Foundations of Biochemical Engineering - American Chemical Society

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Growth Characteristics of Microorganisms in Solid State Fermentation for Upgrading of Protein Values of Lignocelluloses and Cellulase Production D. S. C H A H A L Devinder Chahal Enterprises, Inc., 312-1800 Baseline Road, Ottawa, Ontario Canada K2C 3N1

Solid state fermentation (SSF) is considered to r e q u i r e no complex c o n t r o l s , and to have many advantages over l i q u i d state fermentation. Upgrading of p r o t e i n values o f l i g n o c e l l u l o s e s and cellulase production by SSF holds great promise. Pretreatment of l i g n o c e l l u l o s e s with alkali o r steam i s necessary to break lignin and carbohydrate bonds f o r s u c c e s s f u l growth o f microorganisms i n SSF. Filamentous fungi seem to be the most s u i t a b l e organisms f o r SSF. Fungal hyphae can penetrate i n t o plant c e l l lumina through p i t s , c r a c k s , o r by boring holes through the cell w a l l . Once i n s i d e the cell lumen, the fungus utilizes the cell w a l l from i n s i d e . I t is postulated that sequence o f synthesis o f c e l l u l a s e s i n the fungal hyphal t i p is t r i g g e r e d by p h y s i c a l s i g n a l s sent by cellulose. Finally, the substrate is completely utilized to produce fungal biomass rich in p r o t e i n and/or c e l l u l a s e s , depending on the microorganism. " S o l i d S t a t e F e r m e n t a t i o n " (SSF) i s defined as a process whereby a n i n s o l u b l e s u b s t r a t e i s fermented with sufficient moisture, but without f r e e water. I n the l i q u i d s t a t e o r s l u r r y s t a t e f e r m e n t a t i o n , on the o t h e r hand, t h e s u b s t r a t e i s s o l u b i l i z e d o r suspended a s f i n e p a r t i c l e s i n a l a r g e v o l u m e o f w a t e r . I n most l i q u i d s t a t e f e r m e n t a t i o n s ( L S F ) o r submerged f e r m e n t a t i o n s s u b s t r a t e c o n c e n t r a t i o n s r a n g i n g f r o m 0.5 t o 5% a r e u s e d . Now, i n a number o f f e r m e n t a t i o n s t h e c o n c e n t r a t i o n o f t h e s u b s t r a t e h a s gone up t o 1 0 % t o i n c r e a s e t h e p r o d u c t i v i t y p e r u n i t t i m e . Solid s t a t e f e r m e n t a t i o n i s c o n s i d e r e d t o r e q u i r e no complex c o n t r o l s and t o h a v e many a d v a n t a g e s o v e r t h e L S F (J.) · The SSF for upgrading the p r o t e i n values of lignocelluloses, ( a g r i c u l t u r a l , f o r e s t r y and a n i m a l w a s t e s ) i s b e c o m i n g a f o c u s o f a c t i v i t y f o r some r e s e a r c h e r s ( 2 , 3 , 3 a , 4 ) . R e c e n t l y i t has been calulated (5) that high c e l l u l a s e activity p e r u n i t volume o f f e r m e n t a t i o n b r o t h i s t h e most i m p o r t a n t f a c t o r i n o b t a i n i n g s u g a r

0097-6156/83/0207-0421$06.50/0 © 1983 American Chemical Society

In Foundations of Biochemical Engineering; Blanch, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.


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c o n c e n t r a t i o n s o f 20-30% from h y d r o l y s i s o f c e l l u l o s e i n a p r o c e s s for ethanol p r o d u c t i o n f r o m c e l l u l o s i c m a t e r i a l s . I t has a l s o been c o n f i r m e d ( 6 ) t h a t c e l l u l a s e a c t i v i t y per u n i t volume can be i n c r e a s e d by i n c r e a s i n g t h e c e l l u l o s e c o n c e n t r a t i o n i n t h e medium. But i t i s n o t p o s s i b l e to handle more t h a n 6% cellulose in c o n v e n t i o n a l fermenter because o f r h e o l o g i c a l problems. In o r d e r , t h e r e f o r e , t o i n c r e a s e t h e c e l l u l o s e c o n c e n t r a t i o n h i g h e r t h a n 6%, SSF seems t o be t h e most a t t r a c t i v e a l t e r n a t i v e (5)· To yam a (_7) has a l r e a d y shown t h a t s u g a r s y r u p o f 22% c o n c e n t r a t i o n can be o b t a i n e d by h y d r o l y z i n g d e l i g n i f i e d r i c e s t r a w o r b a g a s s e w i t h t h e c o n c e n t r a t e d c e l l u l a s e s o b t a i n e d by T r i c h o d e r m a r e e s e i i n SSF. R e c e n t l y , i n t e r e s t i n t h e p r o d u c t i o n o f a m y l a s e s and c e l l u l a s e s b y SSF i s i n c r e a s i n g b e c a u s e a g r e a t demand o f t h e s e enzymes i s envisaged i n t h e n e a r f u t u r e . These enzymes a r e r e q u i r e d t o c o n v e r t s t a r c h and c e l l u l o s e i n t o g l u c o s e f o r f u r t h e r f e r m e n t a t i o n i n t o e t h a n o l to a l l e v i a t e the s h o r t a g e o f l i q u i d f u e l i n the world 0 5 ) . But t h e s u r v e y o f s u c h l i t e r a t u r e i n d i c a t e d t h a t v e r y l i t t l e is known a b o u t t h e g r o w t h c h a r a c t e r i s t i c s and behavior of m i c r o o r g a n i s m s i n SSF. O r i g i n of S o l i d S t a t e

Fermentation

The o r i g i n o f SSF i s l o s t i n the m i s t o f a n t i q u i t y when r e l a t i o n s h i p of microorganism were e s t a b l i s h e d w i t h t h e i r h o s t (animals or plants; l i v i n g or dead). In nature microorganisms grow i n c l o s e a s s o c i a t i o n w i t h s o l i d s u b s t r a t e s t o o b t a i n t h e i r n u t r i t i o n s a p r o p h y t i c a l l y or p a r a s i t i c a l l y . One o f the earliest records of SSF t r a c e d out b y Chang ( 8 ) was t h e c u l t i v a t i o n o f paddy s t r a w mushroom, V o l v a r i e l l a v o l v a c e a , i n t h e Canton r e g i o n o f C h i n a ' s Kwangtung p r o v i n c e d u r i n g the Chow D y n a s t y , a b o u t 3000 y e a r s ago. However, u n t i l 20 years ago, almost no scientific r e s e a r c h has b e e n done on t h i s s p e c i e s . The o t h e r e a r l y r e c o r d o f SSF was g r o w i n g o f " S h i i t a k e " ( L e n t i n u s edodes) by t h e C h i n e s e and J a p a n e s e on wood l o g s a b o u t 20 c e n t u r i e s ago (9)· The s p o r e s a r e i n o c u l a t e d i n wood l o g s and a r e l e f t t o i n c u b a t e f o r many months before the mushrooms are harvested for eating. Agaricus c a m p e s t r i s ( A . b i s p o r u s ) , a commonly c u l t i v a t e d mushroom i n Europe and W e s t e r n c o u n t r i e s , h a s b e e n grown i n c a v e s i n F r a n c e s i n c e t h e t i m e o f L o u i s XLV ( 1 6 8 3 - 1 7 1 5 ) ( 1 0 ) . Of g r e a t h i s t o r i c a l r e l e v a n c e t o modern t e c h n o l o g y i s t h e J a p a n e s e " K o j i " p r o c e s s , i . e . g r o w i n g o f A s p e r g i l l u s o r y z a e on r i c e ( o r o t h e r c e r e a l s ) i n s o l i d state. P r o d u c t i o n o f " K o j i " has been i n p r a c t i c e i n J a p a n a t l e a s t s i n c e t h e e i g h t h c e n t u r y f o r p r o d u c t i o n o f Saké - the m o s t traditional alcoholic d r i n k i n J a p a n . The K o j i p r o c e s s was i n t r o d u c e d t o t h e W e s t e r n W o r l d by Takamine i n 1891 f o r t h e production of fungal diastase on a l a r g e s c a l e (11)· The K o j i p r o c e s s i s now b e i n g e x p l o i t e d f o r t h e p r o d u c t i o n o f a m y l a s e s and c e l l u l a s e s ( 7 , 1 2 ) . A n o t h e r e a r l y s o l i d s t a t e f e r m e n t a t i o n was t h e d i s c o v e r y o f g a l l i c a c i d p r o d u c t i o n i n g a l l n u t s p i l e d i n a heap and moistened w i t h water. I t was v a n Teigham ( 1 8 6 7 ) who f i r s t e s t a b l i s h e d t h a t A s p e r g i l l u s n i g e r was r e s p o n s i b l e f o r t h i s f e r m e n t a t i o n ( 9 ) .



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The records f o r the production of R o q u e f o r t cheese from s h e e p ' s m i l k i n c a v e s o f S o u t h e r n F r a n c e go b a c k a b o u t a thousand years. It was established by Thorn ( 1 3 ) t h a t t h e s p e c i a l c h a r a c t e r i s t i c s o f R o q u e f o r t c h e e s e was due t o the growth o f a f u n g u s , Pénicillium r o q u e f o r t i . T h i s o r g a n i s m grows d e e p i n t o c h e e s e b l o c k s u n d e r l i m i t e d 0^ s u p p l y - a p e r f e c t e x a m p l e o f s o l i d state fermentation. The s p e c i a l f l a v o u r o f Camembert c h e e s e i s due t o t h e g r o w t h o f Pénicillium c a m e m b e r t i on the surface of c h e e s e blocks· Nature of the

Substrate

The SSF can o n l y be a p p l i e d to i n s o l u b l e s u b s t r a t e s l i k e c e r e a l g r a i n s , o i l s e e d s , and l i g n o c e l l u l o s e s ( a g r i c u l t u r a l crop residues and f o r e s t r y wastes). The c e r e a l g r a i n s and o i l s e e d s a r e composed o f e a s i l y a v a i l a b l e f o r m o f c a r b o h y d r a t e s ( s t a r c h e s ) , p r o t e i n s , m i n e r a l s , e t c . Thus a w i d e v a r i e t y o f m i c r o o r g a n i s m s a r e able t o g r o w on these substrates in SSF. However, mild p r e t r e a t m e n t s a r e s t i l l r e q u i r e d t o make them s u i t a b l e s u b s t r a t e s . C e r e a l s l i k e r i c e g r a i n s a r e used a s s u c h w i t h o u t any treatment whereas wheat g r a i n s require p e a r l i n g before use. During t h i s p r o c e s s t h e s u r f a c e o f wheat g r a i n s i s a b r a d e d t o p r o v i d e s u i t a b l e s i t e s f o r c o l o n i z a t i o n by t h e m i c r o o r g a n i s m s . I n c a s e o f c o r n and s o y b e a n the g r a i n s a r e b r o k e n i n t o f i v e o r s i x p i e c e s to provide s u i t a b l e s u r f a c e f o r the growth o f the m i c r o o r g a n i s m s , as the hard seed c o a t s a r e v e r y d i f f i c u l t f o r many m i c r o o r g a n i s m s t o c o l o n i z e . O a t s h a v e t o be d e h u l l e d f o r b e t t e r g r o w t h . A l l t h e s e g r a i n s a r e very s u i t a b l e f o r the production o f a f l a t o x i n s and various fermented o r i e n t a l foods ( 1 ) . The most s e r i o u s problem encountered i n SSF of grains, especially rice, i s that t h e s u b s t r a t e s become s t i c k y and form compact m a s s e s i n w h i c h movement o f a i r i s r e s t r i c t e d . The g r a i n s a l s o a g g l o m e r a t e i n t o compact m a s s e s due t o f u n g a l g r o w t h o n t h e i r s u r f a c e s . T h e r e f o r e , t i m e l y and p r o p e r a g i t a t i o n i s r e q u i r e d to b r e a k s u c h a g g l o m e r a t e s and p r o v i d e b e t t e r a e r a t i o n . P r o d u c t i o n o f a f l a t o x i n s and fermented oriental foods on grains i s much e a s i e r than the use of l i g n o c e l l u l o s e s . The carbohydrates of l i g n o c e l l u l o s e s are not easily available and d r a s t i c pretreatments are r e q u i r e d . Moreover, l i g n o c e l l u l o s e s are a l s o n o t a s r i c h i n p r o t e i n s and o t h e r n u t r i e n t s a s are grains. L i g n o c e l l u l o s e s are very c o m p l e x s u b s t r a t e s and a r e composed o f t h r e e m a j o r c o m p o n e n t s : h e m i c e l l u l o s e s , c e l l u l o s e , and lignin. Only a few microorganisms, l i k e white-rot fungi (Polyporus v e r s i c o l o r ) , are able t o g r o w on such m a t e r i a l s (14). The white-rot f u n g i h a v e c o m p l e t e enzyme s y s t e m s t o u t i l i z e a l l t h r e e c o m p o n e n t s . S i m i l a r l y , i t has b e e n shown t h a t P l e u r o t u s o s t r e a t u s (15 ) and Stropharia rugosoannulata (4) can u t i l i z e a l l three components o f wheat s t r a w . On the o t h e r h a n d , many m i c r o o r g a n i s m s c a n n o t u t i l i z e h e m i c e l l u l o s e s ; o f t h o s e t h a t c a n , most a r e u n a b l e to metabolize c e l l u l o s e .



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L i g n i n i n the p l a n t c e l l w a l l n o t o n l y e n c r u s t s t h e c e l l u l o s e m i c r o f i b r i l s i n a s h e a t h - l i k e manner, b u t i s bonded p h y s i c a l l y and c h e m i c a l l y t o t h e p l a n t p o l y s a c c h a r i d e s (16)« Lignin-carbohydrate bonds form m e t a b o l i c b l o c k s that greatly limit the a c t i o n o f m i c r o b i a l h e m i c e l l u l a s e s and c e l l u l a s e s . P h y s i c a l l y , l i g n i n f o r m s a b a r r i e r s u p p r e s s i n g t h e p e n e t r a t i o n by p o l y s a c c h a r i d e - d i g e s t i n g enzymes ( 17 ) . U n l e s s t h e l i g n i n i s d e p o l y m e r i z e d , s o l u b i l i z e d , o r removed, t h e cellulose and the hemicelluiοses cannot be metabolized by most microorganisms· As m e n t i o n e d e a r l i e r , o n l y w h i t e - r o t and b r o w n - r o t f u n g i ( B a s i d i o m y c e t e s ) a r e a b l e t o u t i l i z e carbohydrates from l i g n o c e l l u l o s e s but t h e y a r e v e r y s l o w growing (4,14,15,18,19). Therefore, these f u n g i are not v e r y promising, from the economic p o i n t o f v i e w as candidates to ferment l i g n o c e l l u l o s e s f o r the p r o d u c t i o n o f a n i m a l feed or cellulases. The case is s i m i l a r with other basidiomycetes especially mushrooms. A g a r i c u s bisporus, Lentinus edodes, Volvariella v o l v a c e a , and P l e u r o t u s o s t r e a t u s t a k e a l o n g t i m e (1-2 months) t o p r o d u c e f r u i t i n g b o d i e s ( m u s h r o o m s ) . Mushroom g r o w i n g , however, i s j u s t i f i e d b e c a u s e o f t h e i r economic v a l u e a s f o o d d e l i c a c i e s . Pretreatment

of L i g n o c e l l u l o s e s

Pretreatment o f l i g n o c e l l u l o s e s i s the f i r s t requirement f o r t h e 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 w h i c h a r e u n a b l e t o g r o w on the untreated s u b s t r a t e but w h i c h , o t h e r w i s e , g r o w r a p i d l y and make c e r t a i n products i . e . Trichoderma r e e s e i f o r c e l l u l a s e s ; and Chaetomium cellulolyticum for single-cell protein (SCP) production. Pretreatments t h a t i n c r e a s e the d i g e s t i b i l i t y of lignocelluloses f o r production of SCP and c e l l u l a s e s h a v e b e e n d i s c u s s e d i n d e t a i l by v a r i o u s w o r k e r s ( 2 0 , 2 1 , 2 2 ) . Only those p r e t r e a t m e n t s w h i c h c o u l d make t h e s u b s t r a t e most s u i t a b l e f o r SSF are d i s c u s s e d v e r y b r i e f l y below: Grinding/Ball-Milling. Grinding/ball-milling of ligno celluloses to a v e r y s m a l l p a r t i c l e s i z e r e s u l t s i n the exposure o f more s u r f a c e a r e a f o r the g r o w t h o f m i c r o o r g a n i s m s and also r e d u c e s c r y s t a l l i n i t y (23 ) . The m a i n p r o b l e m i n u s i n g f i n e powder o f l i g n o c e l l u l o s e f o r SSF i s t o s u p p l y good a e r a t i o n , a s t h e m o i s t f i n e powder o f l i g n o c e l l u l o s e w o u l d f o r m a compact mass b y i t s own weight. Improper a e r a t i o n w i l l reduce the growth o f the organism considerably. Frequent agitation and f o r c e d a e r a t i o n may be n e c e s s a r y t o k e e p up t h e g r o w t h o f t h e o r g a n i s m . Alkali. Sodium h y d r o x i d e and aqueous ammonia c a u s e e x t e n s i v e s w e l l i n g and s e p a r a t i o n o f s t r u c t u r a l e l e m e n t s , and lead to the f o r m a t i o n o f c e l l u l o s e I I whose X - r a y p a t t e r n d i f f e r s c o n s i d e r a b l y t h a t o f f r o m c e l l u l o s e I . F i v e t o s i x grams o f sodium hydroxide per 100 grams o f wood seem t o be n e c e s s a r y f o r t h e maximum e f f e c t (24,25). This l e v e l of a l k a l i i s e s s e n t i a l l y e q u i v a l e n t to the combined a c e t y l and c a r b o n y l c o n t e n t s o f wood. T h i s l e a d s t o the



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p o s t u l a t e t h a t the main consequence o f a l k a l i treatment is the saponification of i n t e r m o l e c u l a r e s t e r b o n d s , t h u s p r o m o t i n g the s w e l l i n g o f wood beyond w a t e r - s w o l l e n d i m e n s i o n s and favouring increase enzymatic and m i c r o b i o l o g i c a l penetrati> η into the c e l l - w a l l f i n e s t r u c t u r e ( 2 2 ) . B o t h s o d i u m h y d r o x i d e and ammonia treatments proved t o be v e r y u s e f u l i n i n c r e a s i n g the i n v i t r o d i g e s t i b i l i t y o f l i g n o c e l l u l o s e s ( 2 1 ) as w e l l a s t h e i r u t i l i z a t i o n as carbon source f o r SCP production i n SSF (26 ) . These pretreatments a l s o r e t a i n the f i b r o u s s t r u c t u r e of c e l l u l o s e . The fibrous structure i s very conducive f o r the growth o f the o r g a n i s m s because o f e a s y p e n e t r a t i o n o f enzymes, f u n g a l hyphae and a i r i n t h e s u b s t r a t e . S t e a m . Steam treatment of l i g n o c e l l u l o s e s under high p r e s s u r e ( 2 7 - 3 0 ) i s now b e c o m i n g an i m p o r t a n t p r e t r e a t m e n t t o make t h e s u b s t r a t e e a s i l y a c c e s s i b l e t o h y d r o l y t i c enzymes (31-33). The s u b s t r a t e i s a l s o s t e r i l i z e d , and changed i n t o a f i b r o u s f o r m . A f i b r o u s s u b s t r a t e i s b e t t e r t h a n t h e powdered f o r m f o r SSF. The chemical changes i n steam-treated wood depend on t h e temperature, pressure, and time of exposure to steam. Hemicelluloses a r e h y d r o l y z e d to s o l u b l e sugars by o r g a n i c a c i d s , m a i n l y a c e t i c a c i d d e r i v e d from a c e t y l a t e d p o l y s a c c a r i d e s present in wood ( 2 8 ) . Under more d r a s t i c c o n d i t i o n s , s e c o n d a r y r e a c t i o n s occur which r e s u l t i n the formation of furfural, hydroxymethyl f u r f u r a l , and their precursors by d e h y d r a t i o n o f p e n t o s e s and hexoses, respectively. It has been reported (34 ) that phenolic-like compounds increased from 0.43 t o 5.3% in steam-pretreated bagasse. Since phenolic-like compounds and f u r f u r a l s are u s u a l l y t o x i c t o most m i c r o o r g a n i s m s (35 , 3 6 ) , s u c h t r e a t e d l i g n o c e l l u l o s e s may not be good substrates for SSF, because the toxic compounds a r e i n a c o n c e n t r a t e d f o r m when t h e s u b s t r a t e i s i n m o i s t f o r m . T h e r e f o r e , w a s h i n g out o f the toxic compounds f r o m s t e a m - t r e a t e d s u b s t r a t e s w i l l be n e c e s s a r y b e f o r e u s i n g them f o r SSF. Toxic compounds, on the other hand, are diluted w i t h water i n s l u r r y s t a t e f e r m e n t a t i o n and have l i t t l e e f f e c t on t h e g r o w t h o f m i c r o o r g a n i s m s . T h e r e was no adverse effect of these toxic compounds o n Τ. reesei for cellulase p r o d u c t i o n i n s l u r r y o f 4% steam t r e a t e d wood ( 3 2 ) . Sodium Chlorite ( N a C l O ^ ) . Sodium chlorite, a strong o x i d i z i n g a g e n t , h a s b e e n used f o r removing l i g n i n during the preparation of " H o l o c e l l u l o s e " , the t o t a l carbohydrate p o r t i o n of l i g n o c e l l u l o s e (37 ) . G e o r i n g and Van S e o t ( 3 8 ) d e m o n s t r a t e d that in vitro digestibility of straws i s increased with NaClO^ treatment. Chahal et a l . (39) reported t h a t p r o t e i n p r o d u c t i v i t y increased c o n s i d e r a b l y on NaC102 ~ d e l i g n i f i e d wheat straw fermented w i t h C o c h l i o b o l u s s p e c i f e r . T h i s t r e a t m e n t , by removing lignin, exposes the s u r f a c e o f h e m i c e l l u l o s e s and c e l l u l o s e f o r e n z y m a t i c a t t a c k and a l s o c r e a t e s c a p i l l a r i e s i n the substrate cell w a l l f o r d e e p p e n e t r a t i o n o f enzymes and h y p h a e . However, t h i s process of d e l i g n i f i c a t i o n i s not economically attractive because of the high cost of chemicals involved (38). Sodium


426

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c h l o r i t e t r e a t e d s u b s t r a t e , on t h e other h a n d , i s composed of h e m i c e l l u l o s e s and c e l l u l o s e , w h i c h w o u l d be a good s u b s t r a t e f o r the production of hemicellulases ( e s p e c i a l l y xylanases) and cellulases s i m u l t a n e o u s l y i n m o n o c u l t u r e or mixed c u l t u r e . There is a great future f o r s u c h an enzyme s y s t e m ( m i x t u r e of h e m i c e l l u l a s e s and c e l l u l a s e s ) f o r the complete h y d r o l y s i s o f l i g n o c e l l u l o s e s i n t o monomer s u g a r s . Choice of

Microorganism

In n a t u r e , b a c t e r i a g r o w b e s t o n l y when i n a l i q u i d p h a s e , o r at least when t h e n u t r i e n t s are i n free water. Likewise, s i n g l e - c e l l e d f u n g i , t h e y e a s t s , g r o w w e l l when t h e n u t r i e n t s are in a s o l u b l e f o r m . Such m i c r o o r g a n i s m s may n o t be a b l e t o g r o w s u c c e s s f u l l y i n SSF w h e r e s u b s t r a t e c a r b o n i s n o t available in soluble form. A limited success i n converting l i g n o c e l l u l o s i c m a t e r i a l s i n t o a n i m a l f e e d by u s i n g b a c t e r i a (Cellulomonas sp., Alcaligenes faecalis) or yeasts ( A u r e o b a s i d i u m p u l l u l a n s and C a n d i d a u t i l i s ) has b e e n r e p o r t e d i n s e m i - s o l i d s t a t e f e r m e n t a t i o n ( 3 , 3a) . Low protein yields i n the final product m i g h t be a t t r i b u t e d to the fact that i n such system the unicellular o r g a n i s m s ( b a c t e r i a and y e a s t s ) were u n a b l e t o p e n e t r a t e d e e p i n t o the t i s s u e f o r complete u t i l i z a t i o n o f the s u b s t r a t e . Streptomycetes have not been t r i e d f o r SSF so f a r but H e s s e l t i n e (_1) has r e p o r t e d t h a t he has seen a preparation in w h i c h S t r e p t o m y c e s a u r e o f a c i e n s was grown on m i l l e t s e e d s , t o be used i n s w i n e f e e d m i x e s i n t h e U.S.S.R. On the o t h e r h a n d , f i l a m e n t o u s f u n g i t y p i c a l l y g r o w i n n a t u r e on s o l i d s u b s t r a t e s , s u c h a s , wood, s e e d s , s t e m s , r o o t s and l e a v e s of p l a n t s , w i t h o u t t h e p r e s e n c e o f f r e e w a t e r (_1) . H i g h p r o t e i n c o n t e n t s o f 20-24% i n t h e f i n i s h e d p r o d u c t h a v e b e e n r e c o r d e d by Chahal et a l . (2) i n SSF of corn stover with Chaetomium cellulolyticum. High p r o t e i n content i n the final product has b e e n a t t r i b u t e d t o t h e f a c t t h a t t h e hyphae o f C. c e l l u l o l y t i c u m h a v e t h e power t o p e n e t r a t e deep i n t o the intercellular and i n t r a c e l l u l a r spaces f o r b e t t e r u t i l i z a t i o n o f the s u b s t r a t e ( 4 0 ) . Most f i l a m e n t o u s f u n g i h a v e s u c h i n t r u s i o n power. A i s t (41) has given a good r e v i e w o f t h e p e n e t r a t i o n o f f u n g a l h y p h a l t i p s i n t o p l a n t c e l l w a l l s . He concluded that t h e mechanism o f fungal penetration i n t o s u b s t r a t e s c o u l d be m e c h a n i c a l a n d / o r e n z y m a t i c . I t i s , t h e r e f o r e , e v i d e n t t h a t the c h o i c e of the microorganisms f o r s u c c e s s f u l SSF i s l i m i t e d t o f i l a m e n t o u s o r g a n i s m s - f u n g i and actinomycetes. Mechanism o f F u n g a l G r o w t h D u r i n g

Solid State

Fermentation

F u n g a l hyphae a r e s e p t a t e , t u b u l a r , u n i s e r i a t e f i l a m e n t s , on a v e r a g e 10-15 ym i n d i a m e t e r . The g r o w t h i n l e n g t h o c c u r s a t t h e t i p and i s c o n f i n e d t o t h i s a r e a , so t h a t i n s e p t a t e hyphae when a cell i s cut o f f from the apex i t i s no l o n g e r c a p a b l e o f a n y



20.

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Solid State

Fermentation

427

s i g n i f i c a n t increase i n length. T h e r e i s t h u s no increase in i n t e r s e p t a l d i s t a n c e s , a l t h o u g h t h e r e may be i n c r e a s e s i n d i a m e t e r and w a l l t h i c k n e s s . The o l d e r p o r t i o n o f the hypha d i s i n t e g r a t e s , whereas the apex c o n t i n u e s t o g r o w i n t o new sites in the substrate. A u t o r a d i o g r a p h s made o f hyphae w h i c h h a v e b e e n fed with b r i e f pulses o f t r i t i a t e d w a l l precursors demonstrated that i n c o r p o r a t i o n i s h i g h e s t i n t h e a p i c a l 1 ym and f a l l s o f f r a p i d l y after the first 5 ym, however, there is still appreciable i n c o r p o r a t i o n f r o m 5-75 ym b e h i n d the t i p ( 4 2 ) . H y p h a l w a l l s a r e u s u a l l y two l a y e r e d w i t h an i n n e r l a y e r o f m i c r o f i b r i l l a r m a t e r i a l s , u s u a l l y c h i t i n o r c e l l u l o s e , o r b o t h and an outer l a y e r o f amorphous g l u c a n . The c h i t i n i s composed o f 3-1, 4 l i n k e d N - a c e t y l glucosamine residues and cellulose i s composed o f (3-1,4 l i n k e d g l u c o s e u n i t s . The g l u e an s o f t h e o u t e r l a y e r s o f c e l l w a l l s a r e composed o f h i g h l y b r a n c h e d (3-1, 3 l i n k e d g l u c a n w i t h (3-1, 6 l i n k e d b r a n c h e s . These compounds g i v e r i g i d c e l l w a l l s t o the h y p h a e . The r i g i d n a t u r e o f t h e w a l l b e h i n d the apex and the complex system of b r a n c h i n g ensure t h a t the o l d e r r e a r w a r d p a r t o f t h e hypha i s f i r m l y a n c h o r e d i n t h e s u b s t r a t e and enable the hyphal t i p to e x e r t very considerable forward m e c h a n i c a l p r e s s u r e a s i t e x t e n d s u n d e r t u r g o r (42 ) . C o u p l e d w i t h the production o f e x t r a c e l l u l a r enzymes and the much b r a n c h e d hyphal system, the f u n g i can thus c o m p l e t e l y permeate even the hardest substrate. Fungal Growth i n S o l i d Substrate i n N a t u r e . In n a t u r e , a c c o r d i n g to t h e s t u d y o f C o w l i n g (43 ) , f u n g a l g r o w t h on solid substrate can be of three general t y p e s e x h i b i t e d by wood-inhabiting fungi: Wood-Staining F u n g i . These f u n g i a r e o f two t y p e s : (a) s u r f a c e m o l d s , s u c h a s , T r i c h o d e r m a l i g n o r u m , t h a t d e v e l o p on t h e e x t e r n a l s u r f a c e o f t h e s u b s t r a t e ; and (b) p e n e t r a t i n g f u n g i , C e r a t o c y s t i s spp. t h a t d e v e l o p deep w i t h i n wood, most c o n s p i c u o u s l y i n r a y p a r e n c h y m a . These f u n g i j u s t g r o w on r e s e r v e f o o d and do n o t a f f e c t t h e s t r e n g t h of the substrate cell w a l l s . They m e r e l y p r o d u c e s t a i n s i n wood due t o the presence o f pigments i n t h e i r hyphae. Soft-Rot Fungi. These f u n g i a t t a c k t h e e x p o s e d s u r f a c e s o f wood and wood p r o d u c t s t h a t a r e more o r l e s s c o n s t a n t l y saturated with w a t e r . These f u n g i a r e confined to c y l i n d r i c a l c a v i t i e s c r e a t e d by t h e m s e l v e s w i t h i n t h e s e c o n d a r y w a l l s o f wood cells. B a i l e y and Vestal (44) reported f o r the f i r s t time t h a t the s o f t - r o t f u n g i a t t a c k t h e wood p a r a l l e l to the o r i e n t a t i o n of cellulose molecules in the secondary w a l l s of the cell. S i m i l a r l y , s p i r a l c r a c k i n g along the angle of the fibrils of cellulose i n t h e s e c o n d a r y w a l l o f D o u g l a s f i r wood c e l l s c a u s e d by Fomes p i n i was o b s e r v e d b y P r o c t o r ( 4 5 ) . Soft-rot fungi such as Chaetomium spp. and X y l a r i a s p p . a r e a l i t t l e more a g g r e s s i v e than wood-stain f u n g i , i n that they are able to penetrate deeper into the s u b s t r a t e , but they remain r e s t r i c t e d along the



428

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o r i e n t a t i o n o f the c e l l u l o s e f i b r i l s . They a r e unable to cause complete decay o f the a f f e c t e d t i s s u e because o f t h e i r poor c e l l u l a s e systems. T y p i c a l Wood-Destroying F u n g i . Two g r o u p s o f t h e s e f u n g i a r e d i s t i n g u i s h e d on the b a s i s o f c o l o r and texture of the decayed wood: w h i t e - r o t f u n g i , s u c h as P o l y p o r u s v e r s i c o l o r , produce l i g h t c o l o r e d wood m a i n l y b e c a u s e o f the utilization of lignin along with p o l y s a c c h a r i d e s , w h i l e brown-rot f u n g i , such as, P o r i a monticola, p r o d u c e brown c o l o r e d wood mainly because of utilization of polysaccharides although some l i g n i n i s also degraded · B o t h w h i t e - and b r o w n - r o t f u n g i i n h a b i t t h e wood c e l l l u m i n a and p e n e t r a t e f r o m one c e l l t o a n o t h e r , e i t h e r through n a t u r a l o p e n i n g s , s u c h as pits, or d i r e c t l y by b o r i n g a h o l e . Proctor ( 4 5 ) r e p o r t e d t h a t t h e f u n g a l hyphae p e n e t r a t e t h e wood c e l l wall by a n e n z y m a t i c mechanism r a t h e r t h a n by m e c h a n i c a l f o r c e b u t A i s t ( 4 1 ) s u p p o r t e d the i d e a t h a t t h e p e n e t r a t i o n o f f u n g a l h y p h a l t i p s into plant c e l l wall could be a m e c h a n i c a l and/or enzymatic m e c h a n i s m . The b o r e - h o l e s a r e formed b e c a u s e o f t h e c l o s e c o n t a c t of surface of hyphal t i p a g a i n s t wood c e l l w a l l s u r f a c e . The c e l l u l a s e system produced a t the t i p i s a b l e to solubilize wood cell w a l l f i b e r s , t h e r e b y m a k i n g room f o r f u r t h e r p e n e t r a t i o n by the hyphal t i p . T h i s process c o n t i n u e s t i l l the hyphal t i p e n t e r s i n t o the n e x t c e l l l u m e n . The f u n g a l a t t a c k g o e s on f r o m one c e l l to t h e o t h e r t i l l the w h o l e t i s s u e i s d e c a y e d . The highly localized nature o f the d i s s o l u t i o n i n v o l v e d i n the formation of these bore-holes by the white-rot fungus, P o l y p o r u s v e r s i c o l o r , i s i l l u s t r a t e d i n F i g u r e 1 ( 4 3 ) . The hyphae r e s p o n s i b l e f o r f o r m a t i o n of the h o l e s are a u t o l y z e d as soon as t h e t i p o f t h e hypha r e a c h e s t h e lumen where i t g e t s more s u i t a b l e c o n d i t i o n s f o r f u r t h e r d e v e l o p m e n t . That c o u l d be t h e m a i n r e a s o n why t h e d i s s o l u t i o n o f wood c e l l w a l l r e m a i n s r e s t r i c t e d t o a s i z e a l i t t l e b i g g e r t h a n t h e d i a m e t e r o f p e n e t r a t i n g h y p h a . Where t h e hypha s t a y s f o r long t i m e , d i s s o l u t i o n beyond the b o r e - h o l e i s n o t i c e d , a s shown i n F i g u r e 2 (43 ) . The r e m n a n t s o f hypha are also seen i n the b o r e - h o l e . Figure 2 shows t h a t d i s s o l u t i o n e x t e n d s more l o n g i t u d i n a l l y than t r a n s v e r s a l l y . This i n d i c a t e s that i t i s e a s i e r f o r the c e l l u l a s e s y s t e m t o move a l o n g the o r i e n t a t i o n o f the f i b r e s i n t h e c e l l w a l l . Figure 2 also shows more d i s s o l u t i o n on t h e e x t r e m e l e f t and e x t r e m e r i g h t s i d e s o f c e l l w a l l s , the s u r f a c e s o f the lumina of two adjacent cells. This d i s s o l u t i o n i s due t o the c e l l u l a s e s y s t e m p r o d u c e d b y t h e hyphae g r o w i n g i n t h e cell lumina. The development o f hyphae within the wood c e l l lumen shown i n F i g u r e 3 ( 4 6 ) a l s o shows d i s s o l u t i o n of secondary w a l l along the hyphae f o r m i n g erosion troughs, which i n d i c a t e s that i n i t i a l d i s s o l u t i o n of c e l l u l o s e o c c u r s v e r y c l o s e to the s u r f a c e o f f u n g a l hyphae. A s i m i l a r type o f g r o w t h b e h a v i o r ( p e n e t r a t i o n o f hyphae t h r o u g h c e l l w a l l , v i a p o r e s o f t r a c h a i d s and ray c e l l s ) was also shown by another wood-inhabiting fungus, Phanerochaete chrysosporium ( 4 7 ) .



20.

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Solid State

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I t i s c l e a r from these o b s e r v a t i o n s (43,45,46) t h a t the most active cellulase s y s t e m i s p r o d u c e d on t h e s u r f a c e o f t h e f u n g a l hypha w h i c h i s a b l e t o p r o d u c e a b o r e - h o l e when i t i s p e n e t r a t i n g the cell w a l l t r a n s v e r s e l y and where t h e enzyme s y s t e m has r e s t r i c t e d movement, and p r o d u c e s e r o s i o n t r o u g h s i n i t i a l l y along the hyphae when a t t a c k i n g the i n n e r s u r f a c e o f t h e c e l l l u m e n . But i n t h e l a t t e r s i t u a t i o n t h e enzyme s y s t e m i s a b l e t o p e n e t r a t e q u i c k l y and deeply along the o r i e n t a t i o n of fibrils i n the s e c o n d a r y w a l l s , ahead of the hyphal surface and to cause c o n s i d e r a b l e d e c a y . The t a n g e n t i a l s e c t i o n i n F i g u r e 4 shows a l m o s t c o m p l e t e d i s s o l u t i o n o f s e c o n d a r y w a l l s o f t h e two a d j a c e n t fiber trachied c e l l s , l e a v i n g behind o n l y the middle l a m e l l a , which are r e s i s t a n t to c e l l u l a s e system, being highly lignified ( 4 3 ) . The f u n g a l hypha i n c r o s s s e c t i o n i s a l s o s e e n on t h e r i g h t s i d e o f the F i g u r e 4. Figure 4 clearly i n d i c a t e s that i t is e a s i e r f o r the o r g a n i s m t o u t i l i z e the c e l l w a l l s t a r t i n g f r o m the i n s i d e s u r f a c e o f t h e c e l l lumen t o w a r d s t h e o u t s i d e , t h e middle lamella. It i s inferred from the growth b e h a v i o r of the typical wood-inhabiting fungi that t h e m o s t a c t i v e c e l l u l a s e s y s t e m was p r o d u c e d on t h e s u r f a c e o f f u n g a l hypha when i t was i n close contact with c e l l u l o s e . The c e l l u l a s e s y s t e m p r o d u c e d under s u c h c o n d i t i o n s was able to hydrolyze native cellulose with high c r y s t a l l i n i t y , on t h e o t h e r hand t h e enzyme s y s t e m p r o d u c e d by Τ. r e e s e i i n LSF was n o t very effective i n hydrolyzing cellulose until t h e c r y s t a l l i n i t y was reduced by v a r i o u s physical or chemical pretreatments as i n d i c a t e d i n the recent evaluation of enzymatic h y d r o l y s i s o f c e l l u l o s e 05)· I n LSF t h e f u n g a l hyphae and c e l l u l o s e p a r t i c l e s do n o t r e m a i n i n c l o s e c o n t a c t with each other f o r long t i m e due to h i g h a g i t a t i o n i n the c o n v e n t i o n a l fermenter. That m i g h t be t h e r e a s o n t h a t enzyme p r o d u c e d in LSF is not so e f f e c t i v e f o r h y d r o l y s i s o f c r y s t a l l i n e c e l l u l o s e . It i s , t h e r e f o r e , l i k e l y t h a t c e l l u l a s e s y s t e m p r o d u c e d i n SSF will not o n l y h a v e h i g h c e l l u l a s e a c t i v i t y p e r u n i t o f f e r m e n t a t i> η b r o t h 05), b u t will a l s o be m o s t a c t i v e i n h y d r o l y z i n g the crystalline cellulose. Observations on the Fungal Growth in Solid State Fermentation. F u n g a l g r o w t h i n s o l i d s t a t e fermentâti> η o f sodium hydroxide-treated corn s t o v e r has b e e n r e p o r t e d by C h a h a l e t a l . (40) i n d e t a i l . They h a v e r e p o r t e d t h a t Chaetomium c e l l u l o l y t i c u m p r o v e d t o be t h e b e s t o r g a n i s m f o r u p g r a d i n g t h e p r o t e i n v a l u e s o f c o r n s t o v e r f o r a n i m a l f e e d i n g . The o r g a n i s m g r e w p r o f u s e l y on corn stover w i t h i n f i v e days. V i s u a l examination i n d i c a t e d that t h e m y c e l i u m i m p r e g n a t e d the e n t i r e s u b s t r a t e . Microscopic examination of the s u b s t r a t e p a r t i c l e s showed t h a t c e l l s o f t h e s u b s t r a t e t i s s u e were l o o s e l y h e l d t o g e t h e r and tended t o s e p a r a t e e a s i l y when p r e s s e d . I t i n d i c a t e d t h a t sodium h y d r o x i d e t r e a t m e n t s o l u b i l i z e d l i g n i n and weakened the b o n d s of adjacent c e l l s . F i g u r e 5 shows b r o k e n e n d s o f c e l l s on t h e s i d e s



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Figure 1. Bore holes in spruce tracheid made by the hyphae of white rot fungus P o l y p o r u s versicolor (X 20,250). Reproduced, with permission, from Ref. 43. Copyright 1965, Syracuse University Press.

Figure 2. Advanced stage of decay around bore hole and inner side of secondary wall of two fiber tracheids of sweetgum sapwood, X 2700. Reproduced, with permission, from Ref. 43. Copyright 1965, Syracuse University Press.



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Figure 3. Trough formed by decaying of secondary wall of birch vessels in the vicinity of the fungal hyphae of Polystictus versicolor. Reproduced, with permission, from Ref. 46.

Figure 4. Almost complete utilization of secondary walls of two adjacent cells leaving middle lamella intact. Reproduced, with permission, from Ref. 43. Copyright 1965, Syracuse University Press.



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a s w e l l as on t h e e n d s o f t h e s u b s t r a t e p a r t i c l e . Such b r o k e n and exposed c e l l s were t h e f i r s t s i t e s t o be a t t a c k e d by t h e o r g a n i s m . F i g u r e 6 shows m y c e l i a l g r o w t h on s u c h b r o k e n e x p o s e d c e l l s o n t h e l a t e r a l s i d e o f the s u b s t r a t e p a r t i c l e . When s u c h s u b s t r a t e p a r t i c l e s , c o l o n i z e d by the organism, were p r e s s e d slightly under a cover s l i p , they d i s i n t e g r a t e d easily. The d i s i n t e g r a t e d m a t e r i a l c o n t a i n e d some s u b s t r a t e c e l l s s h o w i n g p e n e t r a t i o n by hyphae and d e v e l o p m e n t o f m y c e l i a l g r o w t h i n the c e l l lumina a t v a r i o u s s t a g e s . In F i g u r e 7 p e n e t r a t i o n of a s i n g l e hypha i n t o the l u m e n o f a t h i n l o n g i t u d i n a l f i b e r c e l l i s c l e a r l y s e e n . As s o o n a s t h e hypha e s t a b l i s h e s i t s e l f in the lumen a t h i c k m y c e l i a l mass i s d e v e l o p e d w i t h i n t h e c e l l ( F i g u r e 8). I t was c o n c l u d e d f r o m t h e s e o b s e r v a t i o n s t h a t t h e hyphae of C. c e l l u l o l y t i c u m entered i n t o the c e l l lumen t h r o u g h n a t u r a l openings, mechanical breaks, or spaces ( c r e a t e d by s o l u b i l i z a t i o n o f hemic e l l u i ο se s and l i g n i n d u r i n g sodium h y d r o x i d e - t r e a t m e n t ) i n the c e l l w a l l of the substrate t i s s u e . Once i n s i d e t h e cell lumen, the hypha d i g e s t e d the c e l l w a l l s t a r t i n g f r o m the i n s i d e towards the o u t s i d e . U l t i m a t e l y , the cell collapsed leaving behind m y c e l i a l biomass r i c h i n p r o t e i n . Reese ( 4 8 ) has a l s o r e p o r t e d t h a t i n a t t a c k i n g c o t t o n , many f u n g i p e n e t r a t e d through t h e f i b e r w a l l i n t o t h e lumen and d i d most o f t h e i r d i g e s t i n g f r o m within (Figure 9). S i m i l a r l y B r a v e r y ( 4 6 ) has r e p o r t e d the g r o w t h o f P o l y s t i c t u s v e r s i c o l o r i n t h e wood c e l l lumen and has shown t h a t d i g e s t i o n of the c e l l i s i n i t i a t e d from i n s i d e to outside (Figure 3). P o s t u l a t e d Mechanism o f F u n g a l G r o w t h i n S o l i d

State

M i c r o o r g a n i s m s may posses the p o t e n t i a l a b i l i t y to perform many m e t a b o l i c activities which are not obligatory for the maintenance of c e l l u l a r f u n c t i o n and w h i c h come i n t o p l a y o n l y u n d e r c e r t a i n s p e c i a l e n v i r o n m e n t a l c o n d i t i o n s . Such activities are t y p i c a l l y concerned with energy-yielding metabolism ( 4 9 ) . L i b e r a t i o n o f h y d r o l y t i c enzymes i s an a c t i v e f u n c t i o n o f living fungal cells (50). D u r i n g g r o w t h on c e l l u l o s e , enzymes are liberated, c h i e f l y at the hyphal t i p . They d i f f u s e t o the substrate and d i g e s t i t . The h y d r o l y s i s p r o d u c t s e n t e r i n t o t h e f u n g u s c y t o p l a s m . The hypha t h e n grows i n t o the d i g e s t e d region and m a i n t a i n s c o n t i n u a l i n t i m a t e c o n t e n t w i t h t h e s u b s t r a t e ( 4 8 ) . Whether c e l l u l a s e s a r e a d a p t i v e o r c o n s t i t u t i v e enzymes has n o t b e e n r e s o l v e d so f a r . I t has b e e n r e p o r t e d t h a t T. r e e s e i c a n p r o d u c e c e l l u l a s e s i n t h e p r e s e n c e o f i n d u c e r s s u c h as c e l l o b i o s e , s o p h o r o s e , c e l l u l o s e ( 5 1 , 5 2 , 5 3 ) . On the o t h e r hand, r e c e n t l y a small quantity of c e l l u l a s e production has been r e p o r t e d by hypereelluiase m u t a n t s o f Τ. reesei while growing i n glucose medium u n d e r n o r m a l g r o w t h c o n d i t i o n s (54 , 5 5 ) * There i s e v i d e n c e (48) t h a t g l u c o s e i s n o t a t r u e i n d u c e r but i s m e t a b o l i z e d t o an inducer, probably a $-glucoside. I t appears that the 3-1, 4 glucosidic l i n k a g e must be p r e s e n t i n s o l u b l e compounds t h a t a c t



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a s i n d u c e r s o f c e l l u l a s e . But i n n a t u r e when t h e f u n g a l t i p makes i t s c o n t a c t w i t h c e l l u l o s e f o r the f i r s t t i m e , no such solutes (inducers) are a v a i l a b l e to t r i g g e r the s y n t h e s i s o f c e l l u l a s e w i t h i n the c e l l . I t i s known ( 4 9 ) t h a t the enzymatic machinery for the performance o f these f a c u l t a t i v e metabolic a c t i v i t i e s i s u s u a l l y s y n t h e s i z e d by c e l l s o n l y i n response to a specific c h e m i c a l s i g n a l from the e n v i r o n m e n t . C e l l u l o s e , b e i n g i n s o l u b l e , c a n n o t e n t e r i n t o t h e c e l l t o send any c h e m i c a l s i g n a l s f o r the synthesis of c e l l u l a s e s . Under s u c h c o n d i t i o n s ( i n t h e a b s e n c e o f s o l u b l e i n d u c e r s ) , i t i s p l a u s i b l e t h a t a mere p h y s i c a l c o n t a c t of cellulose with the f u n g a l h y p h a l t i p i s e n o u g h t o send some s o r t o f p h y s i c a l ( i n s t e a d of chemical) s i g n a l s through the w a l l of the fungal cell to the nucleus t o s y n t h e s i z e s p e c i f i c RNA t o produce the r e q u i r e d i n d u c e r ( p r o b a b l y a B - g l u c o s i d e ) as mentioned earlier (48). I t has a l s o been p o i n t e d out e a r l i e r t h a t a c l o s e c o n t a c t b e t w e e n c e l l u l o s e and the o r g a n i s m i s n e c e s s a r y t o t r i g g e r c e l l u l a s e synthesis (56,57). As e a r l y a s 1960, i t was p o i n t e d out b y M a n d e l s and Reese ( 5 2 ) t h a t s o l u b l e p r o d u c t s o f enzyme a c t i o n are natural inducers of the enzymes t h a t a t t a c k i n s o l u b l e s u b s t r a t e s . On t h i s t h e o r y t h e y assumed t h a t s m a l l amounts of i n d u c i b l e enzymes ( c e l l u l a s e s ) a r e p r o d u c e d even i n t h e a b s e n c e o f i n d u c e r ( c e l l o b i o s e ) . When t h e i n s o l u b l e s u b s t r a t e ( c e l l u l o s e ) i s p r e s e n t , i t i s h y d r o l y z e d and the s o l u b l e products (especially cellobiose) thus e n t e r the cell and induce more enzymes (cellulases). The above assumption a l s o supports the p o s t u l a t e t h a t a mere p h y s i c a l c o n t a c t o f t h e o r g a n i s m w i t h c e l l u l o s e may be r e s p o n s i b l e t o t r i g g e r the s y n t h e s i s o f c e l l u l a s e s . The p o s t u l a t e d mechanism o f m e t a b o l i s m of c e l l u l o s e by a c o m p l e t e c e l l u l a s e complex p r o d u c e d a t t h e t i p o f t h e f u n g a l hypha is based on the observation recorded by various workers ( 4 0 - 5 0 , 5 2 , 5 6 , 5 7 , 5 8 ) . The mechanism given i n Figure 10 is e x p l a i n e d as f o l l o w s : 1.

P l a u s i b l y p h y s i c a l s i g n a l s a r e t r a n s m i t t e d from c e l l u l o s e to the f u n g a l c e l l n u c l e u s t o t r i g g e r the s y n t h e s i s o f the f i r s t enzyme ( E ) , w h i c h causes splitting of fibrils and microfibrils f r o m the c r y s t a l l i n e p o r t i o n o f c e l l u l o s e f i b e r . The a c t i o n o f E- h e r e i s t h e same a s e x p l a i n e d by Reese (48) for C^. The is still a c o n t r o v e r s i a l enzyme and i s c o n f u s e d w i t h e x o - 1 , 4 - $ - D - g l u c a n c e l l o b i o h y d r o l a s e . The most important r o l e of C^ to s p l i t g l u c o s e polymer c h a i n s from c r y s t a l l i n e c e l l u l o s e p r o p o s e d i n 1950 ( 5 9 ) i s s t i l l retained here as w e l l as b y Reese ( 6 0 ) . T h e r e f o r e , t h e r e i s e v e r y p o s s i b i l i t y t h a t c l o s e c o n t a c t o f c e l l u l o s e and the organism i s most i m p o r t a n t t o p r o d u c e C^(E^) enzyme, i f n o t , f o r o t h e r components o f c e l l u l a s e s ( e x o - and e n d o - g l u c o n a s e s ) . 1

2.

Subsequent s y n t h e s i s o f the s e c o n d enzyme ( E ~ ) i s triggered. E~ i s composed o f t h r e e c o m p o n e n t s : ( i ) Endo 1, 4 - $ - D - g l u c a n glueanohydrolase ( e n d o - g l u e a n a s e ) w i t h random attack on



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Figure 5. Growth of C h a e t o m i u m cellulolyticum in NaOH-treated corn stover. Substrate particles showing broken cells, first sites for attack by the organism (X 94.5). From Ref. 40.

Figure 6.

Hyphal growth on sites like those shown in Figure 5. From Ref. 40.



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Figure 7.

Penetration by a single hyphae into cell lumen through the broken end (X 94.5). From Ref. 40.

Figure 8.

Mycelial biomass developed in the cell lumen (X

94.5). From Ref. 40.



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Figure 9. M e m n o n i e l l a echinata in the lumen of cotton fiber showing utilization of secondary wall from inside towards outside. Reproduced, with permission, from Ref. 49. Copyright 1959, University of Washington Press.

Figure 10.

Utilization of secondary wall from the inside of the lumen of wood cell, as explained in the text.



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glucose polymer c h a i n s to break I t Into smaller chains (oligomers). Some g l u c o s e u n i t s a r e a l s o r e l e a s e d d u r i n g t h i s reaction, ( i i ) Exo-1, 4-3-D-glucan glueοhydrolase ( e x o - g l u e a n a s e ) removes g l u c o s e u n i t s f r o m the non-reducing end of glucose polymer c h a i n s , ( i i i ) Exo-1,4-3-D-glucan cellobiohydrolase ( e x o - g l u e a n a s e ) removes c e l l o b i o s e from non-reducing ends o f g l u c o s e polymer c h a i n s . T h i s compound enzyme releases a mixture of oligomers, c e l l o b i o s e and glucose. The s e q u e n c e o f s y n t h e s i s o f t h e s e t h r e e components o f E^ i s a n o t h e r c o n t r o v e r s i a l p o i n t i n c e l l u i a s e s y s t e m . 3.

C e l l o b i o s e i s a b s o r b e d b y t h e f u n g a l c e l l s where i t t r i g g e r s t h e s y n t h e s i s o f t h e t h i r d enzyme, 3 - g l u c o s i d a s e ( E ~ ) t o b r e a k c e l l o b i o s e into glucose u n i t s . The E^ i s a n i n t r a c e l l u l a r enzyme b u t a s m a l l q u a n t i t y i s a l s o s e c r e t e d o u t s i d e o f t h e fungal c e l l . L a r g e q u a n t i t i e s o f E~ a r e u s u a l l y r e l e a s e d b y the older c e l l s o r a f t e r the a u t o l y s i s o f the older c e l l s . Thus hydrolyzes c e l l o b i o s e into glucose u n i t s i n s i d e as w e l l as o u t s i d e the fungal c e l l s .

Finally some o f t h e g l u c o s e released from c e l l u l o s e i s catabolized to release e n e r g y needed f o r various metabolic processes and some i s m e t a b o l i z e d t o s y n t h e s i z e f u n g a l b i o m a s s , e x t r a c e l l u l a r c e l l u l a s e s and 3 - g l u c o s i d a s e . The f i n a l p r o d u c t may be a f u n g a l b i o m a s s r i c h i n p r o t e i n ( t o be used a s SCP) a s i n t h e c a s e o f C. c e l l u l o l y t i c u m o r i t may b e v e r y little biomass with more e x t r a c e l l u l a r cellulases ( t o b e used f o r hydrolysis of c e l l u l o s i c m a t e r i a l s i n t o g l u c o s e ) a s i n t h e c a s e o f T. r e e s e i . Physiological Aspects T e m p e r a t u r e . D u r i n g SSF t h e t e m p e r a t u r e i n t h e s u b s t r a t e rises due to heat generated b y m e t a b o l i s m . The r i s e i n t e m p e r a t u r e i s d i r e c t l y r e l a t e d t o t h e d e p t h o f t h e s u b s t r a t e and the m e t a b o l i c a c t i v i t i e s o f t h e organisms. I t has been recorded (61) that d u r i n g composting o f straw f o r mushroom g r o w i n g , t h e temperature o f the outer l a y e r o f t h e compost h e a p ( o n e m e t e r h i g h ) i s a r o u n d 37 C, w h i l e i n t h e i n n e r p o r t i o n t h e t e m p e r a t u r e rises as high a s 60-77 C. On t h e o t h e r h a n d , i n t h e i n n e r m o s t region the temperature reaches o n l y 32-49 C b e c a u s e o f l o w m i c r o b i a l a c t i v i t y , due t o a n a e r o b i c c o n d i t i o n s i n t h a t r e g i o n . Edwards ( 6 2 ) had r e p o r t e d t h a t o n e K g ( d r y w e i g h t ) o f o r g a n i c matter ( c e l l u l o s e ) when consumed b y m i c r o o r g a n i s m p r o d u c e d 0.597 K g o f w a t e r , 1.465 K g o f C 0 a n d 14,960 ΒTU. Some o f t h e h e a t (about 59%) i s used to evaporate water produced d u r i n g the metabolic a c t i v i t y while the remaining (41%) i s l e f t i n the s u b s t r a t e t o b e d i s s i p a t e d . T h i s amount o f e n e r g y , i f n o t dissipated immediately, as i t i s released, w i l l reduce the p r o d u c t i v i t y o r may k i l l t h e o r g a n i s m . 2



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ENGINEERING

A e r a t i o n . A e r a t i o n has t h e f o l l o w i n g f u n c t i o n s i n SSF: ( i ) To s u p p l y 0^ f o r a e r o b i c m e t a b o l i s m . ( i i ) To c o n t r o l the t e m p e r a t u r e . (iii) To remove CO^, w a t e r v a p o u r s , and v o l a t i l e m e t a b o l i t e s produced d u r i n g metabolism. The problem of a e r a t i o n w i l l i n c r e a s e c o r r e s p o n d i n g l y w i t h the i n c r e a s e i n the thickness of the substrate layer. Proper aeration i n the SSF a l s o d e p e n d s o n t h e a i r s p a c e s a v a i l a b l e i n t h e s u b s t r a t e . F i b r o u s m a t e r i a l s c a n p r o v i d e more a i r s p a c e s t h a n finely powdered s u b s t r a t e . A e r a t i o n seems t o be a v e r y c r i t i c a l requirement. O c h r a t o x i n p r o d u c t i o n by A s p e r g i l l u s o c h r a c e u s i n a r o t a t i n g drum f e r m e n t e r s t o p p e d when t h e a i r f l o w r a t e was g r e a t e r t h a n 0.1 l i t e r / K g / m i n u t e ( 6 3 ) . I n c o n t r a s t , t h e increase in air flow rate through a column of c o r n i n o c u l a t e d w i t h A s p e r g i l l u s f l a v u s i n c r e a s e d the r a t e o f production of a f l a t o x i n (64-65). S i m i l a r l y i t was reported that 3-galactosidase and i n v e r t a s e p r o d u c t i o n by A s p e r g i l l u s n i g e r on wheat b r a n was increased c o n s i d e r a b l y when t h e a e r a t i o n was i n c r e a s e d . Moisture. In n a t u r e , vegetables and fruits contain about 60-80% m o i s t u r e . T h i s much m o i s t u r e i s s u f f i c i e n t t o e n c o u r a g e t h e g r o w t h o f m i c r o o r g a n i s m s (66,67 ) . But Christensen (68) reported t h a t E u r o t i u m h a l o p h i l i c u m c a n g r o w on wheat g r a i n s a t 13-14% m o i s t u r e . M o r t o n and E g g i n s ( 6 9 ) have a l s o r e p o r t e d that some fungi like A b s i d i a corymbifera, Fusarium s o l a n i and Chaetomium trilatérale can g r o w and p e n e t r a t e i n wood a t a 14% m o i s t u r e l e v e l a t 35°C. Growth o f s o f t - r o t f u n g i i n s i t u a t i o n s o f e x t r e m e d r y n e s s has b e e n r e c o r d e d by Duneun and E s l y n ( 7 0 ) . A b a s i d i o m y c e t e , S e r p u l a l a c r y m a n s i s w e l l known t o c o l o n i z e d r y timber i n buildings causing the c o n d i t i o n s known as " d r y r o t " . T h i s i s a c h i e v e d by the fungus i n i t i a t i n g growth i n timber w i t h a h i g h water content and then using the e n e r g y and n u t r i e n t s gained to produce r h i z o m o r p h s w h i c h c a n e x p l o r e d r y t i m b e r many m e t e r s away f r o m the initial colony. Though s u c h r h i z o m o r p h s c a n transport water, f u r t h e r water i s produced a t the s i t e o f a c t i o n by the u t i l i z a t i o n o f t h e t i m b e r w i t h t h e e v o l u t i o n o f CO- ( 7 1 ) . V a r i o u s l e v e l s o f m o i s t u r e i n SSF have been r e p o r t e d for different products. About 7 5 % m o i s t u r e i n s t r a w was used f o r SCP p r o d u c t i o n (2 ,3,3a) w h e r e a s f o r a f l a t o x i n p r o d u c t i o n i t was 33.3% in rice and 48.4% i n s t r a w (1). Thus m o i s t u r e l e v e l i n t h e SSF d e p e n d s on t h e n a t u r e o f t h e s u b s t r a t e , o r g a n i s m and the type of end product. pH. M o s t f u n g i a r e a b l e t o g r o w i n a w i d e pH r a n g e o f 5-8. A d j u s t m e n t o f pH d u r i n g t h e g r o w t h i n SSF i s v e r y d i f f i c u l t . Some o f t h e s u b s t r a t e ( s t r a w s , g r a i n s ) have good b u f f e r i n g c a p a c i t y and t h e r e may not be any need t o a d j u s t t h e pH d u r i n g SSF. But maintenance of pH a r o u n d 4.5 is very crucial for cellulase p r o d u c t i o n w i t h T. r e e s e i (_5 ) . Osmotic P r e s s u r e . Raising the osmotic potential of a material by a d d i t i o n of s a l t or sugar or both to a l e v e l higher t h a n t h a t o c c u r i n g i n the c y t o p l a s m i s a w e l l t r i e d method of



20.

CHAHAL

Solid State

Fermentation

439

microbial i n h i b i t i o n and i s w i d e l y u s e d i n p r e s e r v a t i o n o f f o o d s , s u c h a s meats and f r u i t s . Sugar c o n c e n t r a t i o n s i n t h e r e g i o n s of 50-70% a r e u s u a l l y a d e q u a t e ; s a l t i s added t o 2 0 - 2 5 % l e v e l ( 7 1 ) . However, t h e r e are some o s m o p h i l i c and halophyllic organisms ( S a c c h a r o m y c e s r o u x i i , S. m e l l i s and A s p e r g i l l u s g l a u c u s s e r i e s ) which can grow i n c o n c e n t r a t e d sugar solutions (71 ) . High concentrations of salts ( n u t r i e n t s ) are used i n SSF f o r SCP p r o d u c t i o n from s t r a w ( 2 ) . T h e r e f o r e , t h e m i c r o o r g a n i s m s capable o f w i t h s t a n d i n g h i g h o s m o t i c p r e s s u r e w i l l be more s u i t a b l e under s u c h c o n d i t i o n s , o t h e r w i s e , t h e r e q u i r e d n u t r i e n t s a r e t o be added in frequent s m a l l doses to a v o i d h i g h osmotic p o t e n t i a l i n the substrate. C h a r a c t e r i s t i c s o f an O r g a n i s m f o r SCP

and C e l l u l a s e P r o d u c t i o n

P r o d u c t i o n o f SCP and c e l l u l a s e s i n SSF on a l a r g e s c a l e w i l l depend on t h e t h o r o u g h u n d e r s t a n d i n g o f g r o w t h c h a r a c t e r i s t i c s and b e h a v i o r o f SCP and cellulase producing organisms under such conditions before the a p p l i c a t i o n o f b i o c h e m i c a l e n g i n e e r i n g to s c a l e up t h e p r o c e s s e s . The o r g a n i s m f o r s u c c e s s f u l SSF should have t h e f o l l o w i n g p r i m a r y q u a l i t i e s : 1. 2.

3. 4. 5. 6. 7. 8.

I t i s a b l e to u t i l i z e mixtures o f v a r i o u s p o l y s a c c h a r i d e s . I t s h o u l d have c o m p l e t e enzyme s y s t e m s t o s w i t c h over i t s metabolism f r o m one p o l y s a c c h a r i d e t o a n o t h e r p o l y s a c c h a r i d e ( f o u n d i n c o m p l e x s u b s t r a t e s ) w i t h o u t any l a g p h a s e . It i s able to penetrate deep i n t o the t h i c k l a y e r o f the s u b s t r a t e as w e l l as i n t o t h e c e l l s o f t h e s u b s t r a t e . I t i s a b l e to grow i n h i g h c o n c e n t r a t i o n s o f n u t r i e n t s . I t grows i n a v e g e t a t i v e f o r m and d o e s n o t sporulate during the f e r m e n t a t i o n t i m e . I t s h o u l d be f a s t g r o w i n g t o a v o i d c h a n c e s o f c o n t a m i n a t i o n by other fast-growing organisms. I t i s a b l e to grow i n low m o i s t u r e content o f the s u b s t r a t e . It i s able t o g r o w i n t h e p r e s e n c e o f p h e n o l i c and t o x i c compounds p r o d u c e d d u r i n g t h e p r e t r e a t m e n t o f t h e s u b s t r a t e .

Acknowledgement The a u t h o r i s v e r y g r a t e f u l t o D r . D.J. K u s h n e r , p r o f e s s o r o f Microbiology, University of O t t a w a , O t t a w a , O n t a r i o , Canada f o r h i s i n v a l u a b l e s u g g e s t i o n s and f o r e d i t i n g t h e m a n u s c r i p t . Literature Cited 1. 2.

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R E C E I V E D June 1,

1982


Foundations of Biochemical Engineering - American Chemical Society

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