Studies on Softwood Lignin and the Mechanismof Lignification

Citation preview

FO R D H A M U N IV E R SIT Y G R A D U A T E SCHOOL

May 1 5

19

50

This dissertation prepared under my direction by

Walter J« Schubert

entitled

STOPIES ON SOFTWOOD LICWIN AND THE MECHANISM OF LIGNIFICATION

has been accepted in partial fulfilment of the requirements for the

Degree o f ..................... D o cto r..o f...P h ilo so p h y .................................................

................ (Faculty A dviser)

~i

r

STUDIES ON SOFTWOOD LIGNIN ADD THE MECHANISE OF LIGNIFICATION

BY WALTER J . SCHUBERT B .S ., Fordham C o lle g e , 1345 M .S ., Fordham U n i v e r s i t y , 1947

DISSERTATION SUBMITTED ID PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN THE DEPARTMENT OF CHEMISTRY AT FORDHAM UNIVERSITY

NEW YORK 1950

ProQuest Number: 10992949

All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is d e p e n d e n t upon the quality of the copy subm itted. In the unlikely e v e n t that the a u thor did not send a c o m p le te m anuscript and there are missing pages, these will be noted. Also, if m aterial had to be rem oved, a n o te will ind ica te the deletion.

uest ProQuest 10992949 Published by ProQuest LLC(2018). C opyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C o d e M icroform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 4 8 1 0 6 - 1346

iii TABLE OF CONTENTS P age LIST OF TABLES............................................................................ LIST OF FIGURES................................................................................

iv .

vi

ACKNOWLEDGMENTS.......................• .......................................................................................

v ii

I.

INTRODUCTION........................................................................................................

1

II.

EXPERIMENTAL METHODS......................................................................................

5

M ic r o b i o lo g ic a l. . . . .......................................................................... A n a ly tic a l. . . . . . . ....................................................................... P r e p a r a t i v e .................................................................................... S p e c t r o s c o p i c .............................................................................................. ;

5 7

A. B. C. D. III.

EXPERIMENTAL RESULTS. .

’.

12

S tu d ie s on L i g n in ........................................................................................ P r e li m in a r y E x p e rim e n ts ..................................................................... N a tiv e f c i i t e ^ c o ts P in e L ig n in ................................. L ig n in from L e n tin u s le o i d e u s -d e c a v e d $ h i t e S c o ts P in e Wood.................................................. L ig n in s from Sound an d L e n tin u s l e p i d e u s -»deca.ved W hite F i r No o d ....................... L ig n in L ib e r a te d E x c lu s iv e ly by th e A c tio n o f P o r ia v a i l l a n t i i ........................................................................ L ig n in L ib e r a te d ^ x & lu s iv e ly fcy th e A c tio n o f L e n z ite s s e o i a r i a . . . ............................................................. C h e m ic a lly P re p a re d L i g n i n s ............................................................ S tu d ie s on ’’I n d u l i n ” ............................................................................. The L ig n in s o f S ch titz and S a r t e n ................................................ A M o d ifie d P ro c e d u re f o r O b ta in in g N a tiv e L ig n in . . . F u r th e r S tu d ie s on Wood fiecay . . . . . . . . . . . . .

12 13 14

52 40 45 47 51 56

S tu d ie s on th e M etab o lism o f L e n tin u s l e o i d e u s ..................... S t e r o l f o r m a t io n ................................................................................ % s s i mi]_a -y_on 0f 0 (_ K e to g lu ta r ic A c id .....................................

62 62 72

DISCUSSION..........................................................................................................

78

SUMMARY.

87

A. 1. 2. 5. 4. 5. 6

.

7. 8. 9. 10. 11. B. 1. 2. IV . V.

8

11

..................................

BIBLIOGRAPHY..............................................................................................................................

16 22 29

89

_J

iv

I,

A lk a li S o l u b i l i t i e s o f White F ir Wood a ft e r S ix Months Decay by Four W ood-Destroying F l u n g i.....................

15

II,

C h aracterization o f N ative White S c o ts Pine L ig n in .. . . .

17

III.

E ffe c t of the A ction o f L entinus le n id eu s on the Compositioi^bf White S c o ts Pine W ood,..................................

19

Y ie ld s o f L ignin I s o la t e d from Sound and from Lentinus le n id e u s -Decayed Wood.................................

21

Comparison o f L ignin s from Sound and from L entinus le p id e u s-Decayed Wood............... ..........

23

E ffe c t o f th e A ction o f L entinus le p id e u s on the Composition o f White F ir W o o d . , . . . ......... • • • • • • ................

26

Comparison o f Methoxyl Contents o f L ignins from Sound and from L entinus lep id e u s-De cay ed Wood.....................

27

E ffe c t o f th e A ction o f P oria v a i l l a n t i i on th e Composition o f White S co ts Pine W ood........................

31

Comparison o f N ative and P oria v a i l l a n t i i -L ib erated L ignin s o f White S c o ts Pine Wood« . . . ............................

33

E ffe c t o f th e A ction o f L en zites s e p ia r ia on the Composition o f White S c o ts Pine Wood....................................

3&

Y ie ld s o f L ignin I s o la t e d from Sound and from Decayed White S c o ts Pine Wood........................

37

Comparison of Methoxyl Contents o f L ignih s from Sound and Decayed White S c o ts Pine Wood........................

3^

Comparison o f A n a ly tic a l Data o f Chem ically Prepared and N ative White S c o ts Pine L ig n in s......................................

1|2

Comparison o f P r o p e r tie s o f Chem ically Prepared and N ative White S c o ts P ine L ig n in s . ....................................

43

IV, V. V I. V II. V II I . IX. X, X I. X II. X III. XIV.

v

XV. XVI. XVII. 1_

Comparison o f Compositions of In d u lin A and ’’P u rified " In d u lin w ith N ativ e White S c o ts Pine L i g n i n . . . . . . . . . .

4&

Comparison o f Com positions o f L ignin s According to Sch&tz and Sarten w ith N ative White S co ts Pine L i g n i n . . . . . . .

49

Composition o f N ativ e L ig iin Obtained by M odified Procedure and I t s D e r i v a t i v e s .................................................

53

V

T a b le X V III. XIX.

XX.

XXI.

X X II.

Page " 1 E f f e c t o f t h e A c tio n o f S o n io n h o ra c e r e b e lla . on th e C o m p o sitio n o f #lh i t e S c o ts P in e Wood................................... . . . E f f e c t o f t h e A ctio n o f Fomes r o b u s tu s on th e C o m p o sitio n o f W hite ^ c o ts P in e Wood..................................

59

E f f e c t o f t h e A c tio n o f M vrothecium v e r r u c a r i a on th e C o m p o sitio n o f W hite ^ c o ts P in e Wood...........................................

61

C om parison o f D ata o f S t e r o l from L e n tin u s le o id e u s w ith E r g o s t e r o l . . ........................................................................................

64

F o rm atio n o f S t e r o l by L e n tin u s le p id e u s on E th y l A lc o h o l-C o n ta in in g M ed ia .......................................................

69

'X X III. E f f e c t o f I n c r e a s i n g C o n c e n tr a tio n s o f j^imedon on th e M etab o lism o f L e n tin u s l e o i d e u s . .............................................. X^V.

57

The D i s s i m i l a t i o n o f O j- K e to g lu ta r ic A cid by L e n tin u s le o id e u s .....................................................................

71

vi

r

LIST OF FIGURES

F ig u r e

E&ge

I,

U l t r a v i o l e t A b s o rp tio n S p e c tr a o f N a tiv e W hite S c o ts P in e L ig n in and D e r i v a t i v e s . . ..........................................

II.

Com parison o f th e U.V. S p e c tr a o f L ig n in s from Sound and L e n tin u s l e o i d e u s -D ecaved W hite ^ c o ts P in e . ...................

24

C om parison o f the U.V. Spectra o f d e r i v a t i v e s o f L ig n in s from Sounh and L e n tin u s l e o i d e u s -D ecaved W hite S c o ts P in e

25

Com parison o f th e UIV. S p e c tr a o f L ig n in s from Sound and L e n tin u s le o id e u s - D ecaved W hite F i r . . . . . . . . . .

28

Com parison o f t h e U.V. S p e c tr a o f N a tiv e and P o r ia v a i l l a n t i i - L i b e r a te d w h ite S c o ts P in e L i g n i n s ...................

54

C om parison o f th e U.V. S p e c tr a o f D e r iv a t iv e s o f N a tiv e and P o r ia v a i l l a n t i i - L i b e r a te d W hite S c o ts P in e L i g n i n s . . .

55

C om parison o f t h e U.V. S p e c tr a o f N a tiv e and L e n z ite s s e o i a r i a - L i b e r a t e d W hite u c o t s P in e L i g n in s ........................

59

Com parison o f t h e U.V. S p e c tr a o f N a tiv e and A lk a l i # n i t e Scots P in e L i g n in s ....................................................................

44

C om parison o f th e U.V. S p e c tr a o f N a tiv e an d S e h d tz and S a r te n W hite S c o ts P in e L i g n in s . . . . .................................

50

C om parison o f th e U.V. S p e c t r a o f N a tiv e W hite ^ c o ts P in e L ig n in and l i g n i n I s o l a t e d by M o d ifie d P r o c e d u r e

54

C om parison o f t h e U.V. S p e c tr a o f D e r iv a tiv e s o f N a tiv e W hite tf c o ts P in e L ig n in an d o f L ig n in I s o l a t e d ty M o d ifie d P r o c e d u r e ..................................................................................

55

C om parison o f t h e U.V. S p e c tr a o f S t e r o l from L e n tin u s l e o id e u s on ^ lu c o s e and E th y l A lc o h o l and o f E r g o s te r o l

65

III.

IV .

V.

V I. V II.

V III.

IX .

X.

X I.

X II.

L

n

18

_i

ACKNOWLEDGMENTS I w ish t o d e d i c a te t h i s d i s s e r t a t i o n t o my m other and s i s t e r f o r t h e i r en co u rag em en t w hich p la y e d such an im p o r ta n t p a r t i n th e com ple­ t i o n o f my s t u d i e s .

T hese i n v e s t i g a t i o n s w ere a s s i s t e d by a g r a n t from th e O f f ic e o f N aval R e s e a r c h , and w ere c a r r i e d o u t u n d e r t h e d i r e c t i o n o f D r. F .F . N ord.

r

STUDIES ON SOFTWOOD LIGNIN AND THE MECHANISM OF SIGNIFICATION

L

1

r

I,

INTRODUCTION

i

Wood i s p o t e n t i a l l y one o f t h e m ost im p o r ta n t n a t u r a l s o u r c e s o f o rg a n ic conroounds f o r t h e c h e m ic a l i n d u s t r y , f o r i t i s one o f th e fe?/ raw m a t e r i a l s w hich i s c o n tin u o u s ly b e in g r e g e n e r a te d by n a t u r e . M illio n s o f to n s o f wood a r e ..cu t ev e ry y e a r i n a l l p a r t s o f th e w o rld , b u t o n ly a v e ry s m a ll p o r t i o n o f t h i s i s u s e d by th e c h e m ic a l in d u s t r y . T h is p o r t i o n i s c o n v e rte d by th e p u lp i n d u s t r y i n t o c e l l u l o s e , by th e s a c c h a r i f i c a t i o n i n d u s t r y i n t o sim p le s u g a r s , w hich i n t u r n a r e ferm en­ te d t o a l c o h o l and o th e r p r o d u c ts ,a n d f i n a l l y , a s m a ll am ount i s u t i l i z e d i n th e d i s t i l l a t i o n i n d u s t r y .

Of th e t h r e e , o n ly th e l a s t

makes u s e o f th e t o t a l s u b s ta n c e o f th e wood. o f p in e

I n d e e d , th e p r e s e r v a t io n

t r e e s was re c o g n iz e d a s o f econom ic im p o rta n c e on t h i s c o n t i ­

n e n t a s e a r l y a s 1710 ( 6 7 a ) . F o r many d e c a d e s , wood was c o n s id e r e d t o be a u n ifo rm c h e m ic a l compound.

H owever, w ith th e d ev elo p m en t o f o r g a n ic c h e m ic a l te c h ­

n iq u e s o v e r one h u n d red y e a r s a g o , c h e m is ts su cced ed in s e p a r a t i n g wood i n t o i n d i v i d u a l co m p o n en ts.

The f i r s t s e r io u s a tte m p t t o s tu d y

th e c h e m ic a l - c o m p o sitio n o f th e c e l l w a ll o f woody p l a n t s was made in 18S8 by

A nselm e Payen ( 6 7 b ) . He t r e a t e d wood a l t e r n a t e l y

w ith n i t r i c

a c id and c a u s t i c s o d a , and o b ta in e d c e l l u l o s e f i b e r s , w hich h e c o n s id ­ e re d to be th e b a s ic s u b s ta n c e o f th e c e l l membrahe.

He fo u n d , how­

e v e r , t h a t a n o th e r m a t e r i a l d i f f e r i n g g r e a t l y from th e c e l l u l o s e was removed d u r in g t h i s t r e a t m e n t .

T h is m a t e r i a l , w hich he f i r s t r e f e r r e d

t o a s ’’m a tie r e i n c r u s t a n t e ’1, h ad a c o n s id e r a b ly h ig h e r c a rb o n c o n te n t a n d 'a somewhat h ig h e r h y d ro g e n c o n te n t th a n th e c e l l u l o s e . d a l l e d th e s u b s ta n c e " l i g n i n 11, L-

He l a t e r

" _j

2

As a r e s u l t o f th e d ev elo p m en t o f th e wood p u lp in g i n d u s t r y , w hereby la rg e - q u a n t i t i e s o f th e " l i g n i n " w ere o b ta in e d a s a b y - p r o d u c t i n th e form o f ns u l f i t e ’1 and " b la c k ” l i q u o r s , i t was l o g i c a l t h a t a u s e sh o u ld be so u g h t f o r th e s u b s ta n c e , and t h u s , P e t e r K laso n i n Sweden i n i t i a t e d th e f i r s t in v e s tig a tio n s o f " lig n o s u lfo n ic a c id ” .

I n 1893, h e b eg an

h i s s e r i e s o f c l a s s i c a l p u b l i c a t i o n s on l i g n i n , K laso n fo u n d t h a t c o n i f e r y l a l c o h o l , u n d e r th e c o n d itio n s o f th e s u l f i t e p u lp in g p r o c e s s , gave a s u l f o n i c a c id w hich p o s s e s s e d p r o p e r ­ t i e s vehy s i m i l a r t o th o s e o f l i g n o s u l f o n i c a c i d , and h e was th u s th e f i r s t t o s u g g e s t t h a t a p h e n y lp ro p a n e d e r i v a t i v e o f th e c o n i f e r y l ty p e m ig h t be th e p a r e n t s u b s ta n c e o f l i g n i n ( 4 9 ) .

He a l s o d e v elo p ed th e

f i r s t m ethod f o r th e d i r e c t d e te r m in a tio n o f l i g n i n i n woody s u b s ta n c e , and was th e f i r s t t o i s o l a t e l i g n i n w ith 72% s u l f u r i c a c i d , a p r o c e s s w hich fo rm s th e b a s i s f o r th e p r e s e n t s ta n d a r d l i g n i n d e t e r m in a tio n . C o n s id e rin g th e n th e trem en d o u s am ount o f wood w hich g o es to th e p u lp m i l l s a l l o v e r th e w o rld e v e ry d a y , and c o n s id e r in g t h a t a b o u t o n e q u a r t e r o f t h i s i s o b ta in e d a s a l i g n i n b y -p ro d u c t and i s d is c a r d e d , i t i s n o t s u r p r i s i n g t h a t th e p u lp and w o o d - s a c c h a r if i c a ti o n i n d u s t r i e s a r e o* th is c o n s t a n t l y a tte m p tin g t o make u s e J o ^ -i t c l w aste l i g n i n J th e p u lp i n d u s tr y h a s a tte m p te d w ith some s u c c e s s t o u t i l i z e a s m a ll p a r t o f i t s s p e n t l i q u o r s i n b i n d e r s , a d h e s iv e s , b r i q u e t t i n g a g e n ts and s y n t h e t i c r u b b e r s . A s m a ll amount o f th e l i g n i n i n s u l f i t e w aste l i q u o r , how ever, i s c o n v e r­ t e d i n t o one h ig h - g r a d e c h e m ic a l, n am ely , v a n i l l i n . The p r i n c i p a l r e a s o n why c o m p le te u t i l i z a t i o n o f l i g n i n h a s n o t y e t b een a c h ie v e d i s . t h a t i t s c h e m ic a l s t r u c t u r e i s v i r t u a l l y unknown.

A l­

th o u g h o v e r one h u n d re d y e a r s h av e p a s s e d s in c e th e d is c o v e r y o f l i g n i n L_

-I

3 r

i n woody m a t e r i a l s , and a lth o u g h a trem en d o u s am ount o f work h a s been

i

done on th e s u b s ta n c e , l i g n i n c h e m is ts a r e s t i l l f a r . f r o m th e e l u c i d a ­ t i o n o f th e s t r u c t u r e o f th e m a t e r i a l . Due t o o u r fra g m e n ta ry know ledge o f t h e s t r u c t u r e o f l i g n i n , i t i s v ery d i f f i c u l t t o d e f in e i t .

T h ere i s l i t t l e

d o u b t t h a t l i g n i n b e lo n g s

t o a c l a s s o f compounds c o m p le te ly d i f f e r e n t from t h e p o ly s a c c h a r id e s , o r t h a t i t i s a ro m a tic i n n a t u r e .

T h at p o r t i o n o f th e wood c a l l e d

" l i g n i n ” can g e n e r a lly be s e p a r a te d from th e o t h e r com pohents o n ly by s tr o n g r e a g e n t s w hich u n d o u b te d ly change th e l i g n i n .

T h is f a c t i s one

o f th e r e a s o n s why l i g n i n p r e p a r a t i o n s i s o l a t e d by d i f f e r e n t m ethods v a ry so i n t h e i r c o m p o s itio n s . The te rm " l i g n i n " t h e r e f o r e c a n n o t be c o n s id e r e d a s th e d e s ig n a tio n o f a. c h e m ic a lly d e f in e d compound, b u t i s r a t h e r a c o l l e c t i v e te rm f o r a group o f h ig h m o le c u la r , am orphous compounds w hich a r e v e ry c l o s e l y r e ­ l a t e d c h e m ic a lly i n th e same way a s a r e many o th e r n a t u r a l h ig h m o le c u la r p r o d u c ts , such a s c e l l u l o s e , ' th e h e m ic e l lu lo s e s , o r s t a r c h . I n th e l i g h t o f p r e s e n t know ledge o f l i g n i n c h e m is tr y , l i g n i n may be d e f in e d a s t h a t i n c r u s t i n g m a t e r i a l o f a p l a n t w hich i s b u i l t up m a in ly o f p h e n y lp ro p a n e b u i l d i n g s to n e s , w hich c o n ta in s th e m a jo r p a r t o f th e m e th o x y l c o n te n t o f th e p l a n t , w hich i s u n h y d ro ly z a b le by a c i d s , b u t w hich i s r e a d i l y o x i d i z a b l e , and s o lu b le i n h o t a l k a l i and b i s u l ­ fite

(11). U n lik e c e l l u l o s e , l i g n i n n e v e r o c c u rs a lo n e in n a t u r e , b u t i s alw ay s

c o e x i s t a n t w ith c e l l u l o s e o r some o th e r c a r b o h y d r a te .

A lth o u g h l i g n i n

a p p e a rs t o be p h y s i c a l l y a s s o c i a t e d w ith th e s e c a rb o h y d ra te 's , t h i s d oes n o t mean t h a t th e l i g n i n and c a r b o h y d r a te s form a c h e m ic a l compound in L th e wood, and in d e e d , j u s t th e o p p o s ite seems to be th e c a s e (5 9 a ;6 3 )._ ,

r

~i D u rin g th e grow th o f a woody t i s s u e , c e l l u l o s e i s form ed f i r s t . Then, th e fo rm a tio n o f l i g n i n b e g in s , a p r o c e s s c a l l e d nl i g n i f i c s t i o n n . I n t h i s p r o c e s s , th e s p a c e s betw een t h e c e l l u l o s e f i b e r s become f i l l e d in w ith l i g n i n .

The p u rp o s e o f l i g n i f i c a t i o n seems t o be tw o - f o ld .

It

nC em entsn th e c e l l u l o s e f i b e r s t o g e t h e r , and a t th e same tim ie, i t s t i f f ­ e n s th em , th e re b y p r o t e c t i n g them from p h y s i c a l dam age.

T h u s, th e com­

p l e t e l y l i g n i f i e d f i b e r no lo n g e r p la y s an a c t i v e r o l e i n th e l i f e o f th e p l a n t , b u t r a t h e r , i t

s e r v e s a s t r u c t u r a l p u rp o s e .

The rem o v al from wood o f th e c e l l u l o s e and o th e r c a r b o h y d r a te s by th e a c t i o n ‘o f c e llu lo s f c - d e ^ r a d in g w o o d -d e s tro y in g fu n g i h a^b een s u g g e s / tecl (4 0 , 65)\ a s a p° s s i b l e m ethod by w hich L ignin i n i t s n a t u r a l form ■"A

m ig h t be i s o l a t e d , bn c o n t r a s t to th o s e p r e p a r a t i o n s w hich a r e o b ta in e d , by c h e m ic a l m eth o d s, and w hich r e p r e s e n t l i g n i n o n ly i n a m o d ifie d form . The i s o l a t i o n o f l i g n i n by such m eans, t o g e t h e r w ith a s tu d y o f th e in te r m e d ia r y m e tab o lism o f one o f th e o rg a n ism s em ployed, c o n s t i t u t e th e s u b je c t-m a tte r of t h i s t h e s i s .

5

r

II.

&.

EXP ERIMENTAL .METHODS

MICROBIOLOGICAL METHODS. The w o o d -d e s tro y in g f u n g i u s e d i n th e s e i n v e s t i g a t i o n s w ere th e

fo llo w in g B a s id io m y c e te s : 1.

C o n io p h o ra c e r e b e l l a

2.

Fomes r o b u s tu s

5.

L e n tin u s le n id e u s

4.

L e n z ite s s e p ia r ia

5.

P o ria v a i l l a n t i i

O rg an ism s 1 , 5 , 4 and

5w ere o r i g i n a l l y , o b ta in e d

B o ta n ic a l G arden th ro u g h th e c o u r te s y

of

from t h e ^ew York

D r. W illia m J . R o b b in s .

Ho.

2

was r e c e iv e d from th e MC e n tr a a lb u r e a u v o o r S c h im m e lc u ltu re s l,, B a a rn , H o lla n d . I n a d d i t i o n , th e c e l l u l o l y t i c A scom ycete, M vrothecium v e r r u c a r i a . was em ployed, and was r e c e iv e d from th e A m erican Type C u ltu r e C o l l e c t i o n , ?/a.sh in g to n , D.C. S to c k c u l t u r e s o f a l l s i x o rg a n ism s w ere m a in ta in e d on a s o l i d nu­ t r i e n t medium o f t h e f o llo w in g c o m p o sitio n : G lu c o s e

2 0 .0

gms.

H e o p e p to n e..................................

1 .0

gm.

KH2 PO4 . . . . . .............................................. 1 .5 gms. MgS04 .7H 20 ..........................

0.5* gm.

T hiam ine h y d r o c h lo r id e

2 .0

mgs

2 0 .0

gms

D if c o - B a c to - a g a r Tap w a te r t o

1 0 0 0 .0

ml

_]

The s to c k c u l t u r e s w ere c u l t i v a t e d on 50 m l. p l a t e s o f t h i s medium i n 125 m l. E rlen m ey er f l a s k s , and w ere in c u b a te d i n t h e d a rk a t 27°C. They w ere r e g u l a r l y t r a n s p l a n t e d a t i n t e r v a l s o f one m onth. L iq u id m ed ia, f o r e x p e rim e n ta l p u r p o s e s , h ad t h e same n u t r i e n t com­ p o s i t i o n a s th e a b o v e , b u t w ith t h e s o l i d i f y i n g a g a r o m itte d .

I n th o s e

e x p e rim e n ts i n w hich t h e d i s s i m i l a t i o n o f some c a rb o n s o u rc e o th e r th a n g lu c o s e was u n d e r i n v e s t i g a t i o n , t h e s u g a r was o m itte d from th e mddium and was r e p la c e d by t h e i n d i c a t e d am ount o f th e new s u b s t r a t e . M edia w ere s t e r i l i z e d u n d e r 15 l b s . p r e s s u r e a t 1 2 0 ° C ., u n le s s s t a t e d o th e r w is e , and each f l a s k was i n o c u l a t e d w ith a s p o r e - m y c e lia l s u s p e n s io n o f t h e o rg a n ism u n d e r i n v e s t i g a t i o n by means o f a s t e r i l e s y r in g e . E x p e rim e n ta l f l a s k s w ere a l s o in c u b a te d a t 2 7 ° C ., and a e r o b ic con­ d i t i o n s w ere u s e d th r o u g h o u t.

P r e p a r a t i o n . S t e r i l i z a t i o n and I n o c u la tio n o f Wood S am p les:

I'h e wood

s p e c ie s i n v e s t i g a t e d i n th e s e - e x p e r im e n ts weae w h ite S c o ts p in e ( P in u s s v l v e s t r i s ) and w h ite f i r ('A b ie s c o n c o lo r ) , b o th o b ta in e d th ro u g h th e c o u r te s y o f D r. L. C. Sw ain o f th e D ep artm en t o f F o r e s tr y o f th e U n iv e r­ s i t y , Durham, N. H. The wood sam p les w ere f i r s t c le a n e d o f t h e i r b a r k , e t c . , and th e n gro u n d t o 60 mesh i n a W iley m i l l .

Ten gram sam p les o f t h e saw d u st w ere

f/eig h ed i n t o each o f S e v e r a l 500 m l. F e rn b a e h -ty p e c u l t u r e f l a s k s , and t o each f l a s k was added a 25 m l. p o r t i o n o f th e n u t r i e n t medium o f th e above c o m p o s itio n , w ith g lu c o s e o m itte d . The f l a s k s w ere p lu g g e d w ith c o t to n , an d i n o r d e r t o a v o id th e rm a l d e s t r u c t i o n o f th e wood, w ere s t e r i l i z e d w ith s tre a m in g steam a t 1 0 0 °C ., f o r 50 m in . on t h r e e s u c c e s s iv e d ay s ( i . e . , s t e r i l i z a t i o n by T y n d a l l i z a tio n ).

A f te r c o o lin g , ea c h f l a s k was in o c u la te d w ith a 5 m l. sp o re

7 r s u s p e n s io n o f one o f t h e o rg a n ism s i n d i c a t e d a b o v e.

The in o c u la te d

n

f l a s k s w ere in c u b a te d i n th e , d a rk a t 2 7 ° C ., and th e p r o g r e s s o f d ecay was fo llo w e d by p e r i o d i c a n a ly s e s o f t h e v/ood r e s i d e s . B.

ANALYTICAL METHODS. 1.

Wood A n a ly s is :

The 60 mesh wood sam p les o r r e s i d u e s w ere a n a ly z e d

in d u p l i c a t e f o r t h e i r c e l l u l o s e and l i g n i n

c o n t e n ts , o r a l k a l i s o l u b i l i ­

t i e s , and a l l r e s u l t s ( e x c e p t p e r c e n ta g e o f

m o is tu r e ) a r e c a l c u l a t e d and

r e p o r t e d on th e b a s i s o f th e v /eig h t o f t h e o v e n -d ry m a t e r i a l . &) M o is tu r e : was d e te rm in e d g r a v i m e t r i c a l l y , by d r y in g t o c o n s ta n t w e ig h t i n an oven a t lOQoC. ( 1 8 ) . h) A l k a l i S o l u b i l i t i e s : One p e r c e n t HaOH s o l u b i l i t y was d e te rm in e d by h e a t i n g th e sam ple w ith 100 m l. o f 1 % NaOH i n a b a th o f b o i l i n g w a te r f o r one h o u r ( 1 8 ) .

Ten p e r c e n t KOH s o l u b i l i t y was d e te rm in e d

in g t h e sam ple w ith 100

m l. o f 10 % KOH f o r 3 h o u rs ( 1 8 ) .

c) L ig n in : was d e te rm in e d by f i r s t rem oving 11e x t r a c t i v e s ” i n l e t a p p a r a tu s w ith

by r e f l u x -

a S oxh-

95 %a l c o h o l, th e n yyith a 1 :2 a l c o h o l- b e n z e n e •s o lu ­

t i o n , and th e n h y d r o ly z in g th e r e s i d u e , f i r s t w ith 40 m l. o f 72 $HgS0^ a t 20°C. f o r 2 h o u r s , th e n w ith 1543 m l. o f 3 % HgS0 4 f o r 3 h o u rs u n d e r r e f l u x , and w e ig h in g t h e r e s i d u e ( 1 8 ) . d) C e l l u l o s e : was d e te rm in e d by h y d r o ly z in g t h e sam ple w ith 10 m l. o f 80 % HgSO^ f o r 2§ h o u r s a t room te m p e r a tu r e , th e n d i l u t i n g w ith 150 m l. o f d i s t i l l e d w a te r and a u to c la v in g a t 15 l b s . f o r one h o u r .

The s o l u t i o n

was f i l t e r e d , n e u t r a l i z e d w ith d i l u t e a l k a l i , an d d i l u t e d t o volume ( 9 0 ) . The p e r c e n t c e l l u l o s e i n th e sam ple was th e n d e te rm in e d by e s ti m a t in g t h e am ount o f g lu c o s e i n an a l i q u o t o f th e above s o l u t i o n by th e U n if ie d Method, o f Munson an d W alker ( 1 9 ) .

8

2.

M etab o lism A n a ly s e s i

&) M y c e lia l W e ig h ts : w ere d e te rm in e d by f i l t e r i n g th e c o n te n ts o f from 4 t o 8 f l a s k s th ro u g h t a r e d alundum c r u c i b l e s and w ash in g w ith d i s ­ t i l l e d w a te r ; th e c r u c i b l e s w ere d r i e d t o c o n s ta n t w e ig h t i n an oven a t 60°C . an d re w e ig h e d . b) G lu c o s e : was d e te rm in e d by a n a ly z in g an a l i q u o t o f f i l t r a t e by th e U n if ie d M ethod o f Munson and W alker ( 1 9 ) . c) E th y l A lc o h o li was d e te rm in e d by th e t i t r i m e t r i c m ethod o f J a n k e and K ro p acsy ( 4 3 ) , a f t e r p o s s i b l e i n t e r f e r i n g s u b s ta n c e s h ad been r e ­ moved ( 8 8 ) . d) O x a lic A cid: was d e te rm in e d by t i t r a t i o n w ith p erm a n g an ate ( 6 ) . e)

oC- K e t o g l u t a r i c A cid : was d e te rm in e d by th e c o l o r i m e t r i c 2 , 4 - d i -

n itr o p h e n y lh y d r a z o n e m ethod o f V illa n o and L a n g e lla ( 8 9 ) . f ) S t e r o l : was d e te rm in e d by e x t r a c t i n g g round sam p les o f n y c e liu m w ith c h lo ro fo rm f o r 24 h o u r s i n S o x h le t e x t r a c t o r s .

A liq u o ts o f t h e

e x t r a c t w ere s u b je c te d t o th e L ieb erm a n n -B u rc h ard r e a c t i o n , and th e i n ­ t e n s i t y o f c o lo r d e v elo p m en t m easu red i n th e E velyn c o l o r i m e t e r ( 2 9 ) , and com pared w ith s ta n d a r d v a lu e s ( 3 0 ) . g)

pH :

o f m edia w ere d e te rm in e d u s in g a C am bridge e l e c t r o n - r a y pH

m e te r .

C.

PREPARATIVE METHODS. 1.

C h e m ic a lly O b ta in e d L ig n in s :

fre e d o f " e x tra c tiv e s ”

I n a l l c a s e s , th e wood was f i r s t

by e x t r a c t i n g th e saw d u st i n a S o x h le t a p p a r a tu s ,

f i r s t w ith a 1 : 2 a lc o h o l- b e n z e n e s o l u t i o n , and th e n w ith w a te r . a ) 72 % S u l f u r i c A cid L ig n in :

T h i r t y grams o f e x t r a c t i v e - f r e e 60

mesh w h ite S c o ts p in e saw d u st w ere t r i t u r a t e d w ith 600 m l. o f 72 %t L.

_j

9

HgSC^ f o r 2 h o u rs*

T h e^ m ix tu re was t r a n s f e r r e d t o an E rlen m ay e r f l a s k ,

d i l u t e d w ith 3 l i t e r s o f w a te r , and r e f l u x e d f o r 5 h o u r s .

The r e s id u e

was f i l t e r e d and ?/ashed w ith h o t w a te r u n t i l th e f i l t r a t e was no lo n g e r a c i d , and d r ie d ( 1 8 ) . b) 70 % S u l f u r i c A cid L ig n in :

E ig h t gram s o f e x t r a c t e d saw dust w ere

added t o 80 m l. o f 70 % HgSO^ a t 1 0 ° C ., and t h e r e a c t i o n was k e p t a t 10°C . f o r 17 h o u r s .

The m ix tu r e was p o u re d i n t o a f l a s k c o n t a in in g 2 l i t e r s

o f c o ld w a te r , and th e n d i l u t e d t o 3 l i t e r s .

The s o l u t i o n was b o il e d

u n d e r r e f l u x f o r 4 h o u r s , f i l t e r e d , w ashed w ith h o t w a te r u n t i l th e wash­ in g s w ere no lo n g e r a c i d , and d r ie d ( 8 1 ) . c) Fuming H y d r o c h lo r ic ^ c id L ig n in : Ten grams o f saw d u st were m ixed w ith 200 ml* o f fum ing HC1 (3 ) a t 0 °C . t o s ta n d w ith o u t c o o lin g f o r 2 ha?s.

The m ix tu re was shaken and a llo w e d

S i x t y - f i v e m l. o f ic e - w a te r w ere

added i n s e v e r a l p o r t i o n s , and th e m ix tu r e a llo w e d t o s ta n d a t room tem p­ e r a t u r e f o r 18 h r s .

I t was d i l u t e d w ith 65 m l. o f w a te r , f i l t e r e d , and

th e r e s i d u e w ashed w ith 1 :1 HC1 s o l u t i o n and w ith w a te r .

The s o l u t i o n

was n e t r a l i z e d w ith d i l . MaOH and b o i l e d f o r 10 m in. Ah e f i l t e r e d p r o d u c t was b o i l e d f o r 10 m in . w ith w a te r , f i l t e r e d and d r i e d ( 4 6 ) . d) A l k a l i L i g n in :

Ten gram s o f e x t r a c t e d saw dust w ere m ixed w ith

200 m l. o f 10 % aq u eo u s NeDH, and a u to c la v e d a t 125°C . f o r 8 h o u r s .

The

u n d is s o lv e d r e s i d u e was f i l t e r e d o f f , and t h e a l k a l i n e h y d r o ly z a te a c i d ­ i f i e d w ith d i l u t e HC1.

The r e s u l t i n g p r e c i p i t a t e was f i l t e r e d , d r i e d ,

end p u r i f i e d by d i s s o l v i n g i n a c e to n e , p r e c i p i t a t i n g i n t o e t h e r , f i l t e r ­ in g and d iy i n g ( 5 5 , 7 0 ).

2.

D e r i v a t i v e s o f H a tiv e L ig n in :

The i s o l a t i o n o f n a t i v e l i g n i n

w i l l be d e s c r ib e d l a .t e r i n t h i s t h e s i s . i_

_j

10

r

a) A c e ta te :

275 m gs. o f n a t i v e l i g n i n w ere d is s o lv e d i n 5 m l. o f n

p y r i d i n e , an d 5 .5 m l. o f a c e t i c a n h y d rid e w ere ad d ed .

The s o l u t i o n was

a llo w e d t o s ta n d f o r 48 h o u r s and was th e n f i l t e r e d o f f , th e p r e c i p i t a t e was w ashed w ith w a te r , an d d r i e d .

The p r o d u c t was d is s o l v e d in d io x a n e ,

p r e c i p i t a t e d i n t o d ry ' e t h e r tw ic e , an d d r i e d ( 1 0 ) . b) P h e n v lh d ra z o n e :

255 mgs. o f n a t i v e l i g n i n w ere d is s o lv e d i n 5

m l. o f d io x a n e , and one m l. o f p h e n y lh y d ra z in e was ad d e d .

The m ix tu re

was h e a t e d u n d e r r e f l u x a t 80°C . f o r 2 h o u r s , and th e n l e t s ta n d o v e r­ n ig h t.

The s o l u t i o n was f i l t e r e d i n t o d ry e t h e r , th e p r e c i p i t a t e f i l ­

t e r e d , d i s s o lv e d i n t o d io x a n e , and p r e c i p i t a t e d i n t o

. e t h e r u n t i l a lemon

y e llo w p r o d u c t was o b ta in e d ( 1 0 ) .

5.

D e r iv a t iv e s o f E r g o s t e r o l :

The i s o l a t i o n o f e r g o s t e r o l from th e

m ycelium o f L e n tin u s le o id e u s w i l l b e d e s c r ib e d l a t e r i n t h i s t h e s i s . a ) A c e ta te : 100 mgs. o f t h e s t e r o l w ere ad d ed t o 5 m l. o f a c e t i c a n h y d r id e , an d a t r a c e o f ZnClg was a d d ed . a b o i l i n g w a te r b a th f o r one h o u r .

The m ix tu re was r e f l u x e d i n

On c o o lin g , a p r e c i p i t a t e c r y s t a l l i z e d

w hich was f i l t e r e d o f f , w ashed w ith 80 % a c e to n e , and r e c r y s t a l l i z e d tw ic e from a 1 :3 e t h e r - a c e t o n e s o l u t i o n ( 7 1 ) . b) B e n z o a te :

100 mgs. o f t h e s t e r o l w ere d i s s o lv e d i n 5 m l. o f p y r i ­

d in e , and 0 .5 m l. o f b e n z o y l c h l o r i d e was a d d e d .

The s o l u t i o n was h e a te d

t o b o i l i n g and p o u red i n t o 1 0 m l. o f w a te r , w hereupon a p r e c i p i t a t e was d e p o s it e d .

was f i l t e r e d o f f , washed w ith 5 % aq u eo u s Ite^COg s o l u t i o n

and w ith w a te r , and r e c i y s t a l l i z e d tw ic e from a b s o lu t e a l c o h o l ( 7 1 ) .

4 . ( A - K e to g lu ta r ic t c i d : T h is compound was p r e p a r e d by a more, r e c e n t m o d if ic a tio n (2 4 ) o f th e o r i g i n a l p ro c e d u re o f N euberg and R in g e r ( 5 7 ) . L. I t h a s a m e ltin g p o in t o f 1 1 0 ° C ., and gave th e f o llo w in g a n a l y s i s :

-*

11 r Found#

D.

C a lc , f o r C5 H0 O5

C

41.25%

41.09%

H

4.20%

4.11%

SPECTROSCOPIC METHODS. 1.

U l t r a v i o l e t A b s o rp tio n S p e c tr a o f L ig n in s and D e r i v a t i v e s :

S o lu ­

t i o n s o f th e l i g n i n sam p les w ere p r e p a r e d f o r s p e c tr o s c o p i c a n a l y s i s byd i s s o l v i n g fro m 3 t o 5 m gs. o f th e sam ple i n 100 m l. o f s o lv e n t (90 p a r t s p u r i f i e d d io x a n e t o 10 p a r t s d i s t i l l e d ?,rater) (57) and d i l u t i n g a s re q u ire d .

The u l t r a v i o l e t a b s o r p ti o n c u rv e s w ere d e te rm in e d w ith th e

BecKman q u a r tz s p e c tr o p h o to m e te r ( 4 ) .

2.

U l t r a v i o l e t A b s o rp tio n S pectrum o f E r g o s te r o l :

S o lu ti o n s o f th e

s t e r o l w ere p r e p a r e d f o r s p e c tr o s c o p i c a n a l y s i s by d i s s o l v i n g a p p ro x . 5 mgs. o f t h e sam ple i n 50 m l. o f a b s o lu t e e t h y l a lc o h o l (7 ) and d i l u t i n g a s re q u ire d .

The u l t r a v i o l e t a b s o r p tio n c u r v e s w ere d e te rm in e d on th e

Beckman q u a r tz s p e c tr o p h o to m e te r .

#The m ie r o - a n a ly s e s r e p o r t e d i n t h i s t h e s i s w ere c a r r i e d o u t by M. B ie r , F . B flh ler and A.A. S ir o te n k o , a l l o f t h i s D e p a rtm e n t, and by %■. J . F . A l i c i n o , o f th e S q u ib b I n s t i t u t e f o r M e d ic a l R e s e a rc h . L.

-1

12

r

III.

A.

EXPERIMENTAL RESULTS

STUDIES ON LIGNIN

Much o f th e d i f f i c u l t y in v o lv e d i n s tu d y in g th e c h e m is tr y o f l i g n i n can be a t t r i b u t e d t o t h e f a c t t h a t , u n t i l r e l a t i v e l y r e c e n t l y , no method was k»own by Yvhich l i g n i n c o u ld be i s o l a t e d i n an u n ch an g ed fo rm ,

f lh a t-

e v e r m ethod was em ployed, a l i g n i n

p r e p a r a t i o n was o b ta in e d w hich was no

lo n g e r i d e n t i c a l w ith l i g n i n a s i t

e x is ts in n a tu re .

..The s o l u t i o n t o th e p ro b lem o f i s o l a t i n g l i g n i n i n i t s n a t u r a l s t a t e t h e r e f o r e r e q u i r e s a m ethod o f e x t r a c t i o n t h a t a v o id s d r a s t i c c o n d i t i o n s , and em ploys n e u t r a l , c h e m ic a lly i n e r t s o l v e n t s .

Brauns* e x t r a c t i o n o f

" N a tiv e L ig n in n from b la c k s p ru c e ( 1 0 ) , w e s te rn hem lock (1 2 ) and a sp e n (2 0 ) w ith 95 % e t h y l a l c o h o l a t room te m p e r a tu r e m et th e s e r e q u ir e m e n ts f o r th e f i r s t ti m e .

H owever, th e f a i l u r e o f a p p ro x im a te ly 97 % o f th e l i g n i n

p r e s e n t t o be so e x t r a c t e d s u g g e s te d t h a t e i t h e r t h i s N a tiv e L ig n in was n o t i d e n t i c a l w ith th e b u lk o f th e l i g n i n o f th e wood, o r t h a t th e u n e x t r a c t a b l e l i g n i n was a s s o c i a t e d e i t h e r c h e m ic a lly o r p h y s i c a l l y w ith th e c e l l u l o s e o f th e wood.

T h u s, th e s e f i n d i n g s in c r e a s e d t h e num erous ex­

i s t i n g s p e c u la tio n s r e g a r d i n g th e n a t u r e o f th e r e s i d u a l l i g n i n and i t s lin k a g e w ith th e c e l l u l o s e i n th e wood ( 9 2 ) . P r i o r t o B ra u n s ’ i s o l a t i o n o f n a t i v e l i g n i n , B a rto n -W rig h t and Bos­ w e ll (5 ) h a d o b ta in e d a l i g n i n p r e p a r a t i o n v e ry s i m i l a r i n c h a r a c t e r t o Brauns* l i g n i n , by e x t r a c t i o n w ith e t h y l a l c o h o l o f N orw egian s p ru c e wood w hich h a d p r e v io u s ly been r o t t e d by th e '’d r y r o t " f u n g u s , M e ru liu s l a c r v m ans. T h u s, th e s e two in d e p e n d e n t f i n d i n g s s u g g e s te d th e p o s s i b i l i t y o f u t i l i z i n g c e l l u l o l y t i c enzyme sy stem s a s a p o s s i b l e m ethod f o r o b ta in in g

15

r a d d i t i o n a l am ounts o f an u n a l t e r e d l i g n i n fro m wood ( 6 5 ) .

~l

S in c e t h e c l a s s i c a l s t u d i e s o f F a lc k ( 2 6 ) , i t i s g e n e r a l l y a c c e p te d t h a t t h e r e a r e two d i f f e r e n t ty p e s o f wood d eca y b ro u g h t a b o u t by f u n g i, nam ely , th e "brow n” and th e " w h ite " r o t s .

I n t h e fo rm e r, p r e f e r e n t i a l

a t t a c k i s made on t h e c a r b o h y d r a te com ponents o f t h e wood, and th e l i g n i n re m a in s u n a f f e c t e d , w ith t h e d e c a y in g r e s id u e t u r n i n g brown i n c o l o r . I n th e seco n d ty p e , l i g n i n seems t o be th e m ain s u b s t r a t e o f th e fu n g u s , and i n th e r e s i d u e , t h e r e a r e p a tc h e s o f a w h ite s u b s ta n c e c o n s id e r e d to be p u re c e l l u l o s e . O bviously* th e n , i f an a t te m p t was to be made t o o b ta in l i g n i n by th e a c t i o n o f f u n g i on wood, o rg a n is m s o f th e f i r s t "group ( i . e . , th e "brovai" r o t s ) m ust be em ployed, s in c e th e s e p o s s e s s a s e l e c t i v e c e l l u l o s e - h y d r o ­ l y z in g enzyme s y ste m , b u t le a v e th e l i g n i n u n a t ta c k e d . 1.

P r e lim in a r y E x p e rim e n ts. I n o r d e r t o d e te rm in e t h e r e l a t i v e d e g r e e s o f w ood-deeay by t h e f o u r

w o o d -d e s tro y in g f u n g i on h a n d , w h ite f i r saw d u st was s u b je c te d t o a ta c k by th e f o llo w in g m o ld s:

C. c e r e b e l l a . L. l e o i d e u s . L. s e p i a r i a and P ,

v a illa n tii. I n view o f t h e c o n s i d e r a t i o n t h a t an i n c r e a s e i n a l k a l i s o l u b i l i t y s e r v e s a s a good in d e x o f c e l l u l o s e d e g r a d a tio n ( 1 7 ) , i t was d e c id e d t o em ploy th e a l k a l i - s o l u b i l i t y m ethod o f a n a l y s i s i n t h i s i n v e s t i g a t i o n . A c c o r d in g ly , a f t e r s i x m onths o f i n c u b a t i o n , t h e fungcCL m y c e lia w ere r e ­ moved from th e d eca y ed saw d u st by d e c a n ta t io n from a w a te r s u s p e n s io n , and t h e r e s i d u a l m a t e r i a l was d r i e d .

The p e r c e n ta g e s o l u b i l i t i e s o f

th e r e s i d u e s in 1% NaOH and \0% KOH w ere d e te rm in e d , an d w ere com pared w ith r e s u l t s on u n ih o c u la te d sam p les a s c o n t r o l s . L.

-J

14

The' r e s u l t s , c o r r e c t e d f o r m o is tu r e c o n t e n ts , a r e p r e s e n te d i n T a b le I .

~1

From th e in c r e a s e d a l k a l i s o l u b i l i t i e s o f th e d ecay ed sam p les

com pared w ith th e u n in o c u la te d c o n t r o l , i t i s o b v io u s t h a t th e s e f u n g i w ere in d e e d d e g ra d in g t h e c e l l u l o s e com ponent o f t h e wood, and a c c o r d in g ­ l y a p p e a re d to be nbrown r o t s ” .

M o reo v er, u n d e r l a b o r a t o r y c o n d i tio n s ,

th e s e o rg a n is m s a t t a c k e d t h e wood i n t h e f o llo w in g o r d e r o f d e c r e a s in g e ffe c tiv e n e s s :

( l ) L. s e o i a r i a and P . v a i l l a n t i i . (2) L . le o id e u s and

(3) C.. c e r e b e l l a . I n o r d e r t o d e te rm in e w h e th e r an a l c o h o l - e x t r a c t a b l e l i g n i n m ig h t be o b ta in e d from d eca y ed wood, th e r e s i d u a l m a t e r i a l was e x t r a c t e d w ith 95% a l c o h o l a t room te m p e r a tu r e f o r sev en d a y s .

Upon e v a p o r a tio n o f

th e s o l v e n t , a cru d e r e s i n o u s p ro d u c t was o b ta in e d w h ich , a f t e r s e v e r a l p r e c i p i t a t i o n s from d io x a n e i n t o w a te r , y i e l d e d a. t a n n i s h pow der w hich gave th e f o llo w in g a n a l y s e s :

C, 6 2 .6 8 $ ;

H, 6 .0 4 $ ; OCHg, 1 2 .7 6 $ .

A c c o r d in g ly , i t a p p e a re d t h a t a l i g n i n - l i f e e s u b s ta n c e c o u ld in d e e d be i s o l a t e d from f u n g a ll y d eca y ed wood.

2.

N a tiv e W hite S c o ts P in e L ig n in . O b v io u s ly , i t was e s s e n t i a l to e s t a b l i s h , f o r c o m p a ra tiv e p u r p o s e s ,

a l i g n i n p r e p a r a t i o n w hich c o u ld s e r v e a s a s ta n d a r d o f r e f e r e n c e , and th e N a tiv e L ig n in o f i r a u n s (.10) was c h o s e n , fcince t h i s s e e m s 'to re s e m b le m ost c l o s e l y l i g n i n " in s i t u ” .

A c c o rd in g ly , th e method o f i s o l a t i o n o f

l i g n i n i s e s s e n t i a l l y th e same asE S rauns* i s o l a t i o n o f n a t i v e b la c k sp ru c e l i g n i n , and i s a s f o llo w s : Sound 60 mesh w h ite S c o ts p in e saw d u st was f i r s t e x t r a c t e d th o ro u g h ly w ith c o ld w a te r and e t h e r .

T hen, th e l i g n i n was i s o l a t e d by e x t r a c t i n g

th e saw d u st w ith 95$ e t h y l a l c o h o l a t room te m p e r a tu r e , i n a p e r c o l a t o r L_

_J

15 r

T a b le I

A lk a li S o l u b i l i t i e s o f W hite F i r Wood a f t e r S ix M onths ^ e c a y by F o u r W o o d -D estro y in g F u n g i

P e rc e n t S o lu b ility in O rganism O n in o c u la te d C o n tr o l '

L

1 $ NaOH

10 % KOH

9 .7

1 6 .0

C.

c e re b e lla

1 0 .4

1 8 .7

L.

le o id e u s

2 7 .1

4 0 .7

L.

s e p ia ria

5 7 .6

4 9 .6

P.

v a illa n tii

5 9 .6

4 8 .7

-J

16

ty p e e x t r a c t o r , w hich w i l l be d e s c r ib e d e ls e w h e re ( 5 8 ) , u n t i l t h e a l c o ­ h o l i c e x t r a c t no lo n g e r re s p o n d e d t o th e p h l o r o g l u c i n o l - t e s t f o r l i g n i n . The a l c o h o l was d i s t i l l e d . o f f a t re d u c e d p r e s s u r e , w hereupon a r e s in o u s m a t e r i a l was o b ta in e d .

T h is was v/ashed w e ll w ith w a te r and e t h e r .

The

r e s u l t i n g pow der was d is s o lv e d i n d io x a n e , and p r e c i p i t a t e d i n t o iioe-w rater. The p r e c i p i t a t e 'was d r i e d , r e d i s s o l v e d in d io x a n e , and p r e c i p i t a t e d i n t o e th e r.

T h is l a t t e r p r e c i p i t a t i o n was r e p e a te d u n t i l th e m e th o x y l c o n te n t

re m a in e d c o n s t a n t .

The l i g n i n so o b ta in e d ¥»as a l i g h t c re a m -c o lo re d pow der.

The p r o d u c t was o b ta in e d i n a y i e l d o f 5.2% o f th e t o t a l l i g n i n , and was c h a r a c t e r i z e d th ro u g h i t s e le m e n ta ry c o m p o s itio n , m e th o x y l c o n t e n t, s o l u b i l i t y , r e d u c in g a b i l i t y , c o l o r r e a c t i o n s and u l t r a v i o l e t a b s o r p tio n s p e c tru m , and th ro u g h th e m e th o x y l c o n te n ts and u l t r a v i o l e t a b s o r p ti o n s p e c t r a o f i t s a c e t a t e and p h e n y lh y d ra z o n e d e r i v a t i v e s .

The a n a l y t i c a l

d a t a and p r o p e r t i e s o f t h e l i g n i n and i t s d e r i v a t i v e s a r e p r e s e n te d in. T a b le ' I I , an d th e t h r e e a b s o r p ti o n s p e c tr a a r e shown i n F ig u r e I .

5.

L ig n in from L e n tin u s l e p i d e u s -d e c a v e d .W hite Scots P in e Wood. Sound sam p les o f 'w hite S c o ts p in e saw dust w ere i n f e c t e d w ith t h e

nbrown r o t ” , L. l e p i d e u s . and th e e f f e c t s o f t h e d ecay o f th e wood by th e o rg an ism w ere p r o g r e s s i v e l y fo llo w e d by c e l l u l o s e and l i g n i n a n a ly ­ se s o f th e r e s id u e s . The r e s u l t s o f th e p e r i o d i c a n a ly s e s o f th e d e c a y in g wood a r e p r e ­ s e n te d i n T a b le I I I .

From t h e s e d a t a , i t i s o b v io u s t h a t th e n e t e f f e c t

o f t h e a c t i o n o f L. le p id e u s on wood i s in d e e d a d e p l e tio n i n th e c e l l u ­ l o s e c o m p o s itio n o f th e wood, c o n c o m ita n t w ith an i n c r e a s e i n th e r e l a t i v e c o n te n t o f l i g n i n .

L_

17

r Table II C h a r a c t e r i z a t i o n o f N a tiv e ^ h i t e ^ c o ts P in e L ig n in

QCH

C

64.0%

H

6.5%

OCH,

14.5%

OCHg o f A c e ta te

10. 1%

o f P h e n y lh y d ra z o n e

1 3 .5 %

S o lu b le i n :

I n s o lu b le i n :

R educing A b i l i t y

E th y l a l c o h o l , M eth y l a l c o h o l , D io x an e, P y r i d i n e , 4% NaOH s o l 1^ , G I. A c e ti c M d W a te r, E t h e r , B enzene, P e tro le u m e f ^ e r

R educes 1

eh liH

s S o lu ti o n

C o lo r R e a c tio n s w ith : P h lo r o g lu c i n o l A n ilin e p -P h e n y le n e d ia m in e D ip h en y lam in e

L-

R e d - v i o le t Y ello w Y ellow Y ellow

-j

F I G- u R E. X NATIVE W HITt 3COTA PINC IIONIN

PMENTLHYOAAIONt

!LOO

f60

I0O

60

J*o

19

r

n

T a b le I I I E f f e c t o f th e A ctio n o f L e n tin u s le o id e u s on th e C o m p o sitio n o f W hite S c o ts P in e Wood P e r io d o f Decay ( i n m onths)

L

C e l lu lo s e $ to

L ig n in P

0

6 1 .7

2 6 .8

4

4 9 .5

5 1 .8

5

4 6 .1

5 7 .4

6

4 6 .0

4 0 .0

7

5 9 .9

4 4 .9

20

T h is " l i g n i n e n r ic h e d " d eca y ed wood t h e r e f o r e seemed t o o f f e r a p o t e n t i a l s o u rc e f o r t h e i s o l a t i o n o f f u r t h e r am ounts o f u n a l t e r e d l i g n i n . As i n d i c a t e d a b o v e, o n ly Z,Z% o f th e l i g n i n c o n te n t o f sound w h ite S c o ts p in e wood can be i s o l a t e d by a l c o h o l i c e x t r a c t i o n .

I t was t h e r e ­

f o r e o f i n t e r e s t to d e te rm in e w h e th e r in c r e a s e d am ounts o f l i g n i n co u ld be so e x t r a c t e d from th e d e c a y e d wood. A c c o rd in g ly , th e w h ite S c o ts p in e -wood sam p les d ecay ed by L. le o id e u s w ere th e n th o ro u g h ly e x t r a c t e d w ith e t h y l a l c o h o l a t room te m p e r a tu r e , and th e y i e l d s o f l i g n i n i s o l a t e d from th e sound and d ecay ed wood sam p les a r e com pared i n T a b le IV . T h u s, a f t e r a sev en month p e r i o d o f d ecay by L . l e o i d e u s . t h e r e was a p p ro x im a te ly a tw o - f o ld i n c r e a s e i n th e y i e l d o f a l c o h o l - e x t r a c t a b l e l i g ­ n in from th e d eca y ed sam ple o v e r and above t h a t o b ta in e d from th e sound wood.

I t was th u s o f im p o rta n c e t o d e te rm in e w h e th e r t h i s l i g n i n , i s o ­

l a t e d i n d o u b le y i e l d from th e d eca y ed wood was s i m i l a r t o o r d i f f e r e n t from t h e n a t i v e l i g n i n i s o l a t e d from t h e sound wood. T h e r e f o re a co m p ariso n was made o f th e l i g n i n i s o l a t e d from th e d e­ cay e d w h ite S c o ts p in e wood w ith t h e n a t iv e l i g n i n o f t h i s wood.

T h is

co m p ariso n in v o lv e d th e a n a l y t i c a l d a t a , p r o p e r t i e s and u l t r a v i o l e t s p e c t r a o f th e l i g n i n j a n d t h e i r d e r i v a t i v e s . The co m p ariso n showed t h a t th e l i g n i n i s o l a t e d from th e d eca y ed w h ite S c o ts p in e wood was a l s o s o lu b le i n m eth y l and e t h y l a l c o h o l s , i n d io x a n e , p y r i d i n e , g l a c i a l a sc e tic a c i d and 4% sodium h y d ro x id e s o l u t i o n , and i n s o l ­ u b le i n w a te r , e t h e r , b en ze n e and p e tro le u m e t h e r .

I t a l s o re d u c e d

F e h l i n g ’ s s o l u t i o n , and gave th e c o rre s p o n d in g c o l o r s ’w ith t h e p h lo r o g lu c i n o l , an d th e a ro m a tic am ine r e a g e n t s . But o f more im p o rta n c e , th e re se m b la n c e e x te n d e d to th e a n a l y t i c a l

21 r

T a b le IV

Y ie ld s o f L ig n in I s o l a t e d from Sound and from L e n tin u s l e p i d e u s -D ecayed Wood

P e r c e n t l i g n i n I s o l a t e d from S p e c ie s o f Wood

W hite S c o ts P in e W hite F i r

L.

Sound Wood

D ecayed Wood

S .2

6 ,9

2*4

4 .9

22

d a t a o f th e l i g n i n s , w hich a r e com pared in T a b le V, and t o th e u l t r a ­ v i o l e t c u r v e s o f th e l i g n i n s , w hich a r e com pared i n F ig u r e I I , and o f t h e i r d e r i v a t i v e s , com pared i n F ig u r e I I I . From th e d a ta and t h e s i m i l a r i t i e s o f th e u l t r a v i o l e t c u rv e s o f th e l i g n i n s and t h e i r d e r i v a t i v e s , i t a p p e a re d t h a t a s f a r a s c o u ld be d e t e r ­ m in ed , t h e n a t i v e l i g n i n o f w h ite S c o ts p i n e , and t h e l i g n i n o b ta in e d from t h i s wood a f t e r d ecay by L . le p id e u s w ere a t l e a s t v e ry s i m i l a r . 4.

L ig n in s from Sound an d L e n tin u s l e p id e u s - d ecay ed W h ite F i r f o o d . I n o r d e r t o i n v e s t i g a t e th e p o s s i b i l i t y t h a t t h e l i g n i n s i s o l a t e d

from w h ite S c o ts p i& ) wood m ig h t r e p r e s e n t a c h e m ic a l e n t i t y w hich i s s i m i l a r i n v a r io u s s p e c ie s o f s o ftw o o d s, a p a r a l l e l co m p ariso n was made o f t h e l i g n i n s o b t a i n a b l e from sound and from L . l e o i d e u s -d e c a v e d w h ite f i r wood. The r e s u l t s o f t h e p e r i o d i c a n a ly s e s o f th e d e c a y in g w h ite f i r wood a r e p r e s e n te d i n T a b le V I, and t h e y i e l d s o f l i g n i n i s o l a t e d from th e sound and d eca y ed sam p les a r e com pared i n T a b le IV . The two l i g n i n s w ere com pared th ro u g h t h e i r m eth o x y l c o n t e n ts and u l t r a v i o l e t s p e c tra .

T h ese m e th o x y l v a lu e s a r e com pared w ith th o s e f o r

th e l i g n i n s from sound and from d ecay ed w h ite S c o ts p in e i n T a b le V II, and t h e i r u l t r a v i o l e t c u rv e s a r e com pared i n F ig u r e IV w ith t h a t o f n a t iv e w h ite S c o ts p in e l i g n i n . From t h e a n a l y t i c a l d a ta ., and th e s i m i l a r i t y o f th e u l t r a v i o l e t s p e c t r a o f th e t h r e e l i g n i n sa m p le s , i t a p p e a re d t h a t th e d eca y ed w h ite f i r l i g n i n was i d e n t i c a l w ith th e n a t i v e l i g n i n o f t h i s s p e c i e s , and m o reo v e r, t h a t th e y w ere b o th v e ry s i m i l a r t o th e n a t i v e l i g n i n o f w h ite S c o ts p i n e .

25

r

T a b le ¥

C om parison o f L ig n in s from Sound and from L e n tin u s le p id e u s D ecayed W hite S c o ts P in e Wood L ig n in from Sound W hite S c o ts P in e

W hite S c o ts P in e

C

6 4 .0 %

6 5 .7 %

H

6 .5 %

6 .3 %

OCH,

1 4 .5 %

1 4 .5 %

10.1 %

1 0 .4 %

1 5 .5 %

1 5 .0 %

0CH5 o f A c e ta te 0CH5 o f P h e n y lh y d ra z one

i_

L ig n in from D ecayed

Fl G-URE.

H

LIGNIN FROM SOUNO WHIT* SCOTS PINE LIGNIN FROn I . LEPf DEl/S OECAYED WHITE SCOTS PINB

I to

I Cm.

ItO

100

EOO

300

FlGrURGl

3H

2, 5

Mo W

9

ACETATE OF tIGWIN FR O li SOUND W S . PIN E

— - B — -ACETATE OF l l G N I N F R o * L. L E P io e u s DECAYED W!S. P IN E

Uo

PHCNYLHYDAA^ONE OF LIGNIN FROM SOUND W 6 . PINE PHENYlHYDAAZONt OF LIGN/N FROM L.LEPIOEUJ DECRYPO W S. PlNB

*00

180

(80

Ito 1% | Ci*« HO

100

80

80

^0

lo

180

300

jlo

380

380

26

T a b le VI

E f f e c t o f th e A ctio n o f L e n tin u s le p id e u s on th e C o m p o sitio n o f W hite j^ ir Wood

L.

P e r io d o f Decay

C e llu lo s e

L ig n in

( i n m onths)

%

%

, 0

6 0 .5

2 4 .9

4

5 1 .2

5 0 .6

5

4 7 .3

5 3 .8

6

46.-5

36.. 4

7

4 5 .8

5 9 .5

27 n

r

T a b le V II

C om parison o f M ethoxyl C o n te n ts o f L ig n in s from Sound an d from L e n tin u s le o id e u s-D ecaved Wood

P e r c e n t M ethoxyl

Sound W hite S c o ts P in e

14.-5

Sound W hite F i r

1 4 .8

D ecayed % i t e S c o ts P in e

1 4 .5

D ecayed W hite F i r

1 4 .5

F» G-URE J5L LI»MN FROM SOUND WHITE SCOTS PINE u g n in f r o m s o u n o w h it e Fi r tIG N IN FRO tt L.LGPIOEUS OtCAYEO WHITE FIR

I3LO

IOO

80

60

40

300

400

29

r I t t h e r e f o r e a p p e a re d t h a t th e s e p r o p e r t i e s w ere c h a r a c t e r i s t i c f o r thfe l i g n i n s o f v a r io u s s o ftw o o d s. 5.

L ig n in L ib e r a te d E x c lu s iv e ly by th e A c tio n o f P o r i a v a i l i a n t i i . I t was a t once o b v io u s t h a t t h e p r e v io u s i n v e s t i g a t i o n s d id n o t p e r ­

m it th e u n e q u iv o c a l s ta te m e n t t h a t t h e p ro d u c t o b ta in e d from th e d ec a y e d wood d id n o t c o n s i s t t o a c e r t a i n e x t e n t o f th e n a t i v e l i g n i n e x i s t i n g i n t h e wood p r i o r t o t h e e n zy m atic d e c a y , f o r th e n a t i v e l i g n i n m ust b e p r e s ­ e n t i n th e a l c o h o l i c e x t r a c t o f t h e d eca y ed r e s i d u e , t o g e t h e r w ith t h e l i g ­ n in l i b e r a t e d by t h e en z y m a tic a c t i o n .

A c l e a r - c u t s e p a r a t io n o f th e s e

two l i g n i n f r a c t i o n s w i l l now be m ade, and t h e i r i d e n t i t y c o n firm e d . I t a p p e a re d t h a t t h e above a m b ig u ity co u ld be c l a r i f i e d by f i r s t ex­ t r a c t i n g th e wood t o be s u b je c te d to d eca y w ith a l c o h o l u n t i l a l l th e n a ­ t i v e l i g n i n was c o m p le te ly rem oved.

The n a t i v e - l i g n i n - f r e e wood c o u ld

th e n be d ecay ed by th e f u n g i, and a f t e r a s u i t a b l e p e r i o d o f d e c a y , th e wood r e s i d u e c o u ld be r e - e x t r a c t e d w ith a l c o h o l .

T h is e x t r a c t th e n w ould

o f n e c e s s i t y c o n ta in o n ly t h a t l i g n i n w hich was l i b e r a t e d from i t s a s s o ­ c i a t i o n i n th e wood by th e e n z y m a tic a c t i v i t y , and m ust be f r e e -of n a t i v e lig n in . The s p e c ie s o f wood i n v e s t i g a t e d i n th e s e e x p e rim e n ts was w h ite S c o ts p i n e , and t h e T,bro?m r o t 1’ o rg a n is m em ployed t o e f f e c t th e d eca y was P o r ia v a illa n tii. As i n d i c a t e d i n T a b le I ? , 3 .2 $ o f th e l i g n i n c o n te n t o f sound w h ite S c o ts p in e wood can be e x t r a c t e d w ith a l c o h o l .

T h is N a tiv e L ig n in was

c o m p le te ly rem oved from a sam ple o f t h i s s p e c ie s o f wood, and i s c h a r a c t e r ­ i z e d i n T a b le I I .

The n a t i v e - l i g n i n - f r e e wood was th e n s u b je c t e d t o decay

fcy £ • v a i l l a n t i i f o r a p e r io d o f f i f t e e n m o n th s, and th e e f f e c t s o f th e

50

r d ecay on th e c h e m ic a l c o m p o sitio n o f th e wood a r e p r e s e n te d in T a b le

fan.

The d eca y ed sam p les w ere th e n th o ro u g h ly e x t r a c t e d w ith e t h y l a lc o h o l a t room te m p e r a tu r e .

As a r e s u l t o f th e g r e a t e r !,l i g n i n e n ric h m e n t" o f

t h e wood, th e y i e l d o f l i g n i n i s o l a t e d from th e P . v a i l l a n t i i - d ecav ed w h ite S c o ts p in e wood now am ounted t o 22,7% o f th e r e s i d u a l l i g n i n .

T h u s,

a f t e r a p r o lo n g e d p e r io d o f d eca y by a c e l l u l o s e - d e g r a d i n g w o o d -d e s tro y in g o rg a n ism , t h e r e i s a s u b s t a n t i a l i n c r e a s e i n th e y i e l d o f a l c o h o l - e x t r a c t a b l e l i g n i n o b t a i n a b l e from th e d ecay ed wood. I t was th e n o f u tm o s t im p o rta n c e t o d e te rm in e w h e th e r t h i s l i g n i n , l i b e r a t e d from i t s a s s o c i a t i o n i n th e wood e x c l u s i v e l y ty th e e n zy m atic a c t i v i t y o f th e f u n g u s , was i n f a c t i d e n t i c a l w ith , o r d i f f e r e n t from , t h e n a t i v e l i g n i n i s o l a t a b l e fro m sound wood. T h e r e f o re a co m p ariso n was made o f th e l i g n i n l i b e r a t e d from w h ite S c o ts p in e wood e x c l u s i v e ly by t h e o rg an ism , P . v a i l l a n t i i . w ith th e n a ­ t i v e lig n in o f t h i s s p e c ie s . T h is co m parison, a l s o in v o lv e d th e e le m e n ta ry c o m p o s itio n s , m eth o x y l c o n t e n t s , s o l u b i l i t i e s , r e d u c in g a b i l i t y , c o l o r r e a c t i o n s and u l t r a v i o l e t s p e c t r a o f t h e l i g n i n s , and t h e m eth o x y ls an d u l t r a v i o l e t s p e c t r a o f th e a c e t a t e s and p h e n y lh y d ra z o n e s . The r e s u l t s showed t h a t t h e l i g n i n l i b e r a t e d e x c l u s i v e ly by th e a c t io n o f P . v a i l l a n t i i . l i k e th e n a t i v e l i g n i n o f w h ite S c o ts p in e , and th e l i g ­ n i n o b ta in e d a f t e r d ecay by L. l e p i d e u s . was s o lu b le in m e th y l and e t h y l a l c o h o l s , i n d io x a n e , p y r i d i n e , g l . a c e t i c a c i d and 4$ flaOH s o l u t i o n , and i n s o l u b l e i n w a te r , e t h e r , b en ze n e and p e tro le u m e t h e r .

M oreover, i t

re d u c e d F e h li n g ’ s s o l u t i o n , and gave a r e d - v i o l e t c o l o r w ith ' p h lo r o g l u c i n o l , and y e llo w c o l o r s w ith t h e a r o m a tic a m in e s.

SI r

n

T a b le V III

E f f e c t o f th e A c tio n o f P o r i a v a i l l a n t i i on th e C o m p o sitio n o f W h i t e ^ c o t s P in e Wood P e r io d o f Decay ( i n m onths)

L

C e llu lo s e %

L ig n in %

0

4 5 .5

5 3 .9

3

4 5 .4

3 4 .9

5

3 3 .4

4 2 .1

8

2 5 .6

4 6 .5

11

1 7 .6 *

5 1 .1

15

1 5 .2

5 2 .5

3£

r

F u rth e rm o re ,, t h e s i m i l a r i t y e x te n d e d t o th e a n a l y t i c a l d a ta o f t h e ”1 l i g n i n s and t h e i r d e r i v a t i v e s , w hich a r e compaired i n T a b le IX , and to th e u l t r a v i o l e t s p e c t r a o f th e l i g n i n s , w hich a r e shown i n F ig u r e V, and t h e i r d e r i v a t i v e s , shown i n F ig u r e V I. From th e a n a l y t i c a l d a t a , and t h e i d e n t i t y o f th e u l t r a v i o l e t s p e c t r a o f th e l i g n i n s an d t h e i r d e r i v a t i v e s , i t m ust be c o n c lu d e d t h a t th e l i g n i n l i b e r a t e d from wood e x c l u s i v e ly by t h e en zy m atic d e g r a d a tio n o f th e vraod c e l l u l o s e , and th e n a t i v e l i g n i n o f w h ite S c o ts p in e wood a r e in d e e d i d e n t i ­ cal • 6.

L ig n in L i b e r a te d E x c lu s iv e ly t y th e A c tio n o f L e n z ite s s e p i a r i a . In o r d e r t o i n v e s t i g a t e t h e p o s s i b i l i t y t h a t th e e n z y m a tic a lly l i b e r ­

a t e d l i g n i n m ig h t h av e been e f f e c t e d i n some way by t h e enzyme sy stem o f th e o rg an ism em ployed in th e p r e v io u s e x p e rim e n t, n am ely , P . v a i l l a n t i i . a p a r a l l e l s tu d y was c a r r i e d o u t on th e l i g n i n l i b e r a t e d from w h ite S c o ts p in e by th e m old, L . s e p i a r i a . N a tiv e l i g n i n was a g a in c o m p le te ly removed from a sam ple o f w h ite S c o ts p in e wood, an d th e n a t i v e - l i g n i n - f r e e wood was s u b je c te d t o d ecay by L. s e p i a r i a f o r t h i r t e e n m o n th s.

The e f f e c t s o f th e decay on th e

c h e m ic a l c o m p o sitio n o f th e wood a r e p r e s e n te d in T a b le X.

The d ecay ed

sam p les w ere a g a in th o ro u g h ly e x t r a c t e d w ith e t h y l a l c o h o l a t room te m p er­ a t u r e , and th e y i e l d o f l i g n i n o b ta in e d i s p r e s e n te d i n T a b le X I. The. l i g n i n o b ta in e d was c h a r a c t e r i z e d th ro u g h i t s m eth o x y l c o n te n t and u l t r a v i o l e t sp e c tru m .

T h is m e th o x y l v a lu e i s com pared w ith th o s e

o b ta in e d f o r th e o t h e r l i g n i n s i s o l a t e d from sound and d ecay ed w h ite S c o ts p in e wood i n T a b le X I I , and th e u l t r a v i o l e t a b s o r p ti o n sp ectru m f o r t h i s l i g n i n i s com pared w ith th o s e o f n a t i v e and P . v a i l l a n t i i - lib o r a tp .d L.

w h ite S c o ts p in e l i g n i n s in F ig u r e V I I .

-

-1

55

r

T a b le IX

C om parison o f N a tiv e and P o r i a v a i l l a n t i i L ib e r a te d L ig n in s o f W hite S c o ts P in e Wood

N a tiv e W hite S c o ts P ih e L ig n in

L.

W hite S c o ts P in e L ig n in

C

6 4 .0 %

6 4 .2 %

H

6 .5 %

6.0 %

1 4 .5 %

1 4 .4 %

QCHg o f J lc e ta te

10.1 %

1 0 .2 %

OCHg o f Ph e n y lh y d ra zone

1 5 .5 %

1 5 .4 %

och 5

l

E n z y m a tic a lly - L ib e r a te d

_1

i n a t i v e W h ite « c o r « « n e u e n i n 1. . a ...... -WHITE SCOT$ PIHE LIONIN LIBERATED : -iBT * VAILLANTII

ISO

~:x

>

ic«. ISO

IOO


MiN! U B ER A rea ! ACETATE # r

M0

e

v a iii^

h t ii

J

;

i

•pHENYLHTOAAZONE ;• OP W- S.A tlGNIN i LIOEAATEO ZY P. WULLANTII

to o

-

'- r

- I -

''

'

.

I Cm. 1*0

100

80

joo

p..

'

■ |—

56 r

T a b le X

E f f e c t o f th e A c tio n o f L e n z ite s j j e o i a r i a on t h e C o m p o sitio n o f W hite S c o ts P in e Wood

P e r io d o f Decay ( i n m onths)

L.

C e l lu lo s e

L ig n in

%

%

0

4 5 .5

5 5 .9

5

5 9 .9

5 7 .9

6

5 0 .1

4 1 .0

9

1 9 .5

4 5 .6

15

1 8 .5

5 0 .1

37 r

“i

T a b le XI

Y ie l d s o f L ig n in I s o l a t e d from Sound and from D ecayed W hite S c o ts P in e Wood

P e r io d o f Decay

Organism

►ercent L ig n in

( i n m onths)

3.2

©

L-

7

L e n tin u s le o id e u s

6.9

13

L e n z ite s s e p i a r i a

18.3

15

P o r ia v a i l l a n t i i

2 2 .7

38 ~i

r

T a b le X II

C om parison o f M ethoxyl C o n te n ts o f L ig n in s from Sound and D ecayed W hite S c o ts P in e Wood

C o n d itio n o f th e Wood E x tr a c te d

Bound

L

P e r c e n t Ite th o x y l

1 4 .5

U n tr e a te d , L . le p id e u s -d e c a y e d

1 4 .5

II a t i ve - L i gn in - f r e e d , ■ Pv e . i l l a n t i i -d e c a y ed

1 4 .4

^ a tiv e -L ig n in -fre e d , L . s e o i a r i a -d e e a v e d

1 4 .5

a*>| NATIVE WHITE SCOTS PINE t l ONI N

WHITE SCOTS P lN e LIGNIN LI6ERATE0 $Y P. VAILLANTII WHITE SCOTS PINE LIGNIN LIBERATED 8 Y L. SEPIA R IA

100

60

So

300

310

380

400

40

r

From th e d a t a , and. th e ag ree m en t o f th e u l t r a v i o l e t s p e c tr a o f th e " 1 t h r e e l i g n i n sam p les, i t a p p e a r s t h a t th e l i g n i n o b ta in e d from th e L. s e p i a r i a -d e c a y e d w h ite S c o ts p in e wood i s i d e n t i c a l w ith b o th th e P . v a i l l a h t i i - l i b e r a t e d l i g n i n and th e n a t i v e l i g n i n o f t h i s wood.

I t w ould

a p p e a r t h e r e f o r e t h a t th e above p r o p e r t i e s a r e c h a r a c t e r i s t i c f o r th e t o t a l l i g n i n c o n te n t o f t h i s s p e c ie s o f wood.

T h is p o i n t w i l l be c o n s id ­

e r e d i n more d e t a i l in th e D is c u s s io n o f t h i s t h e s i s .

7 . ' C h e m ic a lly P r e p a r e d L ig n in s . The i d e n t i t y o f th e l i g n i n l i b e r a t e d from i t s a s s o c i a t i o n w ith th e c e l l u l o s e o f wood e x c l u s i v e l y by en zy m atic a c t i o n , and t h e n a t iv e l i g n i n o f th e wood h a v in g been e s t a b l i s h e d , i t was o f i n t e r e s t to -c o m p a re th e s e c h e m ic a lly u n a l t e r e d l i g n i n s , o b ta in e d by a m ild e x t r a c t i o n p r o c e d u r e , w ith th e s e l i g n i n s o b ta in e d by t h e cu sto m ary d r a s t i c c h e m ic a l t r e a tm e n ts o f wood. The v a r io u s c h e m ic a l m ethods d e s c r ib e d i n th e l i t e r a t u r e f o r th e i s o ­ l a t i o n o f l i g n i n can be d iv id e d i n t o two c l a s s e s :

( l ) Those t h a t depend

on th e re m o v a l, by h y d r o l y s i s , o f th e c e l l u l o s e an d o t h e r wood com ponents, l e a v in g th e l i g n i n a s

an i n s o l u b l e r e s id u e ;

(g ) Those t h a t depend on th e

rem o v a l o f t h e l i g n i n

from t h e c e l l u l o s e and o t h e r s u b s ta n c e s w ith w hich

i t i s a s s o c i a t e d , by r e a g e n ts w hich s e l e c t i v e l y d i s s o l v e th e l i g n i n . Of th e m ethods o f th e f i r s t c l a s s , th e f o llo w in g w ere a p p l i e d t o w h ite S c o ts p in e wood: a c i d m ethod ( 8 1 ) ;

( a ) 72 % S u l f u r i c a c id method. ( 1 8 ) ;

(b ) 70 % O u lf u r ic

(c ) Fuming h y d r o c h lo r ic a c i d m ethod ( 4 6 ) .

Of th e m ethods o f W h itta k e r (70) and o f

th e seco n d c l a s s , th e p r o c e d u r e s o f P o w e ll and M ehta (5 5 ) w ere com bined f o r th e p r e p a r a t i o n o f

A l k a l i L ig n in . L

The e le m e n ta ry c o m p o s itio n s an d m ethoxyl c o n t e n ts o f th e s e l i g n i n s - 1

41

a re .c o m p a re d w ith th o s e o f n a t i v e w h ite S c o ts p in e l i g n i n , w hich w i l l now be u se d a s t h e s ta n d a r d o f r e f e r e n c e , i n T a b le X I I I . I n o r d e r t o o b ta in a more c o m p le te co m p ariso n o f th e s e l i g n i n s w ith th e n a t i v e l i g n i n , th e f o llo w in g p r o p e r t i e s o f t h e p r e p a r a t i o n s w ere a ls o in v e s tig a te d :

s o l u b i l i t i e s , re d u c in g a b i l i t y and c o lo r r e a c t i o n s .

T h ese p r o p e r t i e s a r e sum m arized and com pared i n T a b le XIV. from t h i s T a b le , th e f o llo w in g o b s e r v a tio n s can be made:

(a ) The

l i g n i n s i s o l a t e d by means o f c o n c e n tr a te d a c i d s a r e i n s o l u b l e i n a l l t h e s o lv e n ts te s t e d ;

(b ) The l i g n i n s i s o l a t e d w ith c o n c e n tr a te d a c i d s do n o t

h av e t h e a b i l i t y o f re d u c in g F e h lin g * s s o l u t i o n ;

( c ) None o f th e chem i­

c a l l y p r e p a r e d l i g n i n s re s p o n d s t o th e c o lo r r e a g e n t s i n a m anner a n a lo g o u s t o th e n a t i v e l i g n i n . I t would a l s o h av e been o f i n t e r e s t to d e te rm in e th e u l t r a v i o l e t s p e c tr a o f th e c h e m ic a lly p r e p a r e d l i g n i n s f o r p u rp o s e s o f co m p ariso n w ith n a t i v e w h ite S c o ts P in e l i g n i n .

H ow ever, t h e s p e c tr a o f t h e l i g n i n s i s o l a t e d by

means o f 72 % s u l f u r i c a c i d , 70 % s u l f u r i c a c i d and fum ing h y d r o c h lo r i c a c i d can n o t be d e te rm in e d due t o t h e i r co m p lete i n s o l u b i l i t y i n a l l s o l ­ v e n ts .

H owever, t h e sp ectru m o f t h e a l k a l i l i g n i n i s o l a t e d w ith 10 %

sodium h y d ro x id e s o l u t i o n was d e te rm in e d , and i s com pared i n f i g u r e V III w ith t h a t o f n a t i v e w h ite S c o ts p in e l i g n i n . from th e d i s c r e p a n t a n a l y t i c a l d a ta and t h e d i f f e r e n c e s i n p h y s i c a l p r o p e r t i e s o f th e v a r io u s 'w hite S c o ts p in e l i g n i n s , i t can be s t a t e d t h a t none o f th e s e c h e m ic a lly o b ta in e d l i g n i n s re s e m b le s n a t i v e l i g n i n i n c h a r a c t e r n e a r l y so c l o s e l y a s d o e s th e p r o d u c t o b ta in e d e x c l u s i v e l y by e n z y m a tic l i b e r a t i o n from th e w o o d - c e llu lo s e .

L

42

r

T a b le X I I I

C om parison o f A n a l y t i c a l D ata o f C h e m ic a lly P r e p a r e d and H a tiv e f f n ite S c o ts P in e L ig n in s

L ig n in

L

C ,^ , D e v ia tio n

H ,^., D e v ia tio n

OCHg,%, D e v ia tio n

H s tiv e

6 4 .0

-------------

6 .5

— —

1 4 .5

---------

72 % HgS04

6 4 .8

-|»0.8

5 .5

- 0 .8

1 5 .9

- 0 .6

70 % HgS04

6 5 .4

- 0 .6

5 .8

- 0 .5

1 4 .4

- 0 .1

Fuming HC1

6 2 .1

- 1 .9

. 5 .5

- 1 .0

1 5 .2

- 1 .5

10 % KaOH

6 2 .1

- 1 .9

5 .5

- 1 .0

1 6 .8

4 2 .5

43 n

r

Table XIV C om parison o f P r o p e r t i e s o f C h em ically P r e p a r e d and N a tiv e W hite. S c o ts P in e L ig n in s

N a tiv e

72^ HgSQ4

70%% S 0 4

Fuming HC1

1Q$N&0H

In s o l. In s o l. In s o l. In s o l. In s o l. In s o l. In s o l. In s o l. In s o l. In s o l.

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S tu d ie s on " I n d u l i n 11. A co m m ercial l i g n i n p r e p a r a t i o n w hich h a s been s u g g e s te d a s a

" s t a n d a r d 11 l i g n i n ( 6 8 ) , nam ely " I n d u l i n 11 (p ro d u c e d by th e W est V ir g in i a P u lp & P a p e r C o .) , wa® a l so s t u d i e d f o r c o m p a ra tiv e p u r p o s e s .

T h is i s an

a l k a l i l i g n i n , p r e p a r e d by a c i d p r e c i p i t a t i o n from th e b la c k l i q u o r ob­ t a i n e d by c o o k in g p in e wood w ith sodium h y d ro x id e and sodium s u l f i d e a t 172°C. f o r 5 t o 4 h o u r s ( 4 2 ) .

I t c o n ta in s s u l f u r i n a d d i t i o n to c a rb o n ,

hy d ro g en and o xygen. " I n d u lin " was r e c e iv e d i n two d i f f e r e n t fo rm s:

(l)

" I n d u lin An,

w hich i s a brown powder o b ta in e d by th e a c i d p r e c i p i t a t i o n i n d i c a t e d a b o v e; (2 ) A w et f i l t e r cake w hich i s s t i l l a sodium s a l t o f th e l i g n i n . The I n d u l i n A was a n a ly s e d f o r i t s G, H, QCHg and S c o n t e n t s , w ith t h e r e s u l t s i n d i c a t e d i n T a b le XV.

H owever, s in c e t h i s m a t e r i a l p o s s e s s e d

a d a rk brown c o l o r and a s tr o n g s u lf u r o u s o d o r , p r o p e r t i e s c o m p le te ly f o r ­ e ig n t o n a t i v e l i g n i n , a p u r i f i c a t i o n o f t h e w et f i l t e r cak e o f I n d u li n m e n tio n ed above was a tte m p te d a s f o llo w s : F i'f ty gram s o f th e f i l t e r cak e w ere su sp e n d ed i n 500 m l. o f w a te r . T h is s u s p e n s io n wq.s made a l k a l i n e w ith 50 m l. o f 5% HaOH, and th e r e s u l t i n g s o l u t i o n was f i l t e r e d .

The f i l t r a t e was a c i d i f i e d w ith 40 m l. o f 10% % S 0 4

y i e l d i n g a d a rk brown p r e c i p i t a t e . and d r i e d .

T h is was f i l t e r e d , w ashed w ith w a te r

F iv e gram s o f th e d ry m a t e r i a l w ere d is s o lv e d i n 50 m l. o f

d io x a n e , and th e s o l u t i o n was p r e c i p i t a t e d by f i l t e r i n g i n t o 600 m l. o f e t h e r , c o o le d i n an i c e - b a t h , w ith s t i r r i n g . The r e s u l t i n g p r e c i p i t a t e was f i l t e r e d , w ashed w e ll w ith e t h e r , and d r i e d , w hereby a l i g h t t a n pow der was o b ta in e d , re s e m b lin g n a t i v e .lig n i n in i t s v is ib le p ro p e rtie s a t l e a s t .

T h is m a t e r i a l was a l s o s u b m itte d

f o r G, H, OCHg and S a n a l y s i s , and th e r e s u l t s o f th e s e a n a l y s e s a r e

_j

46 r

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T a b le IV

C om parison o f C o m p o sitio n s o f I n d u li n A and " P u r i f i e d ” I n d u l i n w ith N a tiv e W hite S c o ts P in e L ig n in

In d u lin A

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C o m p a re d i n T a b le XV w ith t h o s e o b ta in e d f o r th e o r i g i n a l I n d u l i n A, anh f o r n a t i v e -white S c o ts p i n e , l i g n i n . Thus w h ile th e m eth o x y l c o n te n t o f th e ’’p u r i f i e d ” I n d u l i n more n e a r l y a p p ro a c h e s t h a t o f n a t i v e l i g n i n th a n d id t h e o r i g i n a l I n d u l i n A, th e ’’p u r i f i e d ” m a t e r i a l s t i l l r e t a i n e d i t s 1 .4 $ o f s u l f u r , and t h i s elem en t t h e r e f o r e seems t o be an i n t r i n s i c p a r t o f th e I n d u l i n m a t e r i a l , n o t r e ­ m ovable from i t by th e above p u r i f i c a t i o n . As a r e s u l t , i t m ust be c o n c lu d e d t h a t t h e com m ercial p r o d u c t ’’I n d u l i n ” i s u n s u ite d f o r c o n s id e r a tio n a s a ’’s ta n d a r d ” r e p r e s e n t a t i v e o f l i g n i n a s i t e x is ts in n a tu re .

9.

The L ig n in s o f S ch iltz and S a r te n . S chiitz and S a r te n hav e r e p o r t e d (78) t h a t i f wood c h ip s a r e h e a te d

w ith w a te r i n an a u to c la v e f o r 5 d ay s a t 1 0 5 ° C ., o r f o r 1 t o 2 d a y s a t 1 2 5 °C ., th e c h ip s g r a d u a lly become d a r k e r i n c o l o r , and on t h e i r s u r f a c e t h e r e i s d e p o s it e d a f i n e l y d iv i d e d brown f i l m .

They c o n c lu d e t h a t t h i s

i s a new ly fo rm ed s u b s ta n c e , w hich was n o t is r e s e n t i n t h e u n a u to c la v e d wood.

F u r t h e r am ounts o f t h i s new s u b s ta n c e w ere o b ta in e d fro m th e r e s u l t ­

in g c l e a r aq u eo u s e x t r a c t by h e a t in g th e l a t t e r f o r s e v e r a l h o u r s w ith 1$ HC1 a t 95 t o 10QOC., o r f o r a few m in u te s w ith 10 t o 15$ HC1.

M oreover,

on e x t r a c t i o n w ith a lc o h o l^ a c e to n e , 90$ a c e t i c a c i d o r d i l u t e N&OH, th e r e s i d u a l wood y i e l d e d th e same s u b s ta n c e from th e i n t e r i o r o f t h e c h ip s , i n an am ount from 5 t o 10$ o f t h e w e ig h t o f t h e wood, d ep e n d in g on th e s p e c ie s em ployed, and th e d u r a t i o n o f th e h e a t i n g .

The a u t h o r s co n clu d ed

t h a t tits s u b s ta n c e was ’’l i g n i n ” , and f u r th e r m o r e , w ere p e rs u a d e d t h a t t h e l i g n i n was n o t a p r e v io u s l y e x i s t i n g c o n s t i t u e n t o f t h e o r i g i n a l un­ t r e a t e d ??ood, b u t r a t h e r an a r t i f i c i a l p r o d u c t. L

-i

48

In an a tte m p t to r e p e a t t h e s e r e s u l t s , 50 gms. o f w h ite S c o ts p in e 1 wood c h ip s w ere p la c e d i n a f l a s k , and 2250 m l. o f w a te r w ere a d d e d . The f l a s k was p lu g g e d , and a u to c la v e d a t 125°C. f o r one d a y .

At t h i s tim e ,

th e wood' c h ip s h ad in d e e d become d a r k e r i n c o l o r , b u t no f il m o f any k in d c o u ld be d e t e c t e d on th e s u r f a c e o f th e c h i p s , o r i n th e aq u eo u s e x t r a c t . H ow ever, a s m a ll amount o f a brown pow der was o b ta in e d from t h e c l e a r aq u eo u s e x t r a c t when one l i t e r o f t h e l a t t e r was r e f lu x e d w ith 550 m l. o f c o n c . HG1 f o r one h o u r .

The p r e c i p i t a t e d m a t e r i a l was w ashed f r e e o f HCl

and a n a ly z e d , w ith t h e r e s u l t s i n d i c a t e d i n T a b le XVI. The a u to c la v e d wood c h ip s w ere th e n g round t o 40 mesh, and 40 gms. o f th e saw d u st w ere e x t r a c t e d w ith e t h y l a lc o h o l i n a S o x h le t a p p a r a tu s o v e r­ n ig h t.

The a l c o h o l was rem oved by d i s t i l l a t i o n , and th e r e s i d u e w ashed

w e ll w ith w a te r and e t h e r .

The m a t e r i a l was d is s o lv e d i n g l . a c e t i c a c id

an d ’ p r e c i p i t a t e d i n t o i c e - w a t e r .

^h e r e s u l t i n g p r e p a r a t i o n was a n a ly z e d

and th e r e s u l t s a r e p r e s e n te d i n T a b le XVI w here th e y a r e com pared w ith th e c o r r e s p o n d in g v a lu e s f o r n a t i v e l i g n i n . M o reo v er, th e u l t r a v i o l e t a b s o r p tio n s p e c ta o f th e two p r e p a r a t i o n s w ere a l s o d e te r m in e d , and a r e p l o t t e d in F ig u r e IX , w here th e y a r e com­ p a re d w ith t h e c u rv e f o r n a t i v e l i g n i n . T hu s, from a co m p ariso n o f t h e a n a l y t i c a l d a t a and th e u l t r a v i o l e t c u rv e s o f th e s e l i g n i n s w ith t h o s e o f n a t i v e w h ite S c o ts p in e l i g n i n , i t a p p e a rs t h a t th e s e p r e p a r a t i o n s a l s o do n o t r e p r e s e n t l i g n i n ”i n s i t u ” ; m o reo v e r, th e i s o l a t i o n o f n a t i v e l i g n i n from sound wood sam p les w ould seem t o r e f u t e th e c la im s o f S c h iitz (7 7 , 7 9 , 80) t h a t l i g n i n a s i t ex­ i s t s i n th e p l a n t c a n n o t be i s o l a t e d .

L

49 r

T a b le XVI

C om parison o f C o m p o sitio n s o f L ig n in s A cco rd in g to S c M tz and S a r te n w ith N a tiv e W h ite S c o ts P in e L ig n in

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r iO . A M o d ifie d -P ro c e d u re f o r O b ta in in g N a tiv e L ig n in .

~1

The p r e p a r a t i o n d e s ig n a te d a s " a l c o h o l - e x t r a c t e d ” i n th e p r e v io u s s e c t i o n was o b ta in e d e s s e n t i a l l y by e x t r a c t i o n o f a u to c la v e d wood c h ip s w ith e t h y l a lc o h o l i n a S o x h le t a p p a r a tu s , and i s o l a t i o n o f th e e x t r a c t e d %

m a te ria l.

H ence, th e r a t h e r low m eth o x y l c o n te n t o f t h i s p r e p a r a t io n

(11.3% ) was s u r p r i s i n g i n view o f th e e s s e n t i a l s i m i l a r i t y betw een th e m ethod o f o b ta in in g t h i s p r e p a r a t i o n , and t h a t o f i s o l a t i n g n a t i v e l i g n i n . On t h e o t h e r h an d , t h i s p r e p a r a t i o n was fo u n d to e x h i b i t s o l u b i l i t i e s , r e d u c in g a b i l i t y and c o l o r r e a c t i o n s i d e n t i c a l t o o u r l i g n i n p r e p a r a t i o n s ( a s i n T a b le I I ) . Now o u r p ro c e d u re in g t h e S ch iitz l i g n i n

can be s a id to d i f f e r fro m t h a t em ployed f o r o b t a i n ­ i n twro r e s p e c t s :

( l ) Our wood was f r e e d o f a l l

11e x t r a c t i v e s ” p r i o r t o i s o l a t i n g th e l i g n i n , by e x t r a c t i n g th e v/ood t h o r ­ o u g h ly w ith w a te r and e th e r ;: w h ile i n th e S ch iitz p r o c e d u r e , t h e wood i s a u to c la v e d w ith w a te r , th e e t h e r s o lu b le m a t e r i a l was n o t removed?

(g)

Our l i g n i n was e x t r a c t e d w ith e t h y l a l c o h o l a t room te m p e r a tu r e , w h ile b o i l i n g a l c o h o l was u s e d t o o b ta in t h e S chfltz p r o d u c t. C o n s e q u e n tly , i t was th o u g h t o f i n t e r e s t to d e te rm in e w h e th e r th e low m e th o x y l c o n te n t o f t h e S ch iitz p r o d u c t was due to a ” c o n ta m in a tio n ” o f th e p r o d u c t w ith th e unrem oved e x t r a c t i v e s , o r r a t h e r to some change i n th e l i g n i n m o le c u le in d u c e d by t h e e l e v a te d te m p e r a tu r e n e c e s s i t a t e d by th e u s e o f th e S o x h le t a p p a r a tu s . A c c o rd in g ly , w h ite S c o ts a p p a r a t u s , f i r s t 'w ith

p in e

w a te r , th e n

woodm eal w ith e t h e r .

was e x t r a c t e d i n a S o x h le t The wood was th e n th o ro u g h ­

l y e x t r a c t e d w ith e t h y l a l c o h o l i n th e S o x h le t f o r 72 h o u r s .

The s o lv e n t

was rem oved, w hereupon a re d d is h -b ro w n r e s i d u e was o b ta in e d .

T h is

m a t e r i a l was p u r i f i e d by d i s s o l u t i o n i n g l a c i a l a c e t i c a c i $ , and p re c ip p .-

52

r

i

t a t i o n i n t o w a te r .

The a c e t a t e and p h e n y lh y d ra z o n e d e r i v a t i v e s o f t h i s l i g n i n w ere p r e ­ p a r e d and w ere c h a r a c t e r i z e d th r o u g h t h e i r m e th o x y l c o n te n ts and u l t r a ­ v io le t s p e c tra .

The a n a l y t i c a l d a t a o f th e l i g n i n and i t s d e r i v a t i v e s

a r e com pared w ith th o s e o f n a t i v e w h ite S c o ts p in e l i g n i n i n T a b le XVII, w h ile th e u l t r a v i o l e t c u rv e s o f th e l i g n i n s a r e com pared i n F ig u r e X, and t h e d e r i v a t i v e s in F ig u r e X I. From th e d a t a , i t a p p e a rs t h a t th e new l i g n i n p r e p a r a t io n i g a n a l y t i ­ c a l l y s i m i l a r t o o u r n a t i v e l i g n i n , i s o l a t e d u n d e r m ild e r c o n d i t i o n s . M o reo v er, t h e s i m i l a r i t y e x te n d e d t o th e s o l u b i l i t y , r e d u c in g a b i l i t y and c o l o r r e a c t i o n s , a l l o f w hich a r e i d e n t i c a l w ith th o s e o f n a t i v e w h ite S c o ts p in e l i g n i n ( a s i n T a b le I I ) . T hese r e s u l t s th e n seem t o e x p la in th e d i s c r e p a n t a n a l y t i c a l d a ta o b ta in e d f o r t h e S e h fifi l i g n i n a s b e in g due t o a rfc o n ta m in a tio n '■ o f th e p r e p a r a t i o n w ith e t h e r - s o l u b l e e x t r a c t i v e s , f o r when th e s e a r e rem oved p r i o r t o th e e x t r a c t i o n o f t h e . l i g n i n , a s a b o v e , th e p ro d u c t i s o l a t e d in t h e S o x h le t a p p a r a tu s w ith b o i l i n g a lc o h o l shows a p p ro x im a te ly t h e same d a t a a s th e p r o d u c t i s o l a t e d a t room te m p e r a tu r e . M oreo v er, th e u s e o f g l a c i a l a c e t i c a c id a s th e l i g n i n s o lv e n t f o r t h e p r e c i p i t a t i o n i n t o w a te r h a s th e a d v a n ta g e o f a v o id in g th e c o l l o i d a l s o l u t i o n s w hich f r e q u e n t l y r e s u l t -when u s in g d i o x a n e .( 1 0 ) . T h is i n v e s t i g a t i o n th e n h a s r e s u l t e d i n t h e dev elo p m en t o f an im proved m ethod f o r i s o l a t i n g n a t i v e l i g n i n , f o r a lth o u g h b o i l i n g a l c o h o l i s u s e d , a * s i m i l a r p r o d u c t i s o b ta in e d a s a t room te m p e r a tu r e .

T h is f a c t th e n

w ould a llo w th e u s e o f th e S o x h le t a p p a r a tu s i n th e e x t r a c t i o n o f n a t iv e l i g n i n , th e r e b y p e r m i tt in g th e e x t r a c t i o n o f l a r g e r am ounts o f saw d u st jw ith c o n s id e r a b le s a v in g i n tim e , s in c e th e e x t r a c t i o n i s co m p le te d mucl}

53

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11.

F u r th e r S tu d ie s on Wood D ecay. S tu d ie s w ere a l s o made of, th e e f f e c t s o f th e d eca y o f w h ite S c o ts

p in e wood by t h e o rg an ism s £ . c e r e b e l l a . F . r o b u s tu s and M. v e r r u c a r i a .

a) Decay by H oniophora c e r e b e l l a . W hite S c o ts p in e saw dust was s u b je c te d to d eca y by £ . c e r e b e l l a . an o rg an ism w hich i s a common c a u s e o f d ecay i n woodwork and i n damp m in e s, and w hich h a s a l s o been r e p o r t e d a s a c a u se o f r o t i n s ta n d in g c o n i f e r s , and th u s a p p e a r s c a p a b ld o f a c t i n g a s a p a r a s i t e u n d e r c e r t a i n c o n d i­ t i o n s (25 b ) . The r e s u l t s o f an e le v e n m onth decay o f t h e wood t y t h i s o rg a n ism a r e shown i n T a b le X V III.

The a n a ly s e s i n d i c a t e d p r a c t i c a l l y no change in t h e

c o m p o sitio n o f th e r e s i d u a l m a t e r i a l from th e s e v e n th to t h e e le v e n th month o f in c u b e ,tio n , and d ecay a p p e a re d t o h av e c e a se d a t t h a t tim e ,

^ liis

r e s u l t th e n w ould seem to v e r i f y t h e c o n c lu s io n re a c h e d a s a r e s u l t o f th e p r e lim in a r y a l k a l i s o l u b i l i t y d e t e r m in a tio n s (T a b le I ) , w hereby i t was con­ c lu d e d t h a t C,. c e r e b e l l a was a p o o r F v o o d -d estro y er u n d e r l a b o r a t o r y c o n d i­ tio n s . b) Decay by ^omes r o b u s t u s . The i n a b i l i t y o f th e w o o d -d e s tro y in g o rg a n is m , -^omes h a r t i g i i . to a t ­ ta c k th e t a n g e n t i a l w a lls o f f i r wood h a s b een a t t r i b u t e d to a low d e g re e o f l i g n i f i c a t i o n (4 7 , 4 8 ) .

F . h a r t i g i i i s d e s c r ib e d (25 a) a s a v a r i e t y

o f th e s p e c ie s F . r o b u s tu s w hich grows on s i l v e r f i r wood.

I n t h e m ost

co m p lete d e s c r i p t i o n o f t h i s o rg a n ism t o be fo u n d i n th e l i t e r a t u r e ( 2 2 ) , i t i s r e f e r r e d to a s a "slo w g ro w in g ” o rg a n ism whose o p tim a l te m p e r a tu r e L -1

57

r

T a b le X V III E f f e c t o f t h e A c tio n o f C o n io p h o ra c e r e b e l l a on th e C o m p o sitio n o f ^ h i t e S c o ts P in e fo o d

P e r io d o f Decay ( i n m onths)

L-

C e l lu lo s e %

L ig n in %

0

4 5 .5

'5 5 .9

5

4 0 .6

5 7 .9

7

5 9 .2

5 8 .9

11

5 9 .1

5 9 .5

-I

58

F l i e s betw een 26 and 51°C.

n

In e x p l o r a to r y e x p e r im e n ts , th e o rg a n ism was c u l t i v a t e d on th e f o llo w ­ in g t h r e e s o l i d m edia: M a lt-e x tra c t a g a r.

(a ) 2 % g lu c o s e - a g a r ; .(b) Czapek-Box a g a r ;

(c )

F la s k s w ere in c u b a te d a t 22 and 27°C.

On a l l t h r e e m ed ia, g ro w th was b e t t e r a t 27 th a n a t 2 2 ° .

Growth on

t h e Czapek-D ox a g a r was p o o r , b u t was a b o u t e q u a lly good on t h e g lu c o s e and m a l t - e x t r a c t a g a r s .

C o n s e q u e n tly , th e u s u a l n u t r i e n t medium (w ith

g lu c o s e o m itte d ) was u s e d i n th e w ood-decay e x p e rim e n ts , and th e c u l t u r e s w ere in c u b a te d a t 27°C. S in c e no i n d i c a t i o n i s t o be fou n d in ' t h e l i t e r a t u r e a s t o w h e th e r £* r o b u s tu s b e lo n g s to th e g e n e r a l c l a s s o f "brown" o r " w h ite " r o t s , i t was th o u g h t o f i n t e r e s t t o a tte m p t to th u s c l a s s i f y t h i s o rg a n is m , t y a s tu d y o f th e c h e m ic a l e f f e c t s o f i t s d ecay o f wood. A c c o rd in g ly , g round w h ite S c o ts p in e wood -was s u b je c te d t o d ecay ty F . r o b u s t u s . and t h e p r o g r e s s o f th e decay was fo llo w e d by p e r i o d i c a n a l y s e s o f t h e r e s u l t i n g wood r e s i d u e s .

xh e r e s u l t s o f a sev en m onth d ecay

a r e p r e s e n te d in T a b le XIX. From th e d a t a , i t a p p e a r s t h a t t h e e f f e c t o f th e d ecay o f w h ite ^ c o t s p in e wood t y F . r o b u s tu s i s a d e p l e t i o n am th e c e l l u l o s e c o m p o sitio n o f th e wood, c o n c o m ita n t w ith an i n c r e a s e i n th e r e l a t i v e c o n te n t o f l i g n i n . The r o t p ro d u ced by t h i s o rg an ism t h e r e f o r e a p p e a rs t o be o f th e "brow n" r o t " ty p e .

c)

Decay by M vrothecium v e r r u c a r i a . I t was hoped tb.- a c h ie v e a r a p i d a p p ro a c h t o th e a t ta in m e n t ‘o f a

" l i g n i n - e n r i c h e d " wood by em ploying t h e c e l l u l o l y t i c m old, M vrothecium v e r r u c a r i a ^ ( sv n . M e ta rrh iz iu m g lu tin o s u m ( 95) ) , s in c e t h i s o rg a n ism i s L.

-i

59 r

T ab le XIX

E f f e c t o f t h e A c tio n o f Fomes r o b u s tu s on th e C o m p o sitio n o f W hite S c o ts P in e Wood

P e r io d o f Decay ( i n m onths)

L.

C e llu lo s e . %

L ig n in %

0

4 5 .5

5 3 .9

1

4 2 .5

3 6 .9

5

3 8 .5

3 8 .1

5

3 7 ,0

3 9 .0

3 5.

3 9 .9

9

60 r

-i r e p o r t e d to be p e r h a p s th e m ost s t r o n g l y c e l l u l o l y t i c s p e c ie s o f fu n g u s y e t d is c o v e r e d ( 9 4 ) , and to p ro d u c e a v e ry a c t i v e c e l l u l o s e enzyme ( 7 6 ) . A c c o rd in g ly , w h ite S c o ts p in e saw d u st was s u b je c te d t o

d ecay by t h i s

o rg a n ism f o r s i x m o n th s, b u t a t t h i s tim e , no m y c e lia l d ev elo p m en t c o u ld be d e t e c t e d , d e s p i t e r e p e a te d a d d i t i o n s o f s t e r i l e m edia and r e i n o c u l a ­ tio n s .

M o reo v er, t h e c o n s ta n c y i n c o m p o sitio n o f t h e wood, i n d i c a t e d i n

T a b le XX, c o n firm e d t h i s v i s u a l o b s e r v a ti o n , an d i n d i c a t e s th e u n s u i t a ­ b i l i t y o f ?/ood a s a s u b s t r a t e f o r t h i s o rg an ism d e s p i t e i t s n o rm a lly h ig h c e l l u l o l y t i c a c t i v i t y on f a b r i c s .

L_

-J

61

r

T a b le XX

E f f e c t o f th e A c tio n o f M vrothecium v e r r u c a r i a on th e C o m p o sitio n o f IH hite S c o ts P in e $ood

P e r io d o f Decay ( i n m onths)

L_

C e l lu lo s e %

L ig n in %

0

4 5 .5

5 5 .9

6

4 5 .5

5 5 .9

62

r B.

1.

STUDIES ON THE METABOLISM OF LBNTINUS LBPIDBUS

n

S t e r o l F o rm a tio n The f o r m a tio n o f s t e r o l s by m ic ro o rg a n ism s i s w e ll e s t a b l i s h e d .

In

y e a s t s and m o ld s, th e s t e r o l , e r g o s t e r o l , o f t e n fo rm s a h ig p r o p o r ti o n o f th e u a n s a p p n ifia b le f r a c t i o n .

A lth o u g h e r g o s t e r o l h a s been d e t e c te d

i n s m a ll am ounts in b o th a n im a ls and p l a n t s , i t i s g e n e r a l l y conceded t h a t t h i s s u b s ta n c e i s th e p r i n c i p a l s t e r o l p r e s e n t i n th e f ila m e n to u s f u n g i ( 7 l ) . T h u s, e r g o s t e r o l h a s been i s o l a t e d from v a r io u s i l s p e r g i l l i and P e n i c i l ­ lin .

F o r exam ple, A s p e r g ill u s f i s c h e r i (71) and P e n i c i l l i u m pub eru lu m (8)

h av e t h e a b i l i t y o f s y n th e s iz in g e r g o s t e r o l when grown on s y n t h e t i c m edia c o n t a in in g g lu c o s e a s th e s o le s o u rc e o f c a rb o n .

M ost r e c e n t l y , i n v e s t i ­

g a t i o n s h av e e s t a b l i s h e d f o r th e f i r s t tim e th e f o r m a tio n o f e r g o s t e r o l t y members o f th e g e n e r a , ^ u sa riu m (2 0 ) and N e u ro sp o ra ( 6 6 ) . In a s e r i e s o f p u b l i c a t i o n s , Z e l ln e r was a b l e t o i s o l a t e e r g o s t e r o l from majpy o f th e h ig h e r f u n g i , in c lu d in g s e v e r a l o f th e w o o d - r o ttin g s p e c ie s , such a s A m anita m u s c a r ia . ( 9 6 a ) , A r m i l l a r i a m a lle a . ( 96b) and P o ly o o ru s a u l f u r e u s ( 9 6 c ) . The i n t e r e s t o f th e l a b o r a t o r y i n th e m e tab o lism o f th e w o o d -d e stro y ­ in g f u n g i p ro m p ted an i n v e s t i g a t i o n o f th e m e ta b o lic p r o d u c ts o f th e o rg a n ism , L e n tin u s l e o i d e u s . and d u r in g th e c o u rs e o f t h i s i n v e s t i g a t i o n , th e p r e s e n c e o f a s t e r o l was d e t e c te d i n t h e m yeelium o f t h i s fu n g u s .

a)

I s o l a t i o n o f th e S t e r o l . One l i t e r , p r o t i o n s o f th e u s u a l 2% g lu c o s e medium, c o n ta in e d i n each

o f s e v e r a l S l i t e r F e rn b a e h f l a s k s , w ere s t e r i l i z e d by a u t o c la v i n g , and each f l a s k was i n o c u la te d w ith a two week o ld mat o f L. L e o id e u s . grown L.

65 r

~i on 50 m l. o f medium i n 1 2 t m l. E rlen m ey er f l a s k s .

A f te r two t o t h r e e

m onths o f i n c u b a t i o n , d ep e n d in g on th e r a t e o f g lu c o s e c o n su m p tio n , th e r e s u l t i n g m ats w ere f i l t e r e d o f f , w ashed w ith d i s t i l l e d w a te r and d r i e d . The ground m ats w ere e x t r a c t e d w ith c h lo ro fo rm i n a S o x h le t a p p a r a tu s f o r 48 h o u r s .

The s o lv e n t was rem oved by d i s t i l l a t i o n , and th e f a t t y r e s i d u e

s a p o n if i e d tw ic e w ith a l c o h o l i c KOH.

J'h e e t h e r e a l s o l u t i o n o f th e u n s a p -

o n i f i a b l e f r a c t i o n was e v a p o r a te d t o d r y n e s s , and th e r e s id u e r e c r y s t a l ­ l i z e d s e v e r a l tim e s from a 2 :1 a lc o h o l- b e n z e n e s o l u t i o n , and fro m a b s o lu te a lc o h o l.

The r e s u l t i n g c r y s t a l l i n e m a t e r i a l m e lte d a t 1 5 8 -6 0 ° C ., and

re s p o n d e d d e c i s i v e l y to th e c o l o r r e a c t i o n s c h a r a c t e r i s t i c o f s t e r o l s .

b)

C h a r a c t e r i z a t i o n o f th e S t e r o l . A m ixed m e ltin g p o i n t o f t h e s t e r o l i s o l a t e d from L . le p id e u s w ith an

a u t h e n t i c sam ple o f e r g o s t e r o l showed no d e p r e s s io n .

A c c o rd in g ly , th e

d a t a o b ta in e d f o r t h i s s t e r o l a r e com pared w ith th o s e g iv e n f o r e r g o s t e r o l (4 5 ) i n T ab le XXI, and i t s u l t r a v i o l e t a b s o r p tio n sp ectru m i s com pared in F ig u r e X II w ith t h a t r e p o r te d f o r e r g o s t e r o l ( 7 ) . From th e s e d a t a , and th e e x c e l l e n t ag ree m en t o f th e u l t r a , v i o l e t c u rv e s , i t i s co n c lu d e d t h a t th e s t e r o l p ro d u ced by L. le p id e u s h a s been i d e n t i ­ f i e d w ith e r g o s te r o } ..

c)

Q u a n t i t a t i v e E s tim a tio n o f S t e r o l P r o d u c tio n . The amount o f s t e r o l p r e s e n t i n m ic ro o rg a n ism s i s s a id to v a ry betw een

0 .1 and 1 .7 $ ( 5 1 ) .

F o r c o m p le te n e s s o f in f o r m a tio n , q u a n t i t a t i v e e s tim a te

io n s o f th e t o t a l s t e r o l c o n t e n t o f f u l l y d e v e lo p e d m ats o f L. l e p i d e u s ‘ .were p e rfo rm e d .

The c o l o r i m e t r i c m ethod em p loying th e b lu e c o l o r p ro d u ced

w ith 90$ t r i c h l o r o a c e t i c a c id (2 1 ) was r e p la c e d by t h a t u t i l i z i n g th e L L ieb erm a n n -B u rc h ard r e a c t i o n ( 2 9 ) .

*

64

r Table XXI C om parison o f D ata o f S t e r o l from L e n tin u s le p id e u s w ith E r g o s te r o l E r g o s te r o l

S t e r o l from L. le p id e u s

160-1°C .

M e ltin g P o in t

1 5 8 -6 0 °C .

M. P . o f A c e ta te

1 7 2 -5 o c .

M. P . o f B en zo ate

1 6 8 -9 °G.

1751 168-

•

C o lo r R e a c tio n s :

L.

1)

S alk o w sk i

Red c o lo r

Same

2)

L ieb erm a n n -B u rc h ard

Red —fb lu e —*gr een

Same

5)

T @ rte lli-J a ffg

Y e llo w —*green

Same

4)

R o sen h eim -C allo w

P in k - ^ b lu e - g re e n

Same

5)

C h lo r a l H y d ra te

Red —>gre en —Vblu e

Same

6)

T r i c h l o r o a c e t i c A cid

R ed -> b lu e

Same

7)

ZnClg and AcgO

8)

ZnClg and AcgO i n HOAc

, P in k ^ g re e n --

.

r

Same

Ye llo w —*gr e en —Vbrown

Same

-J

Fi & u r e . X U

STEROL PRODUCED BY L.LCPJOCUS ON 0 LUCOS£ 5TER01 PROOUCEO 0T L. LBPIDEUS ON ETHYL ALCOHOL 10,000

6000

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7000 M:

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66

A f t e r t h r e e m onths o f c u l t i v a t i o n on t h e 2 % g lu c o s e medium, th e f u l l y grown m ats o f L . le p id e u s w ere f i l t e r e d o f f , and th e n , d r i e d , gro und and w eig h ed sam p les o f th e s e n y c e l i a w ere e x t r a c t e d w ith c h lo ro fo rm f o r 24 h o u rs i n S o x h le ts .

A liq u o ts o f th e e x t r a c t s w ere s u b je c t e d t o th e

L ieb erm a n n -B u rc h ard r e a c t i o n , and th e i n t e n s i t y o f c o l o r d e v e lo p e d was m easured w ith th e E v ely n c o l o r i m e t e r , and com pared w ith s ta n d a r d v alu es(S O ) The f o llo w in g a r e a v e ra g e r e s u l t s o b ta in e d from f o u r d i f f e r e n t sam p les o f m y c e lia l

S . 21 m gs. s t e r o l p e r gm. o f n y c e liu m

-

5 .2 5 mgs.

»

"

”

H

"

5 .2 1 mgs.

»

"

"

M

"

“

"

11

,r

"

3 .5 1

mgs.

A c c o rd in g ly , i t i s e s tim a te d t h a t s t e r o l i s p r e s e n t t o th e e x t e n t o f a p p ro x im a te ly 0 .3 ^ o f th e w e ig h t o f th e f u l l y d e v e lo p e d mat o f L. l e p i d e u s .

d)

M echanism o f F o rm a tio n o f t h e S t e r o l . E v id en ce e x i s t s t h a t e r g o s t e r o l i s s y n th e s iz e d by m ic ro o rg a n ism s from

some t¥ /o -carb o n compound ( 5 1 ) .

T h u s, H alden £& a l ( 5 8 , 84) su c c e d e d i n

i n c r e a s i n g th e s t e r o l c o n te n t o f y e a s t a b o u t s i x t y f o l d by k e e p in g t h e y e a s t s u s p e n s io n i n t h i n l a y e r s on a g a r c o n ta in in g s u c r o s e , and s u p p ly in g oxygen and a l c o h o l v a p o r s .

S o n d e r h o f f and Thomas (85) d e m o n s tra te d t h a t

when u t i l i z i n g t r i d e u t e r o a c e t i c a c i d a s s u b s t r a t e , y e a s t c e l l s a c c u m u la te d c o n s id e r a b le d e u te r iu m i n th e u n s a p o n if i a b le f r a c t i o n , t h e r e t y s u g g e s tin g th e s y n t h e s i s o f s t e r o l from t h e a c e t a t e .

M aguigan and W alker ( 5 4 ) , on

t h e b a s i s o f t h e i r e x p e r im e n ts , a l s o c o n clu d ed t h a t a c e t a t e can p r o v id e th e h e c e s s a r y c a rb o n f o r s t e r o l s y n t h e s i s by y e a s t . S tu d ie s on th e m echanism -of s t e r o l fo rm a tio n i n th e fu n g i w ere made by V a n g h e lo v ic i an d S erb an ( 8 ? ) .

T h ese Roumanian i n v e s t i g a t o r s c o n c lu d e d

67

r t h a t s t e r o l s y n t h e s i s i n A s p e r g i l l u s n i e e r m ust p a s s th ro u g h s m a ll c a r to n c h a in fr a g m e n ts , inasm uch a s s t e r o l was form ed from Cg to Cg s u b s t r a t e s . E v id en ce f o r th e p a r t i c i p a t i o n o f a ld e h y d ic compounds was g a in e d by i n h i b ­ i t i n g s t e r o l s y n t h e s i s i n t h i s o rg a n ism by means o f b i s u l f i t e io n . - f i n a l l y , e v id e n c e h a s been p r e s e n te d w hich i n d i c a t e s t h a t th e tw o c a rb o n compound a c e ta ld e h y d e s e r v e s a s a key in t e r m e d i a te in t h e en zy m atic s y n t h e s i s o f th e a ro m a tic e s t e r , m e th y l-p -m e th o x y c in n e m a te , p ro d u c e d by le p id e u s ( 6 1 ) , and th u s t h a t t h i s s u b s ta n c e c o n s t i t u t e s a c o n n e c tin g l i n k i n th e d i s s i m i l a t i o n o f c a r b o h y d r a te and t h e s y n t h e s i s o f th e e s t e r by t h i s o rg an ism ( 6 2 ) . T hese o b s e r v a ti o n s s u g g e s te d th e p o s s i b i l i t y t h a t a c e ta ld e h y d e m ig ht a l s o r e p r e s e n t an e s s e n ti a ,! tw o -c a rb o n in t e r m e d i a te c o n n e c tin g th e c a t a ­ b o lis m o f c a r b o h y d r a te and t h e a n a b o lism o f l i p i d s by J^, l e p i d e u s .

T hus,

i t was c o n c e iv a b le t h a t t h i s s u b s ta n c e m ig h t p la y some r o l e i n th e form a­ t i o n o f s t e r o l by t h i s o rg a n is m . A c c o rd in g ly , L. le p id e u s was c u l t i v a t e d on a medium i n w hich e t h y l a l c o h o l r e p la c e d g lu c o s e a s c a rb o n s o u rc e .

In . th e s e e x p e r im e n ts , 20 m l.

o f 95% e t h y l a l c o h o l w ere ad d ed a s e p t i c a l l y t o one l i t e r p o r t i o n s o f s t e r i l i z e d p e p to n e -s a lt s o lu tio n .

The f l a s k s w ere in o c u la te d w ith two

week o ld m ats o f t h e o rg a n is m , and a f t e r a t h r e e month p e r io d o f in c u b a ­ t i o n , th e m y c e lia w ere f i l t e r e d o f f , and t h e u n s a p o n if i a b le f r a c t i o n i s o ­ l a t e d a s from th e mold when grown on g lu c o s e .

A c r y s t a l l i n e m a t e r i a l was

o b ta in e d w hich was p ro v en by m e ltin g p o i n t , m ixed m e ltin g p o i n t , a b s o r p tio n sp ectru m ( s e e F ig u r e X II) t o be i d e n t i c a l w ith t h e s t e r o l o b ta in e d on t h e g lu c o s e medium. T h is f i n d i n g th e n s u g g e s te d t h a t some tv ra -c a rb o n compound d id in d e e d ta k e p a r t i n t h e f o r m a tio n o f s t e r o l by t h i s o rg a n is m .

^

68 ”

**"1

In o r d e r to i n v e s t i g a t e t h e p o s s i b i l i t y t h a t t h i s s u b s ta n c e m ig h t

be a c e ta ld e h y d e , an a tte m p t was made t o t r a p t h e a ld e h y d e form ed by dehy­ d ro g e n a tio n o f th e e t h y l a l c o h o l o f th e medium, em ploying th e a ld e h y d e jb ra p p in g r e a g e n t dimedon ( d im e tly L d ih y d r o r e s o r c in o l) , th e e x p e rim e n t to c o n s i s t i n a q u a n t i t a t i v e co m p ariso n o f th e s t e r o l form ed on th e dim edonsu p p lem en ted and th e u n su p p lem en ted m ed ia, by p e r io d i c a n a l y s e s .

The

medium em ployed was t h a t u s e d i n t h e i s o l a t i o n o f th e s t e r o l from th e o r ­ ganism grown on a l c o h o l, and i n th e i n t e r c e p t i o n e x p e rim e n ts , 0 .5 gm. o f dimedon was added p e r l i t e r o f medium. F i f t y m l. p o r t i o n s o f th e s t e r i l i z e d s a l t - p e p t o n e medium, t o w hich w ere added a s e p t i c a l l y one m l. o f e t h y l a l c o h o l , w ere in o c u la te d w ith one m l. s u s p e n s io n s o f L. l e n i d e u s .

The f l a s k s w ere in c u b a te d a t 2 7 ° C ., and t h e i r -

c o n te n ts w ere a n a ly z e d a t th e end o f 0 , 2 , 5 , 4 and 6 w eeks, w ith t h e r e s u l t s shown i n T a b le X X II. T h is T a b le shows t h a t th e s t e r o l c o n te n ts o f th e m y c e lia form ed on b o th t h e d im ed o n -su p p lem e n ted and u n su p p lem en ted m edia a r e e q u i v a l e n t . H ow ever, th e c o n c e n tr a ti o n o f dim edon em ployed ( 0 .5 gm. p e r l i t e r ) was v e ry low i n co m p ariso n to th e am ount w hich w!o u ld be r e q u i r e d t o t r a p a l l th e a c e ta ld e h y d e a v a i l a b l e fro m th e a lc o h o l p r e s e n t , th e r e b y e n a b lin g m ost o f th e a ld e h y d e t o engage i n i t s n o rm al m e ta b o lic f u n c t i o n s , a s i s e v id e n t from t h e p r o g r e s s i v e e q u iv a le n c y o f th e m y c e lia l w e ig h ts i n th e two p a r a l ­ l e l e x p e r im e n ts . I t was t h e r e f o r e o f im p o rta n c e to f i n d th e m axim al c o n c e n tr a ti o n o f dimedon w hich w ould p e rm it g ro w th o f th e o rg a n ism on t h i s medium, and to d e te rm in e w h e th e r s t e r o l was form ed a t t h i s l e v e l o f tr a p p i n g a.g en t. H ence, to s e p a r a t e p o r t i o n s o f th e u s u a l p e p t o n e - s a l t medium, i n c r e a s L_

in g am ounts o f dim edon w ere added t o c o rre s p o n d t o th e f o llo w in g

-J

69 r

~i

Table XXII F o rm a tio n o f S t e r o l by L e n tin u s le p id e u s on E th y l A lc o h o l-C o n ta in in g M edia

P e r io d o f I n c u b a tio n ( i n w eeks)

M y c e lia l W eight (m gs./lO Q m l.)

A lc o h o l C o n ten t o f Medium (m g s ./m l.)

S t e r o l C o n te n t o f M ycelium (rag s./g m .)

pH o f ' Medium

ALCOHOL MEDIUM, UNSUPPLEMENT ED 0

0 .0

9 .9

0 .0

5 .7

2

8 .8

8 .8

T rac e

4 .7

5

1 1 .9

6 .8

1 .9

4 .5

4

4 5 .4

4 .5

1 .8

4 .5

6

1 4 4 .4

1 .4

1 .1

4 .2

ALCOHOL MED]CUM, SUPPLEMENTED WITH DIMEDON 0 .0

1 0 .5

o• o

L_

0

4 .3

2

9 .8

9 .0

T rac e

4 .6

1Z

1 5 .6

7 .4

1 .6

4 .5

4

5 9 .1

4 .9

1 .5

4 ,5

6

1 5 5 .0

1 .1

1 .9

c^ • Q i/

70 r

c o n c e n tr a ti o n S e r ie s

C o n c e n tr a tio n o f Dimedon ( i n grams p e r l i t e r )

1

0 .5 1.0

5 4

4 .0

F i f t y m l. p o r t i o n s o f th e s e m ed ia, each c o n ta in in g one m l. e t h y l a l c o h o l , w ere i n o c u la te d a s p r e v io u s l y , and in c u b a te d f o r f o u r w eeks, a t w hich tim e t h e i r c o n t e n ts w ere f i l t e r e d th ro u g h t a r e d c r u c i b l e s .

T hese

w ere d r i e d , washed w ith e t h e r t o rem ove t h e c r y s t a l l i n e a c e ta ld e h y d e m ethone com plex, r e d r i e d , w eig h ed , and e x t r a c t e d w ith c h lo ro fo rm i n S oxhle ts .

The r e s u l t a n t e x t r a c t s w ere c o n c e n tr a te d by e v a p o r a tio n , and t e s t -

ed f o r s t e r o l by th e L ieb erraan n -B u rch a rd r e a c t i o n , w ith th e r e s u l t s i n ­ d i c a t e d i n T a b le X X III. The c o n tin u e d p r o d u c tio n o f s t e r o l a t th e h ig h e r c o n c e n tr a ti o n l e v e l s o f dim edon, would a p p e a r t o i n d i c a t e t h a t a c e ta ld e h y d e , s in c e i t was tr a p p e d by th e dim edon, was n o t a p r e c u r s o r i n th e g e n e s is o f th e s t e r o l .

H ow ever,

i t m ust be em p h asized t h a t th e c o n c e n tr a ti o n o f dim edon i n th e s e e x p e r i ­ m ents i s m eager i n r e l a t i o n to th e am ount w hich w ould be r e q u ir e d s t o i c h i o m e tric a lly .

T h e r e f o r e , w h ile i t a p p e a r s im p o s s ib le , u n d e r e x p e r im e n ta l

c o n d i t i o n s , to t r a p a l l th e a ld e h y d e o b ta in a b le by d e h y d ro g e n a tio n o f th e a l c o h o l o f th e medium, y e t , th e in v e r s e r a t i o betw een dim edon c o n c e n tr a ti o n and m y c e lia l w e ig h t, a s i s e v id e n t from T a b le X X III, s t i l l s u g g e s ts t h a t a c e ta ld e h y d e m ig h t r e p r e s e n t th e tw o -c a rb o n i n t e r m e d i a te w hich p la y s d \e s s e n t i a l r o l e i n th e o v e r a l l m e tab o lism o f L. l e p i d e u s . L_

-J

71 r

Table XXIII E f f e c t o f I n c r e a s i n g C o n c e n tr a tio n s o f Dimedon on t h e M etab o lism o f L e n tin u s le p id e u s

S e rie s

L_

C o n c e n tr a tio n o f Dimedon

M y c e lia l W eight

(gram s p e r l i t e r )

(m gs./lO O m l.)

S te ro l T est

1

0.5

5 5 .5

P o s itiv e

2

1.0

2 2 .9

P o s itiv e

5

2 .0

1 5 .7

P o s itiv e

.4

4 .0

5 .8

T ra c e

_J

72

2 .* ‘ D i s s i m i l a t i o n o f c ( - K e t o g l u t a r i c A cid . The g e n e r a l p h a se se q u e n c e o f c a r b o h y d r a te d i s s i m i l a t i o n by woodd e s tr o y i n g f u n g i h a s b een e s t a b l i s h e d a s p ro c e e d in g v ia two s u c c e s s iv e p a th w a y s.

The f i r s t o f th e s e in v o lv e s an a l c o h o l i c f e r m e n ta tio n ( 5 9 ) ,

and t h i s i s fo llo v red by an o x id a tiv e p r o c e s s , w hereby o x a l i c a c i d i s form ed a s th e t e r m in a l p r o d u c t o f th e m e ta b o lism o f . t h e s e o rg a n ism s (6 0 , 8 5 ) .,

T h is o x i d a t i v e p r o c e s s in v o lv e s th e f u n c ti o n in g o f v a r io u s

d e h y d ro g e n a tin g enzyme s y ste m s ( 9 1 ) . The fo rm a tio n o f o x a l i c a c i d from g lu c o s e i s t h e r e f o r e c o n s id e r e d t o p ro c e e d v i a th e f o llo w in g tv ra-p ro n g ed m echanism ( 6 5 ) . G lu co se E th y l A lc o h o l A c e ta ld e h y d e A cetic- Acid.

G ly c o lic A cid

S u c c in ic A cid E um aric A cid M alic A cid

O x a lic A cid^I

I t was t h e r e f o r e o f i n t e r e s t to i n v e s t i g a t e th e s ta g e s in t e r m e d i a te

i n th e M a lic A c id

^ O x a l i c A cid c o n v e r s io n , and i t a p p e a re d t h a t th e

i f i r s t s te p i n t h i s t r a n s f o r m a tio n m ig h t be a d e h y d ro g e n a tio n o f hna,lic to o x a lo a c e tic a c id .

C o n s e q u e n tly , i t would h av e been o f i n t e r e s t to i n ­

v e s t i g a t e th e p o s s i b l e f o r m a tio n o f o x a l ic a c i d from o x a l o a c e t i c a c i d by w o o d -d e s tro y in g f u n g i , b u t s u c h an a p p ro a c h i s r e n d e r e d i m p r a c t i c a l by ^ h e ex tre m e i n s t a b i l i t y o f o x a l o a c e t ic a c i d and i t s s a l t s .

£ o r exam ple^

75

r

— j i n a s tu d y o f th e s t a b i l i t y o f o x a l o a c e t ic a c i d w ith r e l a t i o n t o pH and te m p e r a tu r e i t was fo u n d ( 8 6 ) t h a t a pH o f 2 to 5 c a u s e s more th a n 50 % d e c o m p o sitio n i n ^ h o u r , w h ile a t pH’ s 4 t o 6 ( i .

e . , th e o p tim a l pH

ra n g e f o r v fo o d -d e s tro y in g f u n g i) d e c o m p o sitio n i s more th a n 50 % i n one h o u r , and more th a n 50 % in two h o u r s .

F i n a l l y , a c c o rd in g to A lls o p p

( 2 ) , o v e r 80 % d e c o m p o sitio n t a k e s p la c e in 48 h o u r s . I n th e l i g h t o f th e s e c o n s i d e r a t i o n s , i t was d e c id e d i n s t e a d to i n ­ v e s t i g a t e th e d i s s i m i l a t i o n o f t h e n e x t h ig h e r hom olog o f o x a l o a c e t i c a i d , n am ely , th e more s t a b l e O ^ - k e t o g l u t a r i c a c i d , a compound w hich i s an im p o r ta n t in t e r m e d i a te in th e m e ta b o lism o f c a rb o h y d ra te ,, f a t and p ro te in (6 5 ). A c c o rd in g ly , i t was th e n o f i n t e r e s t to d e te rm in e ’w h eth er t h i s com­ pound c o u ld be u t i l i s e d a s carb o n s o u rc e by L^. l e p i d e u s . and i f s o , to d e te rm in e th e c o u rs e o f i t s d i s s i m i l a t i o n .

a)

C o n v e rsio n o f O ^ - K e to g lu ta r ic -^cid to O x a lic A c id . F o r th e above p u r p o s e , th e u s u a l medium (w ith g lu c o s e o m itte d ) was

su p p le m e n te d w ith 10 gram s o f o ( - k e t o g l u t a r i c a c id p e r l i t e r .

The u n su p ­

p le m e n te d s a l t - p e p t o n e medium ’was f i r s t s t e r i l i z e d by a u t o c la v i n g , and th e n , a * s o lu t io n o f d ^ - k e to g lu ta r ic a c i d s t e r i l i z e d s e p a r a t e ly by S e i t z f i l t r a t i o n , was added a s e p t i c a l l y .

(The medium was b ro u g h t to a pH o f 4 .5 w ith d i l u t e

FaOH). The above medium was in o c u la te d w ith 2 m l. o f s u s p e n s io n o f L .le p id e u s p e r 50 m l. o f medium, and th e f l a s k s were in c u b a te d a t 27°C .

On th e

t h i r t y - s e c o n d and s i x t i e t h d ay s o f in c u b a tio n , th e c o n t e n ts o f d u p l i c a t e f l a s k s w ere a n a ly z e d f o r m y c e lia l w e ig h t, o ( - k e t o g l u t a r i c a c i d , e t h y l a l c o h o l, o x a l i c a c id and pH, w ith th e r e s u l t s shown i n T a b le XXIV. L-

O b v io u s ly , h . le o id e u s h a s th e a b i l i t y o f u t i l i z i n g o ( - k e t o g l u t a r i c - 1

74

Table XXIV The D is s i m il a ti o n o f

C ^ - K e to g lu ta r ic A cid by L e n tin n s le p id e u s

52

60

1000

800

750

M y c e lia l f r e ig h t (m gs. p e r 1 0 0 m l.)

0.0

6 2 .0

6 5 .6

E th y l A lco h o l (m gs. p e r 1 0 0 m l.)

0 .0

0 .0

0 .0

O x a lic A cid (m gs. p e r 1 0 0 m l.)

0.0

0.0

11.6

dH

4 .5

4 .7

4 .9

I n c u b a tio n P e r io d ( i n d ay s) - K e t o g l u t a r i c A cid (m gs. p e r 1 0 0 m l.)

L.

o f Medium

.

75

^ c i d a s carb o n s o u r c e , and m o re o v e r, a f t e r a p p ro x im a te ly two m onths o f n c u l t i v a t i o n , o x a l i c a c i d b e g in s to a c c u m u la te a s a m e ta b o lic p r o d u c t.

b)

M echanism o f O x a lic A cid F o rm a tio n . T h ree m echanism s a p p e a re d p o s s i b l e f o r th e c o n v e rs io n o f

-k e to g lu ­

t a r i c a c id to o x a lic a c id . An o x id a tiv e d e c a r b o x y la tio n o f o( - k e t o g l u t a r i c ' a c i d would r e s u l t in s u c c i n i c a c i d , from w hich o x a l ic a c i d c o u ld be p ro d u c e d v ia p r e v io u s ly e s t a b l i s h e d p a th w a y s: COOH CO

POOH

9 ^ 2 ------- ’—

^ co2

CHp 6oO H

+

?Hg

CHg COOH

>900H 'C O O H

S e c o n d ly , a d i r e c t d e h y d ro g e n a tio n o f ^ - k e t o g l u t a r i c a c id m ig h t g iv e r i s e to o x a l o a c r y l i c a c i d , from w hich o x a l i c a c i d co u ld c o n c e iv a b ly a r is e ? POOH

COOH

co

po

C H p --------------- > CH

CHp

'“COOH

7

\\ pH

-,C O O H

F i n a l l y , a h y d r o c l a s t i c s p l i t o f o C - k e t o g lu ta r i c a c i d m ig h t r e s u l t i n p r o p io n ic a c id and o x a l i c a c id d i r e c t l y : COOH COOH o COOH CHp ------------> +

4

CHg

CH„

600H

CH o COOH

The p o s s i b i l i t y o f th e d i r e c t h y d r o c l a s t i c s p l i t o f ^ - k e t o g l u t a r i c a c i d seemed im p ro b a b le due to th e f a c t t h a t th e p r o p io n ic a c id w hich would be p ro d u c e d i s known to be t o x i c t o f u n g i ( 3 5 ) .

F u rth e rm o re , th e s tr o n g

d e h y d ro g e n a tin g sy ste m s o f w o o d -d e s tro y in g fu n g i (9 1 ) made i t seem l i k e l y

76 r t h a t some d e h y d ro g e n a tio n p r o c e s s was ta k in g p l a c e , and i n f a c t ,

“>

o ( - k e t o g l u t a r i c a c id i s known t o u n d erg o an o x i d a t i v e d e c a r b o x y la tio n t o s u c c in i c a c i d and COg when t h e s u c c in i c d e h y d ro g e n a se enzyme i s b lo c k e d by m a lo n a te ( 9 3 ) . I n an a tte m p t t o d i s t i n g u i s h b etw een th e t h r e e p o s s i b l e m echanism ^ i t was c o n s id e r e d t h a t th e d e h y d ro g e n a tio n i n d i c a t o r , r e s a z u r i n , w hich ch an g e s from b lu e t o p in k on i r r e v e r s i b l e r e d u c t io n ( 9 1 ) , m ig h t be o f v a lu e .

A c c o rd in g ly , e x p e rim e n ts w ere s e t up em p lo y in g t h i s i n d i c a t o r i n

m edia c o n t a i n i n g 0 ^ - k e t o g l u t a r i c a c i d and s u c c in i c a c i d a s carb o n s o u r c e s , t o g e t h e r w ith each o f th e s e su p p lem en ted w ith in c r e a s i n g c o n c e n tr a tio n s o f th e s u c c in i c d e h y d ro g e n a se i n h i b i t o r , m a lo n ic a c i d . R e s a z u r in ’was i n c o r p o r a te d to g iv e a c o n c e n tr a tio n o f 1 .5 1 10'n-5 m ole o f i n d i c a t o r p e r l i t e r , i n m edia o f t h e f o llo w in g c o m p o s itio n s . s e r ie s

o C - K e t o g l u t a r i c A cid % C o n c e n tr a tio n

S u c c in ic A cid % C o n c e n tr a tio n

M alonic A cid % C o n c e n tr a tio n

A -l

0 .5

0.0

0 .0 0

A -2

0 .5

0.0

0.2.5

A-3

0 .5

0 .0

0 .5 0

A-4

0 .5

0.0

1.00

B -l

0 .0

0 .5

0 .0 0

B-2

0 .0

0 .5

0 .2 5

B-3

0 .0

0

.§

0 .5 0

B-4

0 .0

0 .5

1.00

-

Each f l a s k was i n o c u la te d w ith one m l. s u s p e n s io n o f L. l e p i d e u s . Now, i f th e r e a c t i o n p r o c e e d s v i a o x id a tiv e d e c a r b o x y la tio n to s u c c in ­ i c a c i d , th e n s e r i e s A -l and B - l s h o u ld show d e f i n i t e d i s c o l o r a t i o n o f

77 r

n th e i n d i c a t o r , w h ile s e r i e s A -2, -3 and - 4 and B -2, - 3 and - 4 s h o u ld show p r o g r e s s i v e l y d e c r e a s in g i n d i c a t i o n s o f d e h y d ro g e n a tio n due t o th e i n ­ c r e a s i n g am ounts o f th e s u c c in i c d eh y d ro g e n a se i n h i b i t o r . H ow ever, i f th e f i r s t s te p in th e r e a c t i o n c o n s is te d o f a d i r e c t d e ­ h y d r o g e n a tio n o f o ( - k e t o g l u t a r i c a c i d , th e n s e r i e s A - l, - 2 , - 5 and -4 sh o u ld a l l show an e q u a lly i n t e n s e d e h y d ro g e n a tio n , r e g a r d l e s s o f th e con­ c e n t r a t i o n o f th e i n h i b i t o r . F i n a l l y , i f th e d i r e c t h y d r o c la s t ic . s p l i t w ere a c t u a l l y ta k in g p l a c e , th e n o n ly s e r i e s B - l , - 2 , - 3 and - 4 sh o u ld sho’w any d is c o lo r e d ,io n o f th e in d ic a to r. A f te r a b o u t one week o f in c u b a tio n i n th e d a r k , s e r i e s A - l and B - l showed an i n t e n s e d i s c o l o r a t i o n o f t h e i n d i c a t o r , w h ile s e r i e s A -2, - 3 , and - 4 an d s e r i e s B -2 , - 3 and - 4 showed v e ry d e f i n i t e p r o g r e s s i v e l y d e­ c re a s in g d is c o lo r a tio n s o f th e r e s a z u r in . T h e r e f o r e , th e p r o d u c tio n o f o x a l i c a c i d from

o(- k e t o g l u t a r i c

a c id

by L. l e o i d e u s can be v i s u a l i z e d a s p ro c e e d in g v ia th e f o llo w in g scheme: COOH

go

COOH fH 2 ------ »C0 2 + $Hg CHg CH COOH ioOH

gooH CH COOH . |l _________. p H O H _________ fOOH 7 CH ^CH2 —*COOH &)0H COOH

T h u s, th e f i r s t s te p i n t h i s seq u en ce p a r a l l e l s t h a t o f th e a c t io n o f th e enzyme w hich was d e s c r ib e d by Ochoa ( 6 5 ) .

L.

78

1

IV .

. DISCUSSION

n

The p u rp o s e o f th e l i g n i n p o r t i o n o f t h i s t h e s i s was th e i s o l a t i o n , in good y i e l d , o f a l i g n i n u n a l t e r e d by any ch e m ic a l t r e a tm e n t ( a s i n th e u s u a l m e th o d s ), th ro u g h t h e s e l e c t i v e c e l l u l o s e - d e g r a d i n g a c t i v i t y o f th e "brown r o t " f u n g i on wood.

I n p r a c t i c e , t h i s p ro d u c t was o b ta in e d by ex­

t r a c t i o n o f th e d ecay ed wood w ith e t h y l a l c o h o l a t room te m p e r a tu r e , a m ild and c h e m ic a lly i n e r t p r o c e d u r e . H ow ever, t h e r e e x i s t s i n wood a s m a ll am ount o f " N a tiv e L ig n in " w hich can be o b ta in e d from sound wood by such an e x t r a c t i o n p r o c e d u r e .

A ccord­

i n g l y , N a tiv e L ig n in was t o be e x p e c te d i n a n a l c o h o l i c . e x t r a c t o f d ecay ed •wood, t o g e t h e r w ith any l i g n i n w hich m ig h t be f r e e d by e n z y m a tic a c t i o n . In d e e d , a m arked s i m i l a r i t y betw een n a t i v e l i g n i n and th e l i g n i n o b ta in e d a f t e r t h e a c t i o n o f f u n g i on wood was o b s e rv e d . T h u s, i t was p o s s i b l e t o o b ta in from e n z y m a tic a lly d eca y ed w h ite LCo t s p in e wood, by t h e m ild e x t r a c t i o n p r o c e d u r e , a l i g n i n p r e p a r a t i o n w hich was v e ry s i m i l a r to n a t i v e l i g n i n .

T h is l i g n i n w a s 'i s o l a t e d i n y i e l d s s u p e r io r

t o th o s e a t t a i n a b l e from so u n d , u n in f e c te d wood.

I t seem ed t h e r e f o r e to

have b een l i b e r a t e d from i t s a s s o c i a t i o n i n th e wood a s a r e s u l t Of th e e n z y m a tic a c t i v i t y o f t h e fu n g u s c a u s in g th e r o t . A c o m p ariso n o f t h i s m a t e r i a l w ith th e s m a ll am ount o f n a t i v e l i g n i n i s o l a t a b l e from sound wood i n d i c a t e d a c l o s e s i m i l a r i t y o f th e two l i g n i n s . T h is f i n d i n g t h e r e f o r e s u g g e s te d t h a t n a t iv e l i g n i n m ig h t in d e e d be i d e n t i ­ c a l w ith th e t o t a l l i g n i n c o n t e n t o f wood. I n th e l a t t e r p e r i e s o f e x p e r im e n ts , ( i . e . , p a r t s I I I , A, 5 and 6 o f t h i s t h e s i s ) by f i r s t rem oving t h e n a t i v e l i g n i n , and th e n s u b j e c t i n g th e wood t o d e c a y , i t was p o s s i b l e t o o b ta in an e x t r a c t w hich c o n ta in e d o n ly L-

_J

79 r t h a t l i g n i n w hich was l i b e r a t e d by th e e n zy m atic a c t i v i t y o f t h e r o t .

~i

T h u s, i t can be s t a t e d t h a t t h i s l i g n i n r e s u l t e d e x c l u s i v e ly from th e a c t i o n o f th e f u n g i on th e wood. & ince t h i s p r o d u c t, i s o l a t e d i n a p p r e c ia b le y i e l d , a p p e a r s t o be i d e n t i c a l w ith t h e n a t i v e l i g n i n o f t h e vraod, i t w ould seem t h a t a l l th e l i g n i n o f t h i s wood i s a u n ifo rm c h e m ic a l e n t i t y , th e g r e a t e r p a r t o f w h ic h , i e . , t h e ne x t r a - n a t i v e ” l i g n i n , i s p h y s i c a l l y a s s o c i a t e d w ith th e c e l l u l o s e o f th e mfo o d , and n o t e x t r a c t a b l e from sound wood by i n e r t s o l v a n t s , such a s e t h y l a l c o h o l. T ak in g th e mean v a lu e o f t h e n a t i v e and th e e n z y m a tic a lly l i b e r a t e d w h ite S c o ts p in e l i g n i n s and t h e i r d e r i v a t i v e s a s b e in g m ost r e p r e s e n t a ­ t i v e , we can assum e th e l i g n i n t o be composed o f 6 4 .1 % carb o n and 6 .1 5 % h y d ro g e n , w ith 1 4 .5 % m e th o x y l.

The m eth o x y l c o n te n t o f th e p h e n y lh y -

d ra z o n e i s 15.4- % an d t h a t o f t h e a c e t a t e , 10.^2 %, Assuming a m o le c u la r w e ig h t o f .848 f o r th e l i g n i n ,!b u i l d i n g - u n i t ” (1 4 , 1 5 ) , (B ra u n s c o n s id e r s i t t o be a p p ro x im a te ly 840 (16) ) , from th e e le m e n ta ry c o m p o s itio n o f th e l i g n i n , th e m o le c u la r fo rm u la f o r o u r l i g ­ n i n b u il d in g u n i t i s c a l c u l a t e d t o b e : S in c e t h e l i g n i n c o n ta in s 1 4 .5 % m e th o x y l, t h e r e m ust be f o u r m eth­ o x y l g ro u p s p e r b u il d in g u n i t , and th e m o le c u la r fo rm u la can be e x te n d e d to :

(CH3 0 ) 4 G4 1 H4 0 0 x 2 S in c e t h e p h e n y lh y d ra z o n e c o n t a in s 1 5 .4 % m e th o x y l, t h e r e can be b u t

one c a r b o n y l g ro u p p e r b u i l d i n g u n i t , and th e fo rm u la can be f u r t h e r ex­ te n d e d t o :

(CHgO)^

^ 4 0 H4 0 ° l l j

i t h a s been shown by G la d in g (57) t h a t th e c a rb o n y l gro u p o f th e n a t ­ i v e l i g n i n b u i l d i n g u n i t i s r e s p o n s i b l e f o r th e p eak a t 550

i n th e

^ a b s o r p t io n s p e c tru m o f th e p h e n y lh y d ra z o n e o f n a t i v e l i g n i n , and t h a t th e

80

r n o n -a p p e a ra n c e o f t h i s band i n th e s p e c t r a o f th e n a t i v e l i g n i n i t s e l f "1 o r o f i t s a c e t a t e i s due t o th e a lm o s t co m p lete e n o l i z a t i o n o f t h i s c a r ­ b o n y l g ro u p i n th e l i g n i n . A c c o rd in g ly , a more c o r r e c t r e p r e s e n t a t i o n o f t h e above fo rm u la w ould b e :

(CHs 0 ) 4

^ 4 0 % 9 ° ll]

C-OH.

F i n a l l y , th e m eth o x y l c o n t e n t o f th e a c e t a t e i n d i c a t e s th e in t r o d u c ­ t i o n o f n in e a c e t y l g ro u p s i n t o t h e l i g n i n b u i l d i n g u n i t .

S in c e one o f

t h e g ro u p s a c e t y l a t e d i s t h e e n o liz e d c a r b o n y l group ( 1 0 ) , th e e x te n d e d fo rm u la f o r th e b u il d in g u n i t o f th e w h ite S c o ts p in e l i g n i n i s c a l c u l a t e d t o b e:

CHgO CH50 GHgQ ch 5 o

( oh) 8

^C-OH ✓

A more d e t a i l e d s tu d y o f t h i s p r e p a r a t i o n c o u ld g r e a t l y en h an ce th e c l a r i f i c a t i o n o f th e c o n s titu tio n o f lig n in as i t e x is ts n a tu r a lly .

A

d e s i r a b l e g o a l f o r f u t u r e r e s e a r c h would be th e i s o l a t i o n o f d im e r ic o r ,,o l i g o n-p h e n y lp ro p a n e p o ly m ers ( 1 3 ) , and t o e s t a b l i s h th e mode o f conden­ s a t i o n o f th e s e m onom eric b u i l d i n g s to n e s to t h e b u i l d i n g u n i t , and f i n a l l y t o th e l i g n i n m o le c u le i t s e l f .

The p o s s i b i l i t y o f o b ta in in g such

fr a g m e n ts by e n z y m a tic means h a s b een s u g g e s te d ( 6 8 c ) , and c o u ld c o n c e iv - . a b ly p r o v id e a v a lu a b le c lu e t o t h e f i n a l e l u c i d a t i o n o f th e s t r u c t u r e o f lig n in . The o b je c tio n h a s been r a i s e d (92) t h a t t h e f a i l u r e o f a l c o h o l to ex­ t r a c t 27% o f t h e l i g n i n p r e s e n t i n sound wood i n d i c a t e s t h a t n s .tiv e l i g ­ n i n 0,c o u ld n o t be i d e n t i c a l w ith t h e b u lk o f th e l i g n i n i n wood.

H ow ever,

t h e i n a b i l i t y to . i s o l a t e th e re m a in in g p a r t o f th e l i g n i n by t h i s m ethod i s u n d o u b te d ly due t o some p h y s i c a l phenom enon, su ch a s an i n t e r p e n e t r a t ­ in g m ix tu r e w ith t h e lo n g c e l l u l o s e c h a in s ( 7 2 ) . L '

-J

81

T h u s, t h e r e i s much e v id e n c e w hich a t t e s t s t o th e p r e s e n c e o f a p h y s i c a l u n io n b etw een th e l i g n i n and c e l l u l o s e a s th e y c o - e x i s t i n wood, i n c o n t r a d i s t i n c t i o n to a t r u e c h e m ic a l l i n k a g e .

F o r exam ple, e l e c t r o n

m ic ro g ra p h s o f wood show t h a t l i g n i n h a s an a m o rp h o u s -g ra n u la r s t r u c t u r e t h a t e x h i b i t s no i n t i m a t e c o n n e c tio n w ith c e l l u l o s e , s in c e th e f i b r i l s a p p e a re d u n changed by e x t r a c t i o n ( 5 6 ) . I n an i n v e s t i g a t i o n o f c o tto n s e e d h u l l s , bm ith and P u rv e s (82) ob­ t a i n e d i n d i r e c t e v id e n c e t o s u p p o r t th e n o n - e x is te n c e o f a l i g n i n - c e l l u l o s e c h e m ic a l l i n k a g e .

T h e ir r e s u l t s i n d i c a t e d t h a t th e c a r b o h y d r a te s and l i g ­

n in a r e p r e s e n t i n c o tto n s e e d h u l l s a s in d e p e n d e n t and s e p a r a t e c h e m ic a l e n titie s . L M tk e (53) fo u n d t h a t many p l a n t m a t e r i a l s o f te n f a i l t o g iv e t y p i ­ c a l c e l l u l o s e r e a c t i o n s b e c a u se o f th e p h y s i c a l s t r u c t u r e o f th e c e l l w a l l s , and n o t b e c a u se t h e c e l l u l o s e e x i s t s i n c h e m ic a l c o m b in a tio n w ith th e accom panying l i g n i n . F i n a l l y , a s a r e s u l t o f i n v e s t i g a t i o n s on l i g n i n i s o l a t e d w ith h y d ra ­ z in e from f i n e l y g round p l a n t m a t e r i a l ( 3 9 b ) , H u rt H ess h a s s t a t e d ( 3 9 a ) : l i r empfinden e s d a b e i a l s von d e r M atur zweckm flssig e i n g e r i c h t e t , wenn w ir e rk e n n e n , d a s s d ie Z ellw an d im S in n e e in e s m e ch an isch en G efflges m it s t a r k e r E n tv /ick lu n g von in n e r e n G re n z fM c h e n und v e r f e s t i g e n d e r W irkung d u rch K itt s u b s ta n z e n a u f g e b a u t i s t . The i d e n t i t y o f th e n a t i v e and th e e n z y m a tic a lly l i b e r a t e d l i g n i n s o f wood a l s o makes im p ro b a b le th e p r e s e n c e o f a c h e m ic a l li n k a g e b etw een l i g ­ n in and t h e c e l l u l o s e , f o r i f such were th e c a s e , one m ig h t e x p e c t some d i f f e r e n c e b etw een t h e n a t i v e and th e ” e x t r a - n a t i v e " l i g n i n s . no such d i f f e r e n c e was o b s e r v e d .

H owever,

A c c o rd in g ly , th e s e r e s u l t s seem to h av e

s u b s t a n t i a t e d th e e x i s t e n c e o f a sim p le p h y s i c a l u n io n b etw een th e s e two hiood co m p o n en ts, th e r e b y h e l p in g to u n d e r s ta n d th e r e s i s t a n c e o f th e

_j

32

r t o t a l l i g n i n c o n te n t o f th e wood to d is s o lv e i n o r g a n ic s o l v e n t s .

T hus,

t h e a ssu m p tio n t h a t d u r in g th e p r o c e s s o f l i g n i f i c a t i o n , c h e m ic a l l i n k ­ a g e s a r e fo rm ed w ith p r e v io u s l y e x i s t i n g p o ly s a c c h a r id e s (5 5 ) seem s t o be u n fo u n d e d . A lth o u g h th e pro b lem o f t h e o r i g i n o f l i g n i n i s s t i l l u n d e r d is c u s ­ s io n t h e r e e x i s t s much e v id e n c e f o r th e p o s s i b i l i t y t h a t i t a r i s e s u l t i ­ m a te ly from c a r b o h y d r a te s ( 6 5 ) .

Many s u g g e s tio n s h av e been advanced t o

e x p l a in th e n a tu r e o f t h e p r e c u r s o r s o f l i g n i n ( 5 2 ) , and th e mechanism o f th e s y n t h e s i s o f l i g n i n i n th e p l a n t ( 4 1 ) .

Most o f th e s e s u g g e s tio n s

a r e e i t h e r p u r e ly s p e c u l a t i v e , o r e l s e a r e b a se d on e v id e n c e o f an i n ­ d i r e c t o r fra g m e n ta ry c h a r a c t e r ( 6 8 a ) .

S e v e r a l i n v e s t i g a t o r s have sug­

g e s te d t h a t l i g n i n i s form ed by th e p l a n t from c e l l u l o s e th ro u g h a p r o c e s s in v o l v in g m e th v la tio n ( 2 5 ) . The q u e s tio n o f th e f o r m a tio n o f l i g n i n i s :

How, by b io c h e m ic a l

r o u t e s , can t h i s a r o m a tic p r o d u c t o f a h ig h d e g re e o f p o ly m e r iz a tio n be form ed fro m s u b s ta n c e s p r e - e x i s t i n g i n th e wood.

F re u d e n b e rg (5 5 ,5 6 ) and

R ic h tz e n h a in (7 5 , 7 # , 75) m a in ta in t h a t t h i s ta k e s p la c e th ro u g h a d e h y d r o g e n a tio n - c o n d e n s a tio n o f th e p h e n o ls inw ood, such a s t h a t o f is o e u g e n o l t o d e h y d r o - d iis o e u g e n o l, by means o f c e r t a i n d e h y d ro g e n a tin g enzym es.

In

a s e r i e s o f p u b l i c a t i o n s , th e y d e s c r ib e th e c o n d e n s a tio n s o f p h e n o ls o f th e g u a i a c y l and s y r in g y l s e r i e s by means o f d ry p r e p a r a t i o n s o b ta in e d by a l c o h o l i c f r a c t i o n a t i o n o f th e p r e s s j u i c e s o f th e m ushroom s, P s a l l i o t a . c a m p e s tr is and A g a ric u s c a m o e s tr is . T h u s, v a n i l l i c a c i d , s y r i n g i c a c i d , f e r u l i c a c i d , d i h y d r o f e r u l i c a c i d and g u a ia c y la c e to n e ( 3 6 ) , 5 -m e th y l p y r o g a l l o l - 1 , 5 -d im e th y l e t h e r ( 7 3 ) , 5 - a l l y 1 - p y r o g a l l o l - l , 3 -d im e th y l e t h e r ( 7 4 ) , 4 -m e th y l ( o r e t h y l , ^ r n - p r o p y l) g u a ia c o l and d ih y d r o c o n i f e r y l a lc o h o l ( 7 5 ) w ere a l l

83

r condensed t o p r o d u c ts c o n t a in in g a p p ro x im a te ly 65 % c a rb o n , 5 % hy d ro g ek and 15 t o 2 0 % m e th o x y l.

M ost r e c e n t l y , th e c o n d e n s a tio n o f c o n i f e r y l

a lc o h o l i s c la im e d (3 5 ) t o y i e l d a p ro d u c t w hich c o rre s p o n d s i n a l l i t s p r o p e r t i e s t o B ra u n s 1 n a t i v e b la c k s p ru c e l i g n i n ( 1 0 ) . ■H i s c la im e d t h a t i n t h e d e h y d ro g e n a tio n o f th e s e p h e n o ls , t h e r e a r e form ed th e same ty p e s o f li n k a g e s a s a r e r e c o g n iz e d to be c h a r a c t e r ­ i s t i c o f l i g n i n , and th e f o r m a tio n o f th e s e p r o d u c ts i s c o n s id e r e d to be a n a lo g o u s t o t h e f o r m a tio n o f in te r m e d ia r y p r o d u c ts i n t h e p r o c e s s o f lig n ific a tio n . H owever, a s e a r l y a s 1 8 9 7 , K laso n (49) h ad e x p re s s e d th e o p in io n t h a t l i g n i n i s a c o n d e n s a tio n o r p o ly m e r iz a tio n p r o d u c t o f c o n i f e r y l a lc o h o l (i).

L a t e r , h e s u g g e s te d t h a t c o n i f e r y l a ld e h y d e ( I I ) m ig h t be t h e b a s i s

building stone of lignin (80).

CM-CH-CH^OM

CM- C H - C H O

OH

OH

I

II

T h ese compounds in d e e d a r e s t r u c t u r a l l y r e l a t e d t o s e v e r a l o f th e known breakdow n p r o d u c ts o f l i g n i n .

T h u s, a few o f t h e num erous f i s s i o n

p r o d u c ts o f th e a r o m a tic s e r i e s w hich have been i s o l a t e d from l i g n i n ( 6 8 b) a r e 5 -m e th o x y -4 -h y d ro x y -p h e n y l-m e th y lg ly o x a l ( I I I ) , a c e to v a n i llo n e ( I V ), v a n i l l i c a c i d (V ), v a n i l l i n ( V I ) , v e r a t r i c a c i d (V II) and a n i s i c a c id ( V I I I ) . CK5 COOH «.-o t'- o %

°h m CKO

0 -^ * 1 OH

. VI

Q O O lj

V II

V III

84

R e fe re n c e i s a l s o made i n t h e l i t e r a t u r e t o th e 'fo rm a tio n o f s e v e r a l m e th o x y la te d a r o m a tic compounds by th e a c t i o n o f th e w o o d -d e s tro y in g fu n g u s L e n tin u s le p id e u s on p in e wood ( 9 ) .

The p r o d u c ts form ed w ere

m e th y l-p -m e th o x y c in n a m a te ( I X ) , m e th y l c in n a m a te (X) and m e th y l a n i s a t e ( X l ) . Ch *CK * H * c k -C O ck.k , c o o c K -, 0

DC*,

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TThe s t r u c t u r a l r e l a t i o n s h i p o f th e s e compounds t o th e b a s ic b u i l d i n g s to n e s o f l i g n i n p o s t u l a t e d by K la s o n , and th e l i g n i n f i s s i o n p r o d u c ts i s a p p a re n t from -*the f o r m u la s .

H en ce, s in c e th e f u n g a l p r o d u c ts a r e s i m i l a r

to th e l i g n i n f i s s i o n p r o d u c ts , one m ig h t h a v e assum ed t h a t th e y a r o s e from t h e l i g n i n p o r t i o n o f th e wood. H ow ever, i t h a s been s in c e d e m o n s tra te d (6 1 ) t h a t L. le p id e u s p ro d u c e s m e th y l-p -m e th o x y c in n a m a te when grown on m e d ia . c o n ta in in g - o n ly g lu c o s e a s c a r b o n 's o u r c e *

T hus, assu m in g a p r e li m i n a r y h y d r o ly s is o f c e l l u l o s e ( 6 4 ) ,

i t a p p e a r s t h a t .th e e s t e r a r o s e from th e c a r b o h y d r a te f r a c t i o n o f th e •wood r a t h e r th a n from t h e l i g n i n . F u r th e r m o re , th e f i n d i n g t h a t th e o rg a n is m , an a l c o h o l i c f e r m e n te r , gave r i s e t o th e e s t e r when grown on x y lo s e ( 6 l ) prom pted t h e c o n s id e r a ­ t i o n t h a t some t r a n s i e n t tw o -carb o n - compound m ig h t be a c t i n g a s a key in t e r m e d i a t e i n th e s y n t h e s i s o f t h e e s t e r . The e a s e w ith w hich e t h y l a l c o h o l , when s e r v in g a s c a rb o n s o u r c e , was d e h y d ro g e n a te d ( 6 .2 ) s u g g e s te d t h a t t h i s l a b i l e t r a n s i e n t compound 'was a c e t ­ a ld e h y d e .

T h is was fo u n d t o be th e c a s e , f o r when th e o rg a n ism was grown

on an e t h y l a l c o h o l - c o n t a i n i n g medium, th e e s t e r was p ro d u ced ( 6 2 ) .

How­

e v e r , when an a ld e h y d e tr a p p i n g a g e n t (dim edon) was in c o r p o r a te d i n a ^ s i m i l a r medium i n a p a r a l l e l e x p e rim e n t, o n ly th e r n e th o n e - d e r iv a tiv e ,

85

r and no m e th y l-p -m e th o x y c in n a m a te was fo rm ed .

The fo rm a tio n o f t h i s i n - 1

s o lu b le compound h ad p r e v e n te d t h e f u r t h e r u t i l i z a t i o n o f th e a c e t a l d e ­ hyde f o r th e s y n t h e s i s o f t h e e s t e r . I t was n o t i c e d t h a t when th e m ate o f L. le p id e u s was a llo w e d t o i n ­ c u b a te f o r lo n g p e r io d s o f tim e ( e . g . , 2 to 3 m o n th s), th e c r y s t a l l i n e e s t e r , form ed w h ile th e o rg a n ism was grow ing on g lu c o s e , g r a d u a l ly d i s ­ a p p e a re d from th e medium (6 2 )'.

T h is o b s e r v a tio n s u g g e s te d th e p o s s i b i l i t y

t h a t a n o th e r e n z y m a tic sy stem was a l s o p r o v id in g f o r th e s u b s e q u e n t b re a k ­ down o f th e w a t e r - i n s o l u b l e e s t e r i n t o ' a s im p le r , more s o lu b le p r o d u c t. I t was c o n c e iv a b le t h a t c e r t a i n enzym es, d is c h a r g e d i n t o t h e medium d u rin g t h e slow d i s i n t e g r a t i o n o f th e c e l l s , h ad h y d ro ly z e d th e i n s o l u b l e p s te r . T h is p o s s i b i l i t y was s tr e n g th e n e d by t h e o b s e r v a tio n t h a t a l i p a s e p r e p a r a t i o n from t h e m o ld .. F u sariu m l i n i B o lle y ( 5 2 ) , h ad t h e a b i l i t y o f h y d r o ly z in g an e m u lsio n o f m e th y l-p -m e th o x y c in n a m a te i n a r e l a t i v e l y s h o r t tim e ( 5 1 ) .

T h is i n d i c a t e d t h a t th e f i r s t s te p i n th e c a ta b o lis m o f

th e e s t e r by L. le p i d e u s m ig h t be th e l i p o l y t i c h y d r o l y s i s o f th e e s t e r t o th e f r e e a c i d ( 5 1 ) .

f u r t h e r en z y m a tic d e g r a d a tio n o f th e p -m eth o x y Z’*'

cin n am ic a c i d c o u ld c o n c e iv a b ly r e s u l t i n t h e f o r m a tio n o f some s im p le r m e th o x y la te d p ro d u c t su ch a s a n i s i c a c i d . CH-CK-cooH

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