Appl. Microbiol. 1975 Han 930 4

8/13/2019 Appl. Microbiol. 1975 Han 930 4

1/6

1975, 30(6):930.Appl. Microbiol.

Y. W. Han and A. W. Anderson

Ryegrass StrawSemisolid Fermentation of

http://aem.asm.org/content/30/6/930found at:Updated information and services can be

These include:

CONTENT ALERTS

morealerts (when new articles cite this article),Receive: RSS Feeds, eTOCs, free email

http://journals.asm.org/site/misc/reprints.xhtmlInformation about commercial reprint orders:

http://journals.asm.org/site/subscriptions/To subscribe to to another ASM Journal go to:

onN

ov

em

ber1

5,2

01

3byguest

http://a

em.a

sm.o

rg/

Downlo

adedfrom

onN

ov

em

ber1

5,2

01

3byguest

http://a

em.a

sm.o

rg/

Downlo

adedfrom
http://http//aem.asm.org/content/30/6/930http://http//aem.asm.org/content/30/6/930http://aem.asm.org/cgi/alertshttp://aem.asm.org/cgi/alertshttp://aem.asm.org/cgi/alertshttp://journals.asm.org/site/misc/reprints.xhtmlhttp://journals.asm.org/site/subscriptions/http://journals.asm.org/site/misc/reprints.xhtmlhttp://journals.asm.org/site/subscriptions/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://journals.asm.org/site/subscriptions/http://journals.asm.org/site/misc/reprints.xhtmlhttp://aem.asm.org/cgi/alertshttp://http//aem.asm.org/content/30/6/930


2/6

A P P L I E D M I C R o B I O L o G Y , D e c . 1 9 7 5 , p . 9 3 0 - 9 3 4C o p y r i g h t 0 1 9 7 5 A m e r i c a n S o c i e t y f o r M i c r o b i o l o g y V o l . 3 0 , N o . 6P r i n t e d i n U . S . A .

S e m i s o l i d F e r m e n t a t i o n o f R y e g r a s s S t r a w 1Y . W. HAN* AND A . W . ANDERSON

A g r i c u l t u r a l R e s e a r c h S e r v i c e , U . S . D e p a r t m e n t o f A g r i c u l t u r e , and D e p a r t m e n t o f M i c r o b i o l o g y , O r e g o nS t a t e U n i v e r s i t y , C o r v a l l i s , O r e g o n 9 7 3 3 1

R e c e i v e d f o r p u b l i c a t i o n 1 0 J u n e 1 9 7 5C a n d i d a u t i l i s , A u r e o b a s i d i u m p u l l u l a n s , a n d T r i c h o d e r m a v i r i d e were

grown on p r e t r e a t e d ryegrass s t r a w . T h e p r e t r e a t m e n t c o n s i s t e d o f h y d r o l y s i s o fs t r a w w i t h 0 . 5 N H 2 S 0 4 ( w a t e r - s u b s t r a t e , 3 : 1 ) a t 1 2 1 C , 1 0 0 C , a n d room t e m -p e r a t u r e a n d a d j u s t m e n t o f t h e h y d r o l y s a t e t o pH 4 . 5 t o 5 . 0 w i t h 5 N NH40H.T h e 1 2 1 C p r e t r e a t m e n t y i e l d e d a m a t e r i a l c o n t a i n i n g 30 sugar a n d 2 . 3 N .T h e f e r m e n t a t i o n was c a r r i e d on s e m i s o l i d s u b s t r a t e m o i s t u r e l e v e l , 7 5 ) i nr o t a t i n g j a r s f o r 2 t o 3 d a y s a t room t e m p e r a t u r e . T h e o r g a n i s m s grew r a p i d l yd u r i n g t h e p e r i o d f r o m 1 8 t o 4 2 h o f i n c u b a t i o n . D u r i n g t h i s p e r i o d t h e n u m b e r o fm i c r o b i a l c e l l s i n c r e a s e d b y 2 0 - t o 2 0 0 - f o l d , a n d t h e l e v e l o f NH3-N d e c r e a s e df r o m 1 . 3 t o 0 . 9 . T h e f e r m e n t a t i o n r e s u l t e d i n a f o u r f o l d i n c r e a s e i n p r o t e i n ,f i v e f o l d i n c r e a s e i n c r u d e f a t , a n d 40 i n c r e a s e i n t h e d i g e s t i b i l i t y o f s t r a w . T h eb e s t r e s u l t i n t e r m s o f i n c r e a s i n g p r o t e i n a n d d i g e s t i b i l i t y o f s t r a w was o b t a i n e dwhen C . u t i l i s was grown on s t r a w p r e h e a t e d a t 1 2 1 C .

M o r e t h a n 2 0 0 m i l l i o n t o n s o f c e l l u l o s i c a g r i -c u l t u r a l w a s t e s a r e p r o d u c e d e a c h y e a r i n t h eU n i t e d S t a t e s . N a t u r a l l y , t h e d i s p o s a l o f s u c ha l a r g e q u a n t i t y p r e s e n t s p r o b l e m s . Much o ft h i s w a s t e i s d i s p o s e d o f b y b u r n i n g , b u t s u c ha m e t h o d h a s b e e n i n c r e a s i n g l y u n d e r c r i t i c i s mb e c a u s e o f t h e a i r p o l l u t i o n t h a t r e s u l t s . M a nyc i t i e s a n d s t a t e s h a v e t o t a l l y o r p a r t i a l l yb a n n e d t h e o p e n b u r n i n g o f s t r a w a n d s i m i l a rc e l l u l o s i c a g r i c u l t u r a l w a s t e s .S t r a w c o n t a i n s c o m p o n e n t s s u c h a s c e l l u l o s ea n d h e m i c e l l u l o s e , w h i c h c o u l d make i t d e s i r a -b l e a s a n a n i m a l f e e d , e s p e c i a l l y f o r r u m i n a n t s .U n f o r t u n a t e l y , i t s l o w d i g e s t i b i l i t y a n d l o w p r o -t e i n c o n t e n t p r e s e n t l y p r e v e n t i t s u s e i n f e e d -l o t s .

V a r i o u s m e t h o d s h a v e b e e n a d v o c a t e d f o re n h a n c i n g t h e d i g e s t i b i l i t y o r t h e n u t r i t i v ev a l u e o f s t r a w . F o r e x a m p l e , d i g e s t i b i l i t y c a nb e i n c r e a s e d b y t r e a t i n g t h e s t r a w w i t h s o d i u mh y d r o x i d e . T h e n u t r i t i v e v a l u e o f s t r a w c a n b es u p p l e m e n t e d b y a d d i n g a n o n p r o t e i n n i t r o g e ns o u r c e ( 1 4 ) . I n a d d i t i o n , m a ny e f f o r t s h a v eb e e n m a d e t o p r o d u c e s i n g l e - c e l l p r o t e i n b ya p p l y i n g s u b m e r g e d m i c r o b i a l f e r m e n t a t i o n t oc e l l u l o s i c s u b s t r a t e s ( 3 , 4 . T h e a b o v e m e t h o d s ,h o w e v e r , h a v e d i s a d v a n t a g e s e i t h e r b e c a u s et h e y a r e t o o e x p e n s i v e o r b e c a u s e t h e y d o n o ty i e l d p r o d u c t s o f a c c e p t a b l e f o o d v a l u e a n dd i g e s t i b i l i t y , o r b o t h . v a r i e t y o f f o o d s a r e p r e p a r e d b y s e m i s o l i df e r m e n t a t i o n i n t h e O r i e n t ; s o y s a u c e , m i s oI T e c h n i c a l p a p e r n o . 3 9 0 6 , A g r i c u l t u r a l ] E x p e r i m e n tS t a t i o n , O r e g o n S t a t e U n i v e r s i t y , C o r v a l l i s .

s a k e , a n d t e m p e h a r e e x a m p l e s . I n t h e U n i t e dS t a t e s t h e s e m i s o l i d f e r m e n t a t i o n t e c h n i q u e h a sb e e n a d a p t e d f o r m y c o t o x i n p r o d u c t i o n 5 , 7 .T h i s p r o c e s s , u n l i k e t h a t o f s u b m e r g e d f e r m e n -t a t i o n , r e q u i r e s n o c o m p l e x f e r m e n t a t i o n c o n -t r o l s o r e x p e n s i v e d e w a t e r i n g f o r t h e p r o d u c th a r v e s t . T h u s , t h e s e m i s o l i d f e r m e n t a t i o np r o c e s s a p p e a r s t o b e i d e a l f o r p r o c e s s i n g g r a s ss t r a w f o r h i g h p r o t e i n a n i m a l f e e d .

I n t h i s i n v e s t i g a t i o n , a s e m i s o l i d f e r m e n t a -t i o n w a s c a r r i e d o u t o n r y e g r a s s s t r a w , a n dc o n d i t i o n s o f p r e t r e a t m e n t , f e r m e n t a t i o n p a t -t e r n s , a n d t h e c h a r a c t e r i s t i c s o f t h e f e r m e n t e dp r o d u c t w e r e s t u d i e d .MATERUILS AND METHODS

M i c r o o r g a n i s m s . S p e c i e s o f C a n d i d a u t i l i s NRRLY - 1 0 8 4 ) , A u r e o b a s i d i u m ( P u l l u l a r i a ) p u l l u l a n sNRRL Y - 6 2 2 0 ) , a n d T r i c h o d e r m a v i r i d e Q M 9 1 2 3 )w e r e u s e d a s t e s t o r g a n i s m s . T h e y w e r e g r o w n a n dm a i n t a i n e d o n y e a s t - m a l t e x t r a c t b r o t h , w h i c hc o n t a i n e d 0 . 3 y e a s t e x t r a c t ( D i f c o ) , 0 . 3 m a l te x t r a c t ( D i f c o ) , 0 . 5 p e p t o n e ( D i f c o ) , a n d 1 . 0 g l u -c o s e i n d i s t i l l e d w a t e r .S u b s t r a t e . A n n u a l r y e g r a s s L o l i u m m u l t i f o r mL a m ) , s u n - d r i e d a n d g r o u n d t o p a s s a 2 0 - m e s hs c r e e n , was u s e d a s t h e s u b s t r a t e . T h e s t r a w w a sf i r s t h y d r o l y z e d ; o n e p a r t o f s t r a w w a s m i x e d w i t ht h r e e p a r t s o f 0 . 5 N H 2 S O 4 a n d h e a t e d a t 1 2 1 o r1 0 0 C f o r 3 0 min o r k e p t a t room t e m p e r a t u r e ( 2 5 C )f o r 6 0 m i n . A l s o , d i f f e r e n t c o m b i n a t i o n s o f a c i d c o n -c e n t r a t i o n a n d w a t e r - t o - s u b s t r a t e r a t i o s were u s e dw i t h t h e v a r i o u s t i m e - t e m p e r a t u r e t r e a t m e n t s . T h eh y d r o l y z e d s t r a w was a d j u s t e d t o pH 4 . 5 t o 5 . 0 w i t hNH3 o r N H 4 O H .F e r m e n t a t i o n . S c r e w - c a p p e d b o t t l e s o f v a r i o u s

9 3 0

onN

ov

em

ber1

5,2

01

3byguest

http://a

em.a

sm.o

rg/

Downlo

adedfrom
http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/http://aem.asm.org/


3/6

SEMISOLID FERMENTATION OF RYEGRASS STRAW 931s i z e s were u s e d a s s m a l l - s c a l e f e r m e n t a t i o n ves-s e l s . F o r l a r g e - s c a l e f e r m e n t a t i o n , a 3 0 - g a l l o n ca -p a c i t y ( c a . 1 1 3 . 6 l i t e r s ) cement m i x e r r o t a t i n g a t 1rpm was u s e d . T h e b o t t l e s were h a l f - f i l l e d w i t hp r e t r e a t e d ( a c i d h y d r o l y z e d a n d a m m o n i a t e d ) s t r a wa n d were h e l d p e r p e n d i c u l a r t o a b o a r d 4 5 b y 6 0c m ) r o t a t i n g v e r t i c a l l y a t 0 . 5 rpm so t h a t t h e s u b -s t r a t e t u m b l e d c o n t i n u o u s l y . A c t i v e l y g r o w i n g c u l -t u r e s were i n o c u l a t e d i n t o t h e s t r a w 3 , v o l / w t ) ,a n d t h e f e r m e n t a t i o n was c o n t i n u e d f o r 1 t o 7 d a y sa t room t e m p e r a t u r e . S t e r i l e c o n d i t i o n s were m a i n -t a i n e d when s c r e w - c a p p e d b o t t l e s were u s e d ,w h e r e a s f e r m e n t a t i o n was c a r r i e d o u t u n a s e p t i c a l l ywhen t h e cement m i x e r was u s e d .A n a l y t i c a l p r o c e d u r e s . T o t a l N was d e t e r m i n e db y t h e m i c r o - K j e l d a h l m e t h o d o f P e r r i n ( 1 1 ) . NH3-Nwas d e t e r m i n e d b y t h e m e t h o d d e s c r i b e d b y J a c k s o n 6 . C r u d e p r o t e i n was d e t e r m i n e d b y m u l t i p l y i n g af a c t o r o f 6 . 2 5 t o t h e d i f f e r e n c e b e t w e e n t o t a l N a n dN H 3 - N . M o i s t u r e was d e t e r m i n e d b y d r y i n g t o ac o n s t a n t w e i g h t a t 1 0 5 C . T h e pH was m e a s u r e d ona s a m p l e b l e n d e d ( 1 : 1 0 ) i n d i s t i l l e d w a t e r . M i c r o b i a lc o u n t s were made f r o m 1 - g s a m p l e s b l e n d e d 3 0 s i n2 0 m l o f d i s t i l l e d w a t e r ; s a m p l e s were s e r i a l l yd i l u t e d a n d p l a t e d on m a l t - y e a s t e x t r a c t agar.

T o t a l sugar was d e t e r m i n e d b y t h e p h e n o l -s u l f u r i c a c i d m e t h o d ( 1 0 ) , a n d r e d u c i n g s u g a r wasd e t e r m i n e d b y t h e d i n i t r o s a l i c i l i c a c i d m e t h o d ( 1 2 ) .C e l l u l o s e , h e m i c e l l u l o s e , l i g n i n , a n d a s h were d e -t e r m i n e d b y t h e m e t h o d o f G o e r i n g a n d Va n S o e s t 2 . U n l e s s o t h e r w i s e s p e c i f i e d , a l l o t h e r c h e m i c a la n a l y s e s were c a r r i e d o u t b y t h e m e t h o d s o f t h eA s s o c i a t i o n o f O f f i l c i a l A g r i c u l t u r a l C h e m i s t s ( 1 ) .D i g e s t i b i l i t y . I n v i t r o rumen d i g e s t i b i l i t y I V R D )was d e t e r m i n e d b y t h e m o d i f i e d m e t h o d o f M e l l e n -b e r g e r e t a l . ( 9 ) : 0 . 5 g o f s u b s t r a t e a n d 3 5 ml o f rumenf l u i d were p l a c e d i n a 5 0 - m l s c r e w - c a p p e d b o t t l e a n di n c u b a t e d f o r 2 t o 3 d a y s a t 3 9 C . Rumen f l u i d waso b t a i n e d f r o m a H o l s t e i n b u l l a n d m i x e d w i t h am i n e r a l - a n d - b u f f e r m i x t u r e d e s c r i b e d b y M c D o u g a l l( 8 ) a t a r a t i o o f 1 : 1 . T h i s i n o c u l u m was g a s s e d w i t hC O 2 a n d warmed t o 3 9 C p r i o r t o i n o c u l a t i o n . Thec o n t e n t s o f e a c h b o t t l e were f i l t e r e d t h r o u g h as i n t e r e d - g l a s s c r u c i b l e , a n d t h e r e s i d u e was d r i e do v e r n i g h t a t 1 0 5 C ; w e i g h t loss was r e p o r t e d a sp e r c e n t a g e o f IVRD.

RESULTS AND DISCUSSIONP r e t r e a t m e n t o f s t r a w . C e l l u l o s e a n d h e m i -c e l l u l o s e were c o n v e r t e d i n t o sugars b y t h eH 2 S O 4 t r e a t m e n t . A s t h e c o n c e n t r a t i o n o f a c i di n c r e a s e d , t h e l e v e l o f s u g a r r e l e a s e d i n c r e a s e da n d r e a c h e d t h e m a x i m u m a t 0 . 5 N H 2 S O 4 ( F i g .1 . T h e p a t t e r n s o f s u g a r l i b e r a t i o n were s i m i l a rf o r t o t a l a n d r e d u c i n g sugars. Y i e l d o f s u g a r

was maximum, a b o u t 3 0 , when t h e s t r a w wash y d r o l y z e d w i t h 0 . 5 N H 2 S O 4 f o r 3 0 min a t1 2 1 C . S i n c e t h e a c i d h y d r o l y s i s y i e l d s a v a r i e t yo f sugars a n d t h e i r d e g r a d a t i o n p r o d u c t s , ac-c u r a t e measurement o f t h e l e v e l o f f e r m e n t a b l esugar i s d i f f i c u l t ; t h e d i n i t r o s a l i c i l i c a c i d m e t h o dd e t e r m i n e s a l l t h e r e d u c i n g groups, t h u s over-s t i m u l a t i n g t h e s u g a r y i e l d , w h e r e a s t h e p h e n o l -

s u l f u r i c a c i d m e t h o d i s r e l a t i v e l y i n s e n s i t i v e t op e n t o s e s .A t a l l a c i d c o n c e n t r a t i o n s , t h e y i e l d o f sugara t 1 2 1 C was 30 h i g h e r t h a n t h e y i e l d a t 1 0 0 C( F i g . 2 ) . A t room t e m p e r a t u r e , s t r a w appar-e n t l y was n o t h y d r o l y z e d when t h e a c i d c o n c e n -t r a t i o n was 1 . 4 N o r l e s s .The w a t e r - s u b s t r a t e r a t i o o f t h e a c i d - s t r a wm i x t u r e a f f e c t e d t h e y i e l d o f sugar ( F i g . 3 . As

t h e w a t e r c o n t e n t was i n c r e a s e d more sugarwas l i b e r a t e d f r o m t h e m i x t u r e , u n t i l t h ew a t e r - s u b s t r a t e r a t i o r e a c h e d 3 : 1 . B e y o n d t h i sp o i n t , no f u r t h e r i n c r e a s e was o b s e r v e d .S t i l l h i g h e r t e m p e r a t u r e s a n d s t r o n g e r a c i dw o u l d i n c r e a s e t h e e x t e n t o f h y d r o l y s i s . T o o b -t a i n more e x t e n s i v e h y d r o l y s i s w o u l d r e q u i r es p e c i a l e q u i p m e n t t o a c c o m m o d a t e t h e severec o n d i t i o n s , a n d t h e r e l e a s e d sugars w o u l d i n -e v i t a b l y d e g r a d e a n d c o n d e n s e t o f o r m i n s o l -u b l e r e s i n s ( 1 3 ) . T h e s e d e g r ad a t i on p r o d u c t sa re o f t e n f o u n d t o b e i n h i b i t o r y t o m i c r o o r g a -n i s m s . I n o t h e r e x p e r i m e n t s , when d i l u t e a c i d( e . g . , l e s s t h a n 1 N H 2 S O 4 ) was u s e d , p r i -m a r i l y h e m i c e l l u l o s e was h y d r o l y z e d , a n d t h e

0 . 4

I )

CD

Dr) 0 . 1

0 0 . 2 0 . 4 0 . 6 0 . 8H2 S04 N )

1 . 0

FIG. 1 . E f f e c t o f a c i d c o n c e n t r a t i o n on sugar y i e l df r o m ryegrass s t r a w . S t r a w was h y d r o l y z e d a t 1 2 1 Cf o r 30 min w i t h a w a t e r - t o - s u b s t r a t e r a t i o o f 3 : 1 .

0 0 . 2 0 . 4 0 . 6 0 . 8H 2 S O 4 N )1 . 0 i . 2 1 . 4

F I G . 2 . E f f e c t o f t e m p e r a t u r e and a c i d c o n c e n t r a -t i o n on s ug ar y i e l d f r o m ryegrass s t r a w . S t r a w wash y d r o l y z e d a t d i f f e r e n t t e m p e r a t u r e s f o r 30 min w i t haw n t e r - t o - s u b s t r a t e r a t i o o f 3 : 1 .

OA 1 2 1 CO 1 0 0 C0 . 3 A ROOM TEMP e

0 . 2 _ - _

0 . 1 US A A A

V O L . 3 0 , 1 9 7 5

I-I II

E TOTAL SUGAR0 REDUCING SUGAR

II I - 1

0 . 3

0 Q 2

iPQ

c r DD i o

onN

ov

em

ber1

5,2

01

3byguest

http://a

em.a

sm.o

rg/

Downlo

adedfrom


4/6

9 32 HAN ND ANDERSON


5/6

SEMISOLID FERMENTATION OF RYEGRASS STRAW 933i n t h e s e s t r a w s . O f t h e t h r e e o r g a n i s m s t e s t e d ,A . p u l l u l a n s p r o v i d e d t h e g r e a t e s t i n c r e a s e i nt h e p r o t e i n c o n t e n t .T a b l e 2 c o m p a r e s t h e IVRD o f f e r m e n t e d a n du n f e r m e n t e d s t r a w s . When g r o w n o n s t r a w h y -d r o l y z e d a t 1 2 1 C , C . u t i l i s i n c r e a s e d IVRDf r o m 3 2 . 6 7 t o 4 6 . 6 5 , m o r e t h a n a 40 i n c r e a s e .O t h e r f e r m e n t e d s t r a w a l s o s h o w e d i n c r e a s e dI V R D , b u t t o a l e s s e r e x t e n t . T h u s , t h e m i c r o -b i a l f e r m e n t a t i o n n o t o n l y i n c r e a s e d p r o t e i nc o n t e n t b u t a l s o t h e d i g e s t i b i l i t y o f s t r a w . T h ed i g e s t i b i l i t y o f s t r a w m i g h t b e i n c r e a s e d a s t h er e s u l t o f c o m b i n e d a c t i o n o f c h e m i c a l t r e a t m e n ta n d m i c r o b i a l f e r m e n t a t i o n , w h i c h i n c r e a s e dt h e l e v e l s o f p r o t e i n , s u g a r s , f a t , m i n e r a l s ,a n d v i t a m i n s i n t h e s t r a w . T h e s e a r e n e c e s s a r yn u t r i e n t s f o r t h e rumen m i c r o o r g a n i s m s .

T a b l e 3 l i s t s t h e c h e m i c a l c o m p o s i t i o n o ft h e f e r m e n t e d s t r a w s . C o m p a r e d t o t h a t o f t h eu n f e r m e n t e d s t r a w , t h e h e m i c e l l u l o s e c o n t e n to f t h e f e r m e n t e d s t r a w s d e c r e a s e d b y 7 5 t o9 7 , w h e r e a s t h e p r o t e i n c o n t e n t h a d i n c r e a s e dT A B L E 2 . IVRD o f f e r m e n t e d s t r a w s

T r e a t m e n t I V R D ( )C o n t r o l 1 b . . . . . . . . . . . . . . . . . . . . . . . . 3 2 . 7 5 . 1C o n t r o l 2 c . . . . . . . . . . . . . . . . . . . . . . . . 3 4 6 3 . 3A . p u l l u l a n s

1 2 1 C , 3 0 min . . . . . . . . . . . . . . . . . . . 4 4 . 4 1 . 01 0 0 C , 3 0 mino o m t e m p e r a t u r e , 6 0 min . . . . . . 3 9 . 7 + 1 . 3C . u t i l i s1 2 1 C , 3 0 min . . . . . . . . . . . . . . . . . . . 4 6 . 7 + 4 . 71 0 0 C , 3 0 min 4 2 . 8 + 2 . 6Room t e m p e r a t u r e , 6 0 min . . . . . . 3 7 . 6 1 . 3T . v i r i d e1 2 1 C , 3 0 min . . . . . . . . . . . . . . . . . . . 4 3 . 8 4 . 91 0 0 C , 3 0 min . . . . . . . . . . . . . . . . . . . 4 1 . 4 2 . 0Room t e m p e r a t u r e , 6 0 min . . . . . . 3 5 . 7 2 . 2a V a l u e s a r e means a n d s t a n d a r d d e v i a t i o n s .b U n t r e a t e d , u n f e r m e n t e d r y e g r a s s s t r a w .c T r e a t e d H 2 S O 4 a n d N H 3 ) , u n f e r m e n t e d s t r a w .

T A B L E 3 . C h e m i c a l c o m p o s i t i o n o f f e r m e n t e d s t r a w( p e r c e n t d r y m a t t e r )R y e g r a a s F e r m e n t e d s t r a ws t r a w

C o m p o s i t i o n u n -t r e a t e d l p u l u - C . u t i l i s T . v i r i d ec o n t r o l ) l a n sC r u d e p r o t e i n 3 . 1 1 3 . 9 1 2 . 4 1 0 . 9C r u d e f a t 0 . 3 8 1 . 8 9 1 . 3 8 1 . 8 5C r u d e f i b e r 3 4 . 7 3 4 . 1 3 3 . 4 3 6 . 0C e l l u l o s e 3 5 . 1 3 8 . 0 3 6 . 3 3 8 . 3H e m i c e l l u l o s e 2 1 . 7 0 . 5 5 . 4 1 . 1L i g n i n 5 . 9 9 . 8 9 . 0 9 . 4A s h 1 . 1 1 . 8 1 . 8 2 . 3

b y 3 0 0 t o 4 0 0 . T h u s , t h e a c i d h y d r o l y s i s c o n -v e r t e d m o s t o f t h e h e m i c e l l u l o s e t o f e r m e n t a b l es u g a r s , w h i c h w e r e u s e d b y t h e m i c r o o r g a n i s m sf o r p r o t e i n s y n t h e s i s . T h e a p p a r e n t i n c r e a s e o fl i g n i n c o n t e n t i n t h e f e r m e n t e d s t r a w s m ay b ed u e t o t h e i n a b i l i t y o f t h e m i c r o o r g a n i s m s t ou s e t h i s c o m p o u n d . M i c r o b i a l f e r m e n t a t i o n a l s oi n c r e a s e d t h e c r u d e f a t c o n t e n t o f t h e s t r a wf r o m 0 . 3 8 t o a m aximu m o f 1 . 8 9 .K i n e t i c s o f s e m i s o l i d f e r m e n t a t i o n . F i g u r e 4s h o w s t h e f e r m e n t a t i o n p a t t e r n o f r y e g r a s ss t r a w b y C . u t i l i s . T h e o r g a n i s m g r e w r a p i d l yd u r i n g 1 8 t o 4 2 h o f i n c u b a t i o n . D u r i n g t h i sp e r i o d , t h e n u m b e r o f m i c r o b i a l c e l l s i n c r e a s e d2 0 - t o 2 0 0 - f o l d a n d t h e r e a f t e r r e m a i n e d i n t h es t a t i o n a r y p h a s e f o r a t l e a s t 2 4 h . D u r i n g t h es a m e p e r i o d , t h e l e v e l o f NH3-N d e c r e a s e df r o m 1 . 3 t o 0 . 9 a n d t h e pH d e c r e a s e d f r o m 3 . 8t o 3 . 3 . T o t a l N r e m a i n e d c o n s t a n t t h r o u g h o u tt h e f e r m e n t a t i o n .T h e v i a b l e c e l l n u m b e r d e c r e a s e d a f t e r 6 6 ho f i n c u b a t i o n , p r o b a b l y d u e t o c e l l d e a t h a f t e re x h a u s t i o n o f o n e o r m o r e l i m i t i n g n u t r i e n t s o rg r o w t h f a c t o r s . S i n c e c a r b o n a n d n i t r o g e n w e r es t i l l i n e x c e s s , t h e l e v e l o f p h o s p h o r u s o r o t h e rm i n e r a l s m i g h t h a v e b e c o m e l i m i t i n g . T h e n a -t u r e o f t h e l i m i t i n g f a c t o r s a n d t h e o p t i m u mc o n d i t i o n s f o r m i c r o b i a l g r o w t h a r e b e i n g i n -v e s t i g a t e d . E v e n t h o u g h t h e e x p e r i m e n t w a sc a r r i e d o u t u n d e r u n a s e p t i c c o n d i t i o n s , n o a p -p a r e n t c o n t a m i n a t i o n o c c u r r e d , a s r e v e a l e d b yu n i f o r m c o l o n y t y p e s o n t h e a g a r p l a t e s .S e m i s o l i d f e r m e n t a t i o n p r o c e s s . T h e s t r a ww a s f i r s t c h o p p e d t o 0 . 2 5 - t o 1 - i n c h ( c a . 0 . 6 3 t o2 . 5 4 c m ) l e n g t h w i t h a hammer m i l l o r k n i f eg r i n d e r , a n d 1 p a r t o f s t r a w was m i x e d w i t h 3p a r t s o f 0 . 5 N H 2 S O 4 . E x c e s s a c i d w o u l d h a v en o a d d e d b e n e f i t , b u t i n s u f f i c i e n t a c i d s h o u l d b ei n a d e q u a t e f o r t h e m axi m u m r e l e a s e o f s u g a r sf r o m t h e s t r a w . T h e s t r a w was h y d r o l y z e d i nt h e p r e s s u r e c o o k e r u n d e r 1 5 l b / i n 2 s t e a m p r e s -s u r e f o r 3 0 m i n . T h e h y d r o l y z e d s t r a w was t h e nm i x e d w i t h ammonia t o o b t a i n pH 4 . 5 . Am-m o n i a g a s o r NH4OH w a s u s e d f o r t h i s p u r p o s e .When NH4OH was u s e d , a b o u t 3 0 m l o f 4 NNH40H w a s r e q u i r e d t o n e u t r a l i z e 1 0 0 g o fs t r a w . S u c h p r e t r e a t e d s t r a w was t h e n t r a n s -f e r r e d t o a f e r m e n t o r a n d i n o c u l a t e d w i t h as u i t a b l e o r g a n i s m , s u c h a s A . p u l l u l k n s , C .u t i l i s , o r T . v i r i d e . A l i q u i d c u l t u r e o r a s m a l lp o r t i o n o f r e c y c l e d f e r m e n t e d s t r a w was u s e d a si n o c u l u m .T h e f e r n e n t a t i o n w a s c a r r i e d o u t i n a r e -v o l v i n g c o n t a i n e r a t r o o m t e m p e r a t u r e . Noh e a t i n g o r c o o l i n g o f t h e f e r m e n t o r w a s n e c -e s s a r y . A f t e r f e r m e n t a t i o n , t h e s t r a w was d r i e dw i t h h o t a i r . B e c a u s e o f t h e l o w p H , l i m i t e dl e v e l o f n u t r i e n t s , a n d t h e l a r g e i n o c u l u m s i z e ,

V O L . 3 0 , 1 9 7 5

onN

ov

em

ber1

5,2

01

3byguest

http://a

em.a

sm.o

rg/

Downlo

adedfrom


6/6

934 HAN AND ANDERSON

6- EL B N J 2 00 1 2~ ~ ~ ~ ~ ~ ~

35 - 1 0 1 . 2~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~0 12 24 48 72 96 12 132 144

F e r m e n t a t i o n t i m e ( h r )F I G . 4 . K i n e t i c s o f s e m i s o l i d f e r m e n t a t i o n o f

ryegrass s t r a w b y C . u t i l i s .t h e process c o u l d b e o p e r a t e d w i t h o u t s t r i c ta d h e r e n c e t o a s e p s i s .A b a s i c a d v a n t a g e o f t h i s process i s t h a t t h ea b s o r p t i v e p r o p e r t i e s o f t h e s t r a w a re u s e d f o rp r o v i d i n g a s u b s t r a t e i n o p t i m u m c o n d i t i o n f o rc a r r y i n g o u t t h e f e r m e n t a t i o n . T h u s , i n t h ea c i d - t r e a t i n g s t e p , t h e p o r t i o n o f t h e s t r a w t h a ti s n o t h y d r o l y z e d r e t a i n s i t s f i b r o u s n a t u r e a n da c t s a s a m a t r i x t o h o l d w a t e r , sugars, a n do t h e r s o l u b l e s o l i d s . W hen t h i s mass i s am-m o n i a t e d i n t h e n e x t s t e p , t h e f o r m e d ammo-n i u m s u l f a t e i s h e l d i n t h e f i b r o u s m a t r i x w i t ht h e o t h e r s o l u b l e c o m p o n e n t s . Thus t h e r e i smade a v a i l a b l e a m a t e r i a l i n p r i m e c o n d i t i o n f o rf e r m e n t a t i o n . I t c o n t a i n s w a t e r , m i n e r a l s a l t s ,a n d sources o f c a r b o n a n d n i t r o g e n r e q u i r e df o r g r o w t h o f t h e m i c r o o r g a n i s m s . M o r e o v e r ,a l l t h e s e c o m p o n e n t s a re h e l d i n t h e m a t r i x o ft h e f i b r o u s s t r a w m a t e r i a l , f o r m i n g a s e m i -s o l i d mass so t h a t t h e f e r m e n t a t i o n can b ec a r r i e d o u t i n s i m p l e f a s h i o n b y t u m b l i n g i nt h e presence o f a i r a n d e l i m i n a t i n g a l l t h ee l a b o r a t e e q u i p m e n t a n d c o n t r o l s t h a t w o u l d b er e q u i r e d i f t h e s u b s t r a t e were a l i q u i d . F o re x a m p l e , t h e u se o f spargers a n d pumps r e -q u i r e d i n s u b m e r g e d l i q u i d f e r m e n t a t i o n s a ren o t n e e d e d . A l s o , pH a n d t e m p e r a t u r e n e e dn o t b e r i g o r o u s l y c o n t r o l l e d . F o a m i n g p r o b l e m sf r e q u e n t l y e n c o u n t e r e d i n s u b m e r g e d l i q u i df e r m e n t a t i o n s d o n o t o c c u r i n t h i s p r o c e s s .A n o t h e r a d v a n t a g e o f t h i s p r o c e d u r e i s t h a t i ty i e l d s a p r o d u c t w h i c h i n i t s e n t i r e t y w i l l prob-a b l y b e u s e f u l a s an a n i m a l f e e d . T h i s i s i nc o n t r a s t t o s y s t e m s o f s u b m e r g e d l i q u i d f e r m e n -

t a t i o n i n w h i c h t h e p r o d u c t s m u s t b e h a r v e s t e df r o m t h e f e r m e n t a t i o n b r o t h b y c e n t r i f u g a t i o no r o t h e r c o s t l y p r o c e d u r e s .ACKNOWLEDGMENT

W e a c k n o w l e d g e t h e t e c h n i c a l a s s i s t a n c e o f P a kLa m Yu a n d S u e M e i Y a n g .LITERATURE CITED

1 . A s s o c i a t i o n o f O f f i c i a l A g r i c u l t u r a l C h e m i s t s . 1 9 7 0 .O f f i c i a l m e t h o d s o f a n a l y s i s , 1 1 t h e d . A s s o c i a t i o n o fO f f i c i a l A g r i c u l t u r a l C h e m i s t s , W a s h i n g t o n , D . C .2 . G o e r i n g , H . K . , a n d P . J . Va n S o e s t . 1 9 7 0 . F o r a g ef i b e r a n a l y s i s . U . S . D e p a r t m e n t o f A g r i c u l t u r eH a n d b o o k n o . 3 7 0 . U . S . D e p a r t m e n t o f A g r i c u l t u r e ,W a s h i n g t o n , D . C .3 . H a n , Y . W . , a n d C . D . C a l l i h a n . 1 9 7 4 . C e l l u l o s e f e r -m e n t a t i o n : e f f e c t o f s u b s t r a t e p r e t r e a t m e n t o n m i -c r o b i a l g r o w t h . A p p l . M i c r o b i o l . 2 7 : 1 5 9 - 1 6 5 .4 . H a n , Y . W . , C . E . D u n l a p , a n d C . D . C a l l i h a n . 1 9 7 1 .S i n g l e c e l l p r o t e i n f r o m c e l l u l o s i c w a s t e s . F o o dT e c h n o l . 2 5 : 3 2 - 3 5 , 5 6 .5 . H e s s e l t i n e , C . W. 1 9 7 2 . S o l i d s t a t e f e r m e n t a t i o n s .B i o t e c h n o l . B i o e n g . 1 4 : 5 1 7 - 5 3 2 .6 . J a c k s o n , M . L . 1 9 6 0 . S o i l c h e m i c a l a n a l y s i s . P r e n t i c e -H a l l , I n c . , E n g l e w o o d C l i f f s , N . J .7 . L i n d e n f e l s e r , L A . , a n d A . C i e g l e r . 1 9 7 5 . S o l i d -s u b s t r a t e f e r m e n t a t i o n f o r o c h r a t o x i n A p r o d u c t i o n .A p p l . M i c r o b i o l . 2 9 : 3 2 3 - 3 2 7 .8 . M c D o u g a l l , E . I . 1 9 4 8 . S t u d i e s o n r u m i n a n t s a l i v a .1 . T h e c o m p o s i t i o n a n d o u t p u t o f s h e e p ' s s a l i v a .B i o c h e m . J . 4 3 : 9 9 - 1 0 8 .9 . M e l l e n b e r g e r , R W . , L . D . S a t t e r , M . A . M i l l e t t , a n dA . J . B a k e r . 1 9 7 0 . An i n - v i t r o t e c h n i q u e f o r e s t i -m a t i n g d i g e s t i b i l i t y o f t r e a t e d a n d u n t r e a t e d w o o d .J . A n i m . S c i . 3 0 : 1 0 0 5 - 1 0 1 1 .1 0 . M o n t g o m e r y , R . 1 9 6 1 . F u r t h e r s t u d i e s o f t h e p h e n o ls u l f u r i c a c i d r e a g e n t f o r c a r b o h y d r a t e s . B i o c h i m .B i o p h y s . A c t a 4 8 : 5 9 1 .1 1 . P e r r i n , C . G . 1 9 5 3 . R a p i d m o d i f i e d p r o c e d u r e f o r t h ed e t e r m i n a t i o n o f K j e l d a h l n i t r o g e n . A n a l . C h e m .2 5 : 9 6 8 - 9 7 1 .1 2 . P e t t e r s o n , G . , a n d J . P o r a t h . 1 9 6 6 . A c e l l u l o l y t i c e n -z y m e f r o m P e n i c i l l i u m n o t a t u m , p . 6 0 3 - 6 0 7 . I n S . P .C o l w i c k a n d N . 0 . K a p l a n ( e d ) , M e t h o d s i n e n -z y m o l o g y , v o l . 8 . A c a d e m i c P r e s s I n c . , New Y o r k .1 3 . S a e m a n , J . F . 1 9 4 5 . K i n e t i c s o f w o o d s a c c h a r i f i c a t i o n :h y d r o l y s i s o f c e l l u l o s e a n d d e c o m p o s i t i o n o f s u g a r si n d i l u t e a c i d a t h i g h t e m p e r a t u r e . I n d . E n g . C h e m .3 7 : 4 3 - 5 2 .1 4 . T i l l m a n , A . D . 1 9 7 3 . E f f e c t o f f o r m s o f n o n p r o t e i nn i t r o g e n a n d m e t h o d s o f p r o c e s s i n g o n t h e i r n u t r i -t i o n a l v a l u e , p . 1 8 0 - 2 0 0 . I n E f f e c t o f p r o c e s s i n g o nt h e n u t r i t i o n a l v a l u e o f f e e d s , P r o c e e d i n g s o f a s y m -p o s i u m . N a t i o n a l A c a d e m y o f S c i e n c e s , W a s h i n g t o n ,D . C .1 5 . W a i m , A . C . , J . G u g g o l z , G . 0 . K o h l e r , H . G . W a l k e r ,J r . , a n d W. N . G a r r e t t . 1 9 7 2 . I m p r o v i n g d i g e s t i b i l i t yo f s t r a w f o r r u m i n a n t f e e d b y a q u e o u s a m m o n i a . J .A n i m . S c i . 3 5 : 1 0 9 - 1 1 2 .

A P P L . M I C R O B I O L .

onN

ov

em

ber1

5,2

01

3byguest

http://a

em.a

sm.o

rg/

Downlo

adedfrom

Documents

Appl. Microbiol. 1975 Han 930 4