Bacterial Degradation of Kraft Lignin - American Chemical Society

Chapter 38 Bacterial Degradation of Kraft L i g n i n

Production and Characterization of Water-Soluble Intermediates Derived from Streptomyces badius and Streptomyces viridosporus 1

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P. F. Vidal , J . Bouchard , R. P. Overend , E . Chornet , H . Giroux , and F. Lamy 3

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Department of Chemical Engineering, University of Sherbrooke, Sherbrooke, Quebec J1K 2R1, Canada National Research Council of Canada, Ottawa, Ontario K1A 0R6, Canada Department of Biochemistry, University of Sherbrooke, Sherbrooke, Quebec J1K 2R1, Canada

Downloaded by CORNELL UNIV on May 9, 2017 | http://pubs.acs.org Publication Date: July 31, 1989 | doi: 10.1021/bk-1989-0399.ch038

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Two Streptomyces strains, S. badius and S. viridosporus, were found to be able to grow on kraft lignin (Indulin ATR) as sole carbon source. The resulting APPL (Acid Precipitable Polymeric Lignin) was characterized by FTIR and elemental analysis for C, H and N, and was found to contain proteins in addition to a relatively demethoxylated lignin component. The proteins were further characterized by amino acid analysis, while the lignin component was separated by solvent extraction and its molecular weight distribution determined by HPSEC. In t h e last t e n years, research o n l i g n i n biodégradation has followed t w o routes: f u n g i a n d b a c t e r i a l d e l i g n i f i c a t i o n . C r a w f o r d a n d co-workers were the first t o s t u d y i n d e t a i l t h e a c t i o n o f two streptomyces, S. badius 252 a n d S. viridosporus T 7 A , o n lignocellulose f r o m different sources. T h e i r w o r k l e d t o the conclusion t h a t the b a c t e r i a l a c t i o n o n aqueous suspensions of these lignocellulosics resulted i n t h e s o l u b i l i z a t i o n o f l i g n i n fragments w h i c h p r e c i p i t a t e u p o n a c i d i f i c a t i o n : A c i d P r e c i p i t a b l e P o l y m e r i c L i g n i n ( A P P L ) (1-10). In t h i s p a p e r , we report o n t h e b a c t e r i a l g r o w t h o f S. badius a n d S. viridosporus w i t h I n d u l i n A T R , a c o m m e r c i a l k r a f t l i g n i n p r a c t i c a l l y free of sugars, as sole carbon source a n d o n t h e c h a r a c t e r i z a t i o n o f the A P P L derived from this degradation. Materials a n d Methods T h e m a t e r i a l s a n d most o f the methods used are described i n previous papers (11,12). T o s u m m a r i z e : - I n d u l i n A T R is a purified f o r m (acidified water wash) o f I n d u l i n A T from Westvaco C o r p . , Charleston Heights, South Carolina. 0097~6156/89/0399-0529$06.00/0 © 1989 American Chemical Society

Lewis and Paice; Plant Cell Wall Polymers ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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PLANT C E L L W A L L P O L Y M E R S

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5. badius a n d S. viridosporus are o b t a i n e d f r o m the A m e r i c a n T y p e C u l t u r e s C o l l e c t i o n , A T C C #39115 a n d 39117, respectively. These s t r a i n s are grown i n I n d u l i n A T R suspensions ( 0 . 5 % w / v ) . N H C 1 , yeast e x t r a c t a n d glucose are used together or i n d e p e n d e n t l y as n u t r i e n t s . T h e D N A content is measured i n order to determine the rate of bac t e r i a l g r o w t h a c c o r d i n g to B u r t o n (13). A P P L is d e t e r m i n e d b y a c i d p r e c i p i t a t i o n ( 1 2 M H C 1 ) u s i n g either t u r b i d i t y measurements (nephelometry: abs. at 600 n m ) or g r a v i m e t r y a c c o r d i n g to C r a w f o r d et al. (4). A l l e x p e r i m e n t s r e p o r t e d i n t h i s p a p e r were c a r r i e d out w i t h u n i n o c u l a t e d controls whose values were always s u b s t r a c t e d . M e t h o x y l content of the different samples were d e t e r m i n e d u s i n g a m o d i f i e d Zeisel procedure (14). A q u e o u s Size E x c l u s i o n C h r o m a t o g r a p h y ( A S E C ) : A P h a r m a c i a sys t e m w i t h superose T M 1 2 gel as p a c k i n g , T R I S - H C 1 50 m M as eluent (flow rate 0.4 m l m i n ) , was used, detection being b y U V at 280 n m . F o u r i e r T r a n s f o r m Infrared ( F T I R ) Spectroscopy: A 5 D X B N i c o l e t s y s t e m w i t h a T G D S detector was used at a r e s o l u t i o n of 4 c m " . T h e samples were m i x e d w i t h pure K B r at a c o n c e n t r a t i o n of 2 % w / w a n d 64 scans were collected. H i g h P e r f o r m a n c e Size E x c l u s i o n C h r o m a t o g r a p h y ( H P S E C ) : A V a r i a n M o d e l 5000 l i q u i d c h r o m a t o g r a p h e q u i p p e d w i t h a v a r i a b l e w a v e l e n g t h U V detector, two c o l u m n s i n series ( P L gel, 300 x 7 . 5 m m , p a r t i c l e size 5μπι, p o r o s i t y of 50 a n d 5 0 0 Â ) , was used, T H F b e i n g the eluent.

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Results and Discussion Bacterial Growth and APPL Production. G l u c o s e used as a secondary c a r b o n source increases A P P L p r o d u c t i o n , t h o u g h o n l y after a l l glucose has been c o n s u m e d . T h e increase is greater for S. badius t h a n for S. viridosporus ( F i g . 1). A s p r e v i o u s l y s h o w n by C r a w f o r d (2), we also observed t h a t an o r g a n i c source of n i t r o g e n such as yeast e x t r a c t was m u c h better t h a n an i n o r g a n i c source such as N H C 1 , the S t r e p t o m y c e s p r o d u c i n g 7 to 9 times more A P P L i n the former case ( F i g . 2). T h e increase of i n i t i a l p H f r o m 7.2 t o 8.8 s l i g h t l y increases A P P L y i e l d , w h i l e a n a d d i t i o n of C u + + , Fe+++, M n or Zn++ has no effect ( d a t a not s h o w n ) . T h e s t u d y of the D N A content, a n i n d i c a t i o n of b a c t e r i a l g r o w t h , shows t h a t S. badius reaches its s t a t i o n a r y phase after 5 days, 7 days p r i o r to S. viridosporus ( F i g . 3). In b o t h cases, A P P L p r o d u c t i o n s t a r t s i m m e d i a t e l y (t = 0) a n d increases l i n e a r l y d u r i n g i n c u b a t i o n , S. badius p r o d u c i n g more t h a n S. viridosporus. A f t e r 35 days, the A P P L y i e l d represents 7% a n d 5 % of the i n i t i a l I n d u l i n A T R weight for S. badius a n d S. viridosporus, respectively. 4

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Separation of Bacterial Extracellular, Membranous and Cytosolic Proteins and their Effect on Indulin ATR. W e first assume a l i g n i n s o l u b i l i z a t i o n c a t a l y z e d b y enzymes. In order to localize the enzymes responsible for the I n d u l i n A T R d e g r a d a t i o n , the cells of each s t r a i n were f r a c t i o n a t e d : e x t r a


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Bacterial Degradation of Kraft Lignin

VIDAL E T A L

0.35

0.30

0.25

0.20


0.15

0.10

g

0.05

C g

S
3 0 0 , 0 0 0 ) is a t t r i b u t e d to the p r o t e i n f r a c t i o n o f the A P P L , such large molecules never being found i n Indulin A T R . Dissociation of the Protein-Polyphenolie Complex and Characterization of the Polyphenolic Fraction. Since I n d u l i n A T R is almost c o m p l e t e l y s o l u b l e i n T H F w h i l e the A P P L ' s are q u i t e i n s o l u b l e i n t h i s solvent, b u t are soluble i n D M F , a sequence of different percentage m i x t u r e s of these two solvents was used i n order t o dissociate the p r o t e i n - l i g n i n complexes for f u r t h e r analyses of the l i g n i n p a r t . D e s p i t e the fact t h a t crude A P P L ' s are t o t a l l y soluble i n D M F , a n i m p o r t a n t residue is o b t a i n e d at the end of the solvent sequence (0:100, T H F : D M F ) i n d i c a t i n g t h a t the p r o t e i n - r i c h fractions require a s s o c i a t i o n w i t h the p o l y p h e n o l i c p a r t for t h e i r s o l u b i l i z a t i o n i n D M F ( T a b l e V I ) . B e cause each A P P L has a different a m i n o a c i d c o m p o s i t i o n , its s o l u b i l i t y d i s t r i b u t i o n is also different, b u t i n b o t h cases, the T H F f r a c t i o n is the m o s t l i g n i n - l i k e , w i t h o n l y 1% n i t r o g e n . T h i s is confirmed b y F T I R a n a l y sis ( F i g . 7). A s the f r a c t i o n a t i o n proceeds w i t h i n c r e a s i n g solvent p o l a r i t y , the l i g n i n c h a r a c t e r i s t i c b a n d s at 1515, 1460, 1265, 1095, 1035, a n d 810 c m " d i s a p p e a r , w h i l e the a m i d e c h a r a c t e r i s t i c bands at 3290 a n d 3080 c m " appear. 1

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T a b l e V I . E l e m e n t a l A n a l y s i s of the F r a c t i o n s O b t a i n e d b y S e q u e n t i a l S o l ubilization Sample

THF:DMF

% of S t a r t i n g Material

% N

%C

%H

%O

APPL S. bad.

100:0 85:15 50:50 0:100 Residue

8.0 21.1 19.0 9.8 42.1

1.0 2.7 4.8 4.3 8.7

67.4 61.7 58.6 57.2 49.6

7.1 6.1 6.4 5.7 6.5

24.5 29.5 30.2 32.8 35.2

APPL S. vir.

100:0 85:15 50:50 0:100 Residue

2.3 4.3 36.3 2.8 54.3

not 2.0 3.3 4.0 9.9

enough s a m p l e 65.8 7.1 25.1 61.7 6.6 28.4 57.2 5.1 33.7 49.6 6.9 33.6

x

Percentage of oxygen is c a l c u l a t e d b y difference. T h e m o l e c u l a r weight d i s t r i b u t i o n o b t a i n e d b y size e x c l u s i o n c h r o m a t o g r a p h y , for b o t h A P P L ' s T H F soluble f r a c t i o n a n d I n d u l i n A T R , show s i m i l a r profiles, the differences b e i n g t h a t the r a t i o of h i g h m o l e c u l a r weight ( D P 5 0 ) to the lower m o l e c u l a r weight ( D P 5 ) is inversed between I n d u l i n A T R a n d b o t h A P P L fractions, the l a t t e r b e i n g richer i n lower m o l e c u l a r weight ( F i g . 8). Because the A P P L ' s represent o n l y 7% of the i n i t i a l m a -



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F i g u r e 6. A q u e o u s size e x c l u s i o n c h r o m a t o g r a p h y of the A P P L derived f r o m S. badius. F e r r i t i n , aldolase, o v a l b u m i n , c h y m o t r y p s i n o g e n A a n d acetone were used as s t a n d a r d s .



3ΘΟΟ

2ΘΟΟ CCM-15

1OOO

1700

1SOO

1300 WAVENUMBERS

1 lOO CCM-1>

900

F i g u r e 7. F T I R s p e c t r a o f t h e fractions a n d o f t h e i n s o l u b l e residue o b t a i n e d b y s e q u e n t i a l solvent s o l u b i l i z a t i o n . T h e f r a c t i o n s are i d e n t i f i e d b y the T H F : D M F r a t i o used. ( R e p r o d u c e d w i t h p e r m i s s i o n f r o m R e f . 12, © 1989, C N R C . )

3400 300D WAVENUMBERS


700



542

F i g u r e 8. H i g h performance size exclusion c h r o m a t o g r a p h y of I n d u l i n A T R a n d of the T H F soluble f r a c t i o n of each A P P L . ( R e p r o d u c e d w i t h p e r m i s s i o n f r o m Ref. 12, © 1989, C N R C . )


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Bacterial Degradation of Kraft Lignin

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terial, this technique does not allow us to conclude whether or not APPL consists of Indulin ATR depolymerized fragments.


Conclusions The experiments reported were all conducted under the important assump tion that enzymes present in the bacteria studied were responsible for the solubilization of the lignin. This assumption is supported by some, but not all of the results obtained, such as the H2O2 activation of certain bacterial protein fractions. Another theory would be to suppose that the solubilizing proteins are, in fact, surfactants. This would explain, for example, the activity of these proteins at high temperatures. We do know, however, that proteins attach to APPL to form a soluble complex. Following the sur factant theory we would consider that Indulin ATR is a complex of lignin fragments insoluble only because of the medium's properties. The bacterial proteins would attach to the Indulin ATR increasing the hydrophilicity of the fragments, some able to become completely soluble in a neutral aqueous solution. To prove that one theory, the other, or even a combination of both is correct, further studies of the purified secreted and cellular proteins from the bacteria will be part of the next series of experiments planned. Literature Cited 1. Antai, S. P.; Crawford, D. L. Appl. Environ. Microbiol. 1981, 42, 37880. 2. Barder, M. J.; Crawford, D. L. Can. J. Microbiol. 1981, 27, 859-63. 3. Crawford, D. L. Biotechnol. Bioeng. Symp. 1981, 11, 275-91. 4. Crawford, D. L.; Pometto, A. L., III; Crawford, R. L. Appl. Environ. Microbiol. 1983, 45, 898-904. 5. Crawford, D. L.; Pettey, T. M.; Thede, B. M.; Deobald, L. A. Biotech nol. Bioeng. Symp. 1984, 14, 241-56. 6. Pettey, T. M.; Crawford, D. L. Appl. Environ. Microbiol. 1984, 47, 439-40. 7. Borgmeyer, J. R.; Crawford, D. L. Appl. Environ. Microbiol. 1985, 49, 273-78. 8. Pometto, A. L., III; Crawford, D. L. Appl. Environ. Microbiol. 1986, 51, 171-79. 9. Pometto, A. L., III; Crawford, D. L. Appl. Environ. Microbiol. 1986, 52, 246-50. 10. Ramachandra, M.; Crawford, D. L.; Pometto, A. L., III. Appl. Environ. Microbiol. 1987, 53, 2754-60. 11. Giroux, H.; Vidal, P. F.; Bouchard, J.; Lamy, F. Appl. Environ. Mi crobiol. 1989, 54, 3064-70. 12. Vidal, P. F.; Bouchard, J.; Overend, R. P.; Chornet, E.; Giroux, H.; Lamy, F. Can. J. Chem. 1989, in press. 13. Burton, K. Biochem. J. 1955, 61, 473-83. 14. Haluk, J. P.; Metche, M. Cell. Chem. Technol. 1986, 20, 31-50. 15. Hergert, H. L. In Lignins; Sarkanen, Κ. V.; Ludwig, C. H., Eds.; Wiley: New York, 1971. RECEIVED May 19, 1989


Bacterial Degradation of Kraft Lignin - American Chemical Society

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