i x p la n t c o n s titu e n ts ; namely, m oisture ( f o r dry b a s i s ) , t o t a l ash ,
e th e r e x tr a c t, crude p r o te in , lig n in , c e ll u lo s e and o th er
carbohydrates.
The m oisture determ ination was n ecessa ry in order t o c a lc u la te the o th er
c o n s titu e n ts from an a ir -d r y b a s is t o a
fr a c tio n was
dry b a s is .
The "other carbohydrate"
determ ined by d iffe r e n c e .
A b r ie f d is c u s s io n o f th e fa c to r s th a t w i l l a f f e c t th e growth and com­
p o s itio n o f range g r a sse s i s perhaps n ecessa ry b efore any in te r p r e ta tio n
r e s u lt s i s attem pted.
of
Atkinson and Woodward 4 / p o in t out th a t many fa c to r s
-GoIn flu en ce th e growth and com position o f p a stu re herbage.
are c lim a te , sea so n a l v a r ia tio n , s p e c ie s , s o i l ty p e, s o i l
l i z a t i o n and frequency o f c u ttin g .
The c h ie f fa c to r s
fe r tility , fe r ti­
Here a g a in , i t became n ecessa ry t o
seg reg a te th e fa c to r o f sea so n a l change and d isc u ss i t in as g rea t d e t a i l
as th e a n a ly s is p e r m its.
Probably o n ly when each fa c to r a ff e c tin g growth
and com position o f forage p la n ts i s understood w i l l i t be p o s s ib le t o have
a com plete p ic tu r e o f p la n t l i f e .
In p r e d ic tin g fe e d in g v a lu es by chem ical a n a ly sis a stu d y o f th e grass
o r fe e d in g m a ter ia l i s o n ly h a lf th e p ic tu r e .
ju s t as im portant.
A study o f th e animal i s
There i s a g rea t d e a l o f v a r ia tio n in anim als o f d i f ­
fe r e n t s p e c ie s in t h e ir a b i l i t y t o d ig e s t c e r ta in p la n t c o n s titu e n ts .
Even
in animal a o f th e same s p e c ie s , a l l do not have th e same a b i l i t y fo r a ssim i­
la t i o n o f food .
When one i s feed in g mixed r a tio n s th e d i g e s t i b i l i t y o f th e
d if f e r e n t c o n s titu e n ts i s sometimes m a te r ia lly changed.
The Crampton and Maynard method fo r determ ination o f lig n in i s s a t i s ­
fa c to r y in th a t i t i s p o s s ib le t o o b ta in reproducible r e s u l t s .
This method
g iv e s approxim ately one hundred per cen t recovery o f lig n in i n th e f e c e s .
One main o b je c tio n t o t h i s method i s th a t i t i s very lo n g and time-consuming
I t i s d o u b tfu l, th e r e fo r e , i f th e determ ination o f lig n in w i l l be put in to
p r a c tic e by th o se running la r g e numbers o f fe e d an alyses u n le s s a more rapid
method w ith s u ita b le accuracy i s found.
I t i s g e n e r a lly
accepted th a t lig n in i s in d ig e s tib le in i t s e l f , and a l
s o , by ln c r u stln g or seme o th er means, i t prevents th e d ig e s tio n o f oth er
p la n t c o n s titu e n ts which otherw ise would be u t i l i z e d .
L ignin may hi nder
I
m
m
-61-
I::-'V
d ig e s tio n by in a c tiv a tin g th e enzymes by some means.
'
I t i s indeed l i k e l y
th a t p h en o lic groups or o th ers p resen t in th e lig n in complex have a to x ic
e f f e c t on th e d ig e s tio n enzymes.
Waksman g V found th a t th e presence o f
o n ly 8 per cen t lig n in was s u f f i c ie n t in ruminants t o reduce th e rate o f c e l ­
lu lo s e decom position by f i f t y per c e n t.
I f lig n in determ inations are to be
o f r e a l value in fe e d a n a ly s is , a g r ea t d e a l o f sim ila r research must be ca rrie d tn .
U n til th e tim e th a t knowledge o f lig n in in r e la t io n t o
fe e d an alyses
i s put on a q u a n tita tiv e b a s is , i t s u s e fu ln e s s i s r e s t r ic t e d .
probable th a t lig n in i s a mixture o f d if f e r e n t compounds.
I t is
When the ehem-
l s t r y o f l ig n in i s fu r th e r e lu c id a te d , th e p a r t th a t lig n in p la y s in r e ­
s t r i c t i n g d i g e s t i b i l i t y can be put on a sound chem ical b a s is .
The crampton and Maynard method f o r c e llu lo s e was not changed in p rin ­
Cf
.
c ip le ; however, by use o f c e n tr ifu g a l sep a ra tio n i t was p o s s ib le t o shorten
th e tim e required f o r each determ ination.
As p r e v io u sly s ta te d ,
A'Ct
p lan t c e llu lo s e i s a m ixture.
The chemical and
n u t r it iv e p r o p e r tie s o f m ixtures are d i f f i c u l t to determ ine a c cu ra tely .
e f f e c t o f lig n in on th e d i g e s t i b i l i t y o f
The
p la n t c e llu lo s e i s a com plicating
fa c to r .
Percentages o f c e ll u lo s e and lig n in were determined by lo s s o f w eight
on ig n it io n .
The percentage o f ash was about I per cen t o f th e t o t a l w eight.
The ash probably co n ta in ed , in main, s i l i c o n d io x id e .
A q u estio n n a tu r a lly
a r is e s as t o whether th e ash presen t was due t o adsorption during a n a ly sis
or was ash o r ig in a lly p resen t as a c o n s titu e n t part o f th e l ig n in and c e l -
.■
—62—
lu lo s e .
I f low -tem perature ig n it io n s were employed t o ash th e c e llu lo s e
and lig n in r e s id u e s , phosphorus might p o s s ib ly be found.
Phosphorus, i f
p resen t in th e lig n in and c e llu lo s e a sh , should he c r i t i c a l l y examined.
The d if f e r e n t sugars p resen t in g r a sse s are o f much in t e r e s t , s in c e
th ey provide th e main source o f energy fo r anim als.
The determ ination o f
th e variou s p en toses and hexoses in th e w a te r-so lu b le h e m ic e llu lo s e , and
c e ll u lo s e f r a c t io n s , might le a d t o v a lu a b le inform ation from a n u tr itio n a l
stan d p oin t
No doubt th e c e llu lo s e in th e g ra sses analyzed contained pen­
to s e s (probably
z y lo s e ) .
Further in v e s tig a tio n o f th e carbohydrates o f
th e s e g r a sse s i s d e s ir a b le .
The determ ination o f c e llu lo s e and lig n in i s a d e f in it e s te p forward;
th e fo reg o in g d is c u s sio n was presented to p o in t out th a t resea rch in t h is
f i e l d i s o n ly in i t s in fa n cy .
,I" '
-6 3 -
SUMMARY
Lignin a n alyses on g r a sse s from G a lla tin V a lley in d ic a te d a s im ila r it y
o f a l l s p e c ie s t e s t e d , during May and June, Ig W .
By th e end o f August,
however, inerm is had reached a high o f 1 7 .8 per cen t and on t h i s b a s is was
probably th e le a s t - d i g e s t i b l e o f th e g r a s s e s .
C ontrasting w ith t h is was
m arginatus, which ended w ith a lig n in co n ten t o f o n ly 13-3 p ercen t.
Standard crista tu m showed a tendency t o con tain more lig n in th a t P a ir way crista tu m , and on t h i s b a s is would be judged s l i g h t l y in f e r io r .
L ignin
v a lu es f o r th e se two g r a sse s f e l l between th o se fo r th e two brcme g r a ss e s.
Pauciflorum showed a d e f in it e s u p e r io r ity over th e o th er Agropyrons
t e s t e d in th a t i t m aintained a low er lig n in content during May and June.
At Ju d ith B asin th e f i v e crista tu m s e le c t io n s sampled matured a month
e a r li e r than crista tu m d id in G a lla tin V a lle y , but fo llo w ed a s im ila r path
o f in c r ea se in l i gnin c o n te n t.
The s p e c ia l s e le c tio n s o f crista tu m con­
ta in e d s l i g h t l y l e s s l ig n in than th e r eg u la r p lo t s .
Here, as in G a lla tin
V a lle y , Fairway con tain ed l e s s lig n in than Standard.
C e llu lo s e ranged from approximately 20 t o W p er c e n t, th e changes in
c e llu lo s e c lo s e ly p a r a lle lin g changes in lig n in content as th e season ad­
vanced.
»64-
ACKKOWL
T
The author w ish es t o tak e t h i s op p ortu n ity to express h is a p p recia tio n
t o Dr. A. R. P a tto n , Dr. W. E. Carlson
and Mr. D. R. McCormick o f th e Mon­
ta n a A g r ic u ltu r a l Experiment S ta tio n f o r tim e , a s sis ta n c e and advice g iv en
him during th e
year w h ile t h i s work was b ein g completed.
-6 5 -
B IB L IO G a R A P H Y
1.
A lle n , R. S . , and C arlson, A. J . , Am. J .
2.
A lm quist, H. J . , J . B io l. Chem. 134. 213-16 (1940)
3.
Anderzen, 0 . , and Holmberg, B ., B er. 5 6 , 2044 (1923)
4.
5.
A tkinson, H. S . , and Woodward, S. C ., Can. Chem. P roc, Inds. 2 2 , 67-71
(1938)
Beckwith and Edythe, P roc. S o c. E x p tl. B io l. Med. 2%, 4 -6 ( 1929)
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