Utilization of waste cellulose-V comparative effects of different pretreatments on the enzymatic hydrolysis of cellulose and ligno-cellulosic substrates

Eur. Polym J. Vol. 22. No. 7. pp. 515-519. 1986 Printed m Great Britain

0014-3057 86 S3.00 ~-0.00 Pergamon Journals I.td

UTILIZATION OF WASTE CELLULOSE--V COMPARATIVE EFFECTS OF DIFFERENT PRETREATMENTS ON THE ENZYMATIC HYDROLYSIS OF CELLULOSE A N D LIGNO-CELLULOS1C SUBSTRATES C. DAVIt), R. FORNASIIzR and Pll. TIIIR',' l.;niversitc Libre de Bruxelles, Facultd des Sciences. CP 206.1 Bd. du Triomphe, 1050 Bruxelles, Belgium ( Receiced 24 December 1985)

Abstract-- Pretreatments based on the oxidation of cellulose and ligno-cellulosic materials by sodium hypochlorite arc compared with classical prelreatments (Fe-' .tI,O> ,-rays. concentrated and dilute NaOH, ('adoxen, mechanical pulping). Substrates studied are paper pulp. wheat straw and spruce wood. The importance of oxidation and of transformation of cellulose I into cellulose 11 are discussed. Pretreatment of spruce wood by NaCIO gives yields of glucose during enzymatic hydrolysis which compare very favourably with those obtained with the more expensive cadoxen and concentrated NaOH treatments. High yields of glucose are also obtained in the case of bagasse and barley strays. The work has been extended t',~ other halogenated oxidizing agents.

INTROI)U(7ION The yield and rate (and hence the cost) of the enzymatic hydrolysis of cellulose and ligno-cellulosic materials into glucose strongly depend on chemical and physical p r e t r e a t m e n t s of the substrates. M a n y publications report and discuss the rate a n d yield of hydrolysis of cellulose substrates [1] but fewer are concerned with ligno-cellulosic c o m p o u n d s . Since these are potentially valuable and renewable energy sources, the present work is concerned with the c o m p a r a t i v e study o f different chemical pret r e a t m e n t s on cellulose a n d ligno-cellulosic compounds. The tina[ purpose of this study is a deep u n d e r s t a n d i n g of the chemical d e g r a d a t i o n of the different c o m p o n e n t s of the substrates in order to p r o m o t e the choice of an efficient a n d e c o n o m i c pretreatment. The substrates chosen are paper pulp (cellulose), wheat and barley straw, spruce wood and bagasse (ligno-cellulosies).

perature. The substratc was then filtered and washed three times with ~ater. Extraction under retlux with water (exp. 10), HCI 0.1M (exp. 11) or NaOH 0.1M (exp. 12) was finally performed for 2 hr. Other samples were treated by NaOH 18% (exp. 13 and 14) or successively transformed into sodium-cellulose, cellulose xanthate and regenerated cellulose b3 the usual methods (exp. 151. The pretreatment with cadoxen was performed without prior extraction of the hemicelluloses. Substrate (1 g) was swelled for 1 hr in 10ml cadoxen. The mixture was then added to [(lO m[ methanol, filtered, washed with acid methanol and washed t~ice with 50 ml water. The amount of cellulose recovered was measured by quantitative saccharilication and glucose analysis by lhe glucose oxidase method. The X-ray diagrams were obtained with a Philips PW 1010 generator equipped with a PW 1050:30 goniometer using K,Cu. The samples were prepared by milling the substrale and compressing it between two glass pkttes. REyII_:I.I"S a n t ~ I ) l s c u S s l O n

L'ff~'C! Q]" d![~erelll p r f l r e a l m e t l l s Oil lhe reactiritt" 0 [ cellulose, wheat straw and .spruce ~tood

I,.XPERIMENI"AI. The enzymatic hydroiyses were performed at 45 in a 0.1 M citrate buffer (pH 4.8) using a 2.5% concentration of substrate and an enzymatic activity (Trichoderma viride Onozuka) of 0.07 IFPU m g ~. Glucose was analysed by the glucose oxidase method [2]. ,'-Irradiations were performed using a C'o'~ gamma cell model 2(hO. The pretreatments with t t 2 0 , . F e " were done by incubation of 1 g of ground cellulose at room temperature in 500 ml of 100 mM acetate bvffer (pH 4.2). Fe"' and H-O_, were respectively 5 x I(I aM and I vol% in the buffer solution. The incubated cellulose wits washed three times for 2 hr with the acetate buffer and three times for 2hr with distilled water. For the pretreatment by NaCIO, 1.5 g substrate was shaken for I hr with 50 ml NaCIO 0.5 M at room ternPaper presented in part at the Americam Chemical Society Annual Meeting (Washington. D.C., August 1983). 515

In this section, the quantity of glucose formed during enzymatic hydrolysis as a function of time will be given for untreated and pretreated paper pulp, spruce wood and wheat straw (Table 1). It has been measured for identical experimental conditions (temperature, substratc and enzyme c o n c e n t r a t i o n ) in order to c o m p a r e the efliciencies o f the different p r e t r e a t m e n t s on these three substrates. It is well k n o w n that, in these conditions, the q u a n t i t y of glucose fc,rmcd as a function of time for pure cellulose tends to a limit which depends mainly on the crystallinity of the substrate. Table 2 (exp. I) indicates that the limiting yield is 4 1 % for the present conditions. The limiting yield is the ratio of the q u a n t i t y of glucose formed after 4 days to the m a x i m u m q u a n t i t y of glucose that could be obtained by quantitative t r a n s f o r m a t i o n of cellulose into glu-

516

et al.

C. DAVID

T a b l e I. E n z y m a t i c h y d r o l y s i s o f p a p e r p u l p , w h e a t s t r a w a n d s p r u c e w o o d a f t e r d i f f e r e n t p r e t r e a t m c n t s Cellulose

.

1.0

1.2

.

.

0.9

1.2

1.5

.

.

1.2

1.6

1.9

1.4

2.0

2.5

1.4

2.0

2.5

I I 1.6 1.7 5.8 4.0 5.0 5.8 6.2 3.3 4.1 3.5 5.4 8.6 9.0 5.5 5.4

.

.

9.2 4.0 6.2 ---17.5 -7.0 -------.

.

.

.

.

.

.

.

.

.

" 1 2 0 . 0.5 hr. ? R o o m t e m p e r a t u r e present conditions.

.

.

.

. .

. .

T a b l e 2. Effect o f d i f f e r e n t p r e t r e a t m e n t s

on

Substrate pretreatment Untreated 7 50 M r H 2 0 2 / F e 2+ Cadoxen NaOH, 0.5%+ N a O H . 5 % . 2 hr~ NaOH, 18%. 18hr§ N a O H , 1 8 % I' N a C 1 0 , 0.5 MI' 9 + H20 extract 9 + HC110 ~M extract 9+NaOHI0 ~Mextract 9 + NaOH, 18%,2 hr 9 + N a O H , 1 8 % , 24 h r 9 -t- C S 2 N a C I O , p H 8, 0.5 M I Mechanical ( < 80 #, d r y ) Mechanical ( 8 0 - 1 5 0 g, d r y ) Mechanical (150- 300/~, d r y ) Mechanical ( 3 0 0 - 5 0 0 ,u, d r y ) Mechanical (uncalibrated wet)

~"

-s

--89 -------------. .

. .

.

.

14.4 14.4 8.5 8.8

. .

. .

for 2 4 h r . § T h i s c o r r e s p o n d s

% c e l l u l o s e in the p r e t r e a t e d substrate?

- c

--76 84 89 86 89 80 79 56 51 45 40 41 45 72 .

.

1.4 2.1 2.7 8.3 5.5 8. I 9.5 9.9 5.8 6.4 5.5 8.2

.

1.9

to 1 6 . 7 m o l / k g in the

p a p e r p u l p (C), w h e a t s t r a w (S) a n d s p r u c e w o o d ( W )

-w

--84 76 80 65 58 54 71 48 43 37 40 38 42 68

2

percent cellulose in the pretreated substrate. The percent cellulose in the pretreated substrate is obtained by quantitative saccharification of the substrate. The cellulose recovered as given in Table 2: % cellulose in the pretreated sample % subst, rec. x % cellulose in the initial sample results from the chemical oxidation of anhy-

Substrate recovered (%)* Experiment

. .

for 2 h r . ++Room t e m p e r a t u r e

cose. In the case of wheat straw and spruce wood, the limiting yields are very low (17 and 14%). The main reason for this is the presence of lignin which limits the accessibility of the cellulosic part of the substrate to the enzymes [3]. Other parameters will be used to characterize the pretreatment and the substrate (Table 2). They are the percent weight of recovered substrate and the

21

5.6 1.9 2.8 3.4 4.2 4.0 3.8 2.6 5.3 11.6 12.4 6.8 5.6

1.5 2.1 2.8 7.8 3.3 4.2 4.8 5.3 6.3 5.9 4.3 7.9 15.5 17.1 11.5 7.3

4

7.7 3.2 4.6 ---10.0 -5.2 --------

21

20

4

1.3 1.4

2

5.3 2.5 2.8 ---7.5 -3.3 -------.

20

19

2

0.9 1.2 1.2 3.5 1.5 1.8 2.3 2.8 2.6 1.9 1.6 3.1 6.0 7.5 4.1 3.6 0.6

1

Untreated 7 50 M r H 2 O J F e 2+ Cadoxen NaOH, 0.5%* NaOH, 5%* NaOH, 18%t N a O H , 18"/0 ~ N a C I O , 0.5 M § 9 + H2Oextract 9 + H C 1 1 0 - ~M e x t r a c t 9+ NaOH l0 ~M 9 + NaOH, 18%,2 hr 9 + N a O H , 1 8 % , 24 h r 9+CS, N a C I O , p H 8, 0.5 M § Mechanical ( < 80 ,u, d r y ) Mechanical ( 8 0 - 1 5 0 g, d r y ) Mechanical (150-300,a, dry) Mechanical (300-500/~, dry) Mechanical (uncalibrated wet)

19

18

I

1.5 2.6 3.5 12.2 7.8 12.0 I 1.0 12.6 9.3 9.5 7.3 ll.6 18.6 19.9 13.1 9.5

Substrate pretreatment

I 2 3 4 5 6 7 8 9 l0 II 12 13 14 15 16

18

Wood

4

Experiment

17

1 2 3 4 5 6 7 8 9 10 II 12 13 14 15 16 17

Straw [ g l u c o s e f o r m e d a f t e r (days)]

100 65 76 ---.-90 83 -------. .

.

s 42 35.5 44.5 53 50 60 58 62.5 53 73 81 96 84.5 82.5 80 55

Cellulose recovered (%)

Yield after 4 days (%)

w

c

s

W

C

S

~V

45 41 48.5 50 48.5 50 46.5 52.5 50 68.5 72.5 85 75.5 76 70 47 46.8

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

--

--

84.5 89 96 95 93 80 80 93 87 86 87 80.5 75 83 91 . .

91 82 93 96 96 92 94 88 85 83 85 68 69 70 75 .

41 25 32.5 ----78 34 -------

17 29 31.5 92 62 80 76 80 57 52 36 47 88 93 65.5 68

14 21 23 62.5 27 34 41 40.5 49 34.5 24 37 82 90 66 62 10

46.8

--

13

.

.

.

.

.

.

46.8

--

16

.

.

.

.

.

46.8

--

21

46.8

--

21

.

.

.

.

.

.

.

* O b t a i n e d by w e i g h i n g . "PObtained by q u a n t i t a t i v e s a c c h a r i f i c a t i o n a n d g l u c o s o x i d a s e d e t e r m i n a t i o n o f g l u c o s e . ~.120 , 0.5 hr. § R o o m t e m p e r a t u r e for 2 hr. 0BRoom t e m p e r a t u r e for 24 hr. IIThis c o r r e s p o n d s to 16.7 m o l / k g in the p r e s e n t c o n d i t i o n s .

Utilization of waste cellulose--V

The results for the enzymatic hydrolysis of different pretreated substrates are summarized in Tables I and 2. Typical results obtained for spruce wood are given in Fig. I. Since cellulose II gives much higher yields of hydrolysis than cellulose I [5], the possibility of transformation of cellulose I into cellulose I1 during the different pretreatments has been considered. The X-ray diagram of different treated and untreated substrates have been recorded; a few examples arc given in Fig. 2. All the diagrams havc been semiquantitatively examined according to two criteria (Table 3): formation of cellulose II as indicatcd by thc presence of peaks at 20 = 21 and 13 and relative importance of amorphous to crystalline phase. The amorphous phase corresponds to the broad halo centred about 20 = 16 . It consists of lignin, hemicellulose and a fraction of the cellulose. Paper pulp consists of cellulose I and a small fraction of amorphous phasc. ? and HeOe/Fe :J ( Tables I and 2, exp. 2 and 3 ). The yield after 4 days is lower for pretreated paper pulp than it is for the initial substrate. A small increase in yield is observed for wheat straw and spruce wood. The percent cellulose recovered indicates that the attack of anhydroglucose units resulting in oxidation is important for paper pulp and probably responsible for the decrease in maximum yield observed after pretreatment; owing to high selectivity of enzymes, the presence of oxidized units has an inhibiting cffcct as already proposed [5]. The higher percentagc of cellulose recovered in the case of wheat straw and spruce wood indicates that oxidation of anhydroglucose units is less important than for paper pulp owing to competitive oxidation of hemicelluloses and

t0 4

~ 86

~

'7t169

~ 4 2 ~o

24 48 I

0

I

96 120

[ 72

[

517

[

(hr)

Fig. I. Enzymatic hydrolysis of differently pretreated spruce wood. Glucose formation as a function of time: (1) untreated; (2) ?-irradiation (50 Mrad); (3) H20:/Fe2+; (4) Cado×en: (5) NaOH, 0.5%; (6) NaOH, 5%; (7) NaOH, 18%; (8) NaC10, 0.5 M: (16) NaCIO, pH 8. droglucose units of solubilized and unsolubilized cellulose chains. Comparison of the percent weight recovered and of the percent cellulose recovered indicates that the weight loss is much more important than the loss of the cellulose. The weight loss is mainly due to oxidation and solubilization of lignin and hemicelluloses. This was confirmed in a detailed investigation of the pretreatment of Eucalyptus wood by HC10-NaCIO [3] by the analysis of the pentoses, hexoses and lignin content of the substrates. Elimination of hemicelluloses with dilute acid without elimination of lignin has been shown to be inefficient in promoting the enzymatic hydrolysis [4].

f ~ x

' . . . . .x,

.

.....

... . _ ~

.\

~ ,

j .--.."~-.. : 7

""-'t 27

\ "

~ - , . .

L

i

26

25

24

23

l

l

J

l

i

K

J

J

i

22

2~

20

19

IO

17

16

15

14

~

I - ~ ' ~ 15

12

_

J 11

28 Fig. 2. X-ray diagram of paper pulp ( ): paper pulp treated with NaOH 18% (-. -); spruce (. . . . . . . . ): spruce treated with NaCIO-HCIO (. . . . . . ); spruce treated with NaOH 18% ( ........ ).

10

C . D A V I D et al.

518

T a b l e 3. E v a l u a t i o n o f the X - r a y d i a g r a m s Substrate

Pretreatment

Paper pulp Paper pulp Cellophane Spruce wood Spruce wood Spruce wood Spruce wood Spruce wood Wheat straw

NaOIt. 18% Cadoxen NaOH, 5% NaOH, 18% N a C I O , 0.5 M NaOH, 18%

Cellulose I

C e l l u l o s e II

+ -~

-

-

+ ~ + +

+ -~ ~- + q. + + +

t + -+ +

Amorphous

phase

+ ~+ + + + t + -~ + + +

( + + ) Large quantity. ( + ) Moderate quantity. ( - ) Absent.

lignin. The net result of the negative effect of the oxidation of glucose and the positive effect of delignification is a small increase in the overall reactivity of pretreated wood and straw. Cadoxen (Tables I and 2, exp. 4). Cadoxen pretreatment performed without prior elimination of the hemicelluloses results in delignification of wood and straw as indicated by the brown colour of the reacting media and washing solvents. The loss of cellulose is small. Transformation of cellulose I into cellulose II occurs, as indicated by the X-ray diagram. The net result ia a very important increase in the yield of enzymatic hydrolysis after 4 days. Cadoxcn pretreatment after elimination of the hemicelluloses has been extensively investigated by Tsao [6] who reported an important increase in the yield of glucose. Dilute NaOH (Tables I and Z exp. 5 and 6). The increase of the yield in glucose after 4 days is very high for straw and lower for wood. Delignification is important in these conditions as demonstrated by the important weight loss without cellulose loss and the decrease in the amorphous contribution in the X-ray diagram (NaOH 5"/0). A recent comprehensive study of the effect of base concentration on the degradation of wood components by NaOH [7] is in agreement with the present results. Transformation of cellulose I into cellulose II does not occur. NaOH 18% (Tables 1 and 2, exp. 7 and 8). The yield after 4 days is high for straw and the weight loss is important. As in the other pretreatments, pretreated wood is less reactive than pretreated straw. Transformation of cellulose I into cellulose II is very important for paper pulp while it is partial for straw and wood. Decrease of the amorphous phase is not apparent in the X-ray diagram. The difference of reactivity of samples pretreated for 2 and 24 hr is unimportant. NaCIO (Tables 1 and 2, exp. 9-16). Table I indicates that the reactivity of pure cellulose decreases after pretreatment with HCIO-NaCIO. Pure cellulose is well known to be oxidized in these conditions [8, 9]. Ketone and acid groups are formed. Chain and cycle breaking have been reported. This effect results in the weight loss and loss of cellulose reported in Table 2. The consequence of the formation of these new functional groups is the observed decrease of reactivity in the presence of the highly selective enzyme catalysts. For wheat straw and spruce wood, the loss of cellulose is lower than for paper pulp but the weight loss (lignin and a small fraction of hemicelluloses) is rather important. An important increase of reactivity results from the pretreatment. It can be assigned to

a preferential oxidation of the lignin fraction of the complex lignin-cellulose network of wood and straw. Lignin is indeed well known to be oxidized by HCIO-NaCIO. Ring opening, acid formation and chain breaking occurs [10, 11]. Transformation of cellulose I into cellulose II is not observed in the X-ray diagram (Fig. 2). The increase of reactivity of wood and straw can thus be assigned to an increase in accessibility of the cellulosic fraction of the substrate due to partial delignification and detachment of the lignin network. It was then attempted to increase further the reactivity of wood and straw pretreated by HCIO-NaCIO either by performing a more complete delignification or by promoting the transformation of cellulose I into cellulose I1 by additional treatments. Elimination of the partly oxidized lignin fragments trapped in the substrate pretreated by HCIO-NaC10 was done by extraction of the substrate with water, dilute HCI or dilute NaOH under reflux (exp. 10, I 1 and 12). These extractions result in a larger weight loss without affecting the percent cellulose recovered. Surprisingly, a decrease of reactivity of the substrate is observed (Table 2). We assign it to a rearrangement of the fibrillar structure of cellulose which accompanies the delignification at 100. This results in decrease of accessibility when compared to the initial wood pretreated with HC10-NaCIO at room temperature. On the contrary, a beneficial effect was observed when the HCIO-NaCIO pretreated wood and straw were subsequently treated with NaOH 18% or transformed into xanthate and regenerated into cellulose II (exp. 13-15). The pretreatment by HCIO-NaCIO can also be performed at controlled pH. The highest yields are obtained for wood and straw if a constant pH value ranging from 7 to 9 is maintained during the pretreatment. This effect has been previously discussed [3, II, 12]. Typical rcsults are given in Tables 1 and 2 for comparative purpose (exp. 16). Increasing the enzyme concentration can lead to nearly quantitative yields of glucose [12]. Mechanical pretreatment (Tables I and 2, exp. 17-21). These pretreatments are inefficient to promote the hydrolysis of glucose (Tables 1 and 2, exp. 17-21). Delignification is well known to be very low in these conditions. Pretreatment o f spruce wood b), hah)genated oxidants The preceding section has shown that an important increase in glucose formation is obtained when spruce wood is pretreated with HC10-NaC10. A systematic investigation of the effect of other halogenated

Utilization of waste cellulose

V

519

Table 4. Enzymatic hydrolysisof spruce wood, barley straw and bagasse after pretreatment with sodium or calcium hypochlorite at pH 8 Substrate % cellulose in Cellulose Glucoseformedafter Yieldafter Initial cone.~' recovered the pretreatment recovered 4 days Substrate Pretreatmcnt (mol:kg) (%) substance (%) I day 2 days 4 days (%) Spruce Spruce Spruce Spruce Barley Barle) Barley Bagasse Bagasse Bagasse

Ca(CIO): NaCIO NaCIO

5 5 15

NaCIO Na('IO -Na('IO NaCIO

2 5 -2 5 --

96 92 72 -80.5 72.2 -64.3 56.6 --

-. . 45 38 39 43

-.

. 85 --92 91

34

47 46 42

72 62 --

2 1.3 4.3 09 3 4 1.9 3.1 6.7 0.9

2.8 1.5 6.6 14 3.7 6.2 2.2 3.7 7.9 1.2

3.5 1.5 8.2 15 4.3 8.2 2.,-1 4.4 9.4 1.8

35.4413.7f 66 I~ 40 68 25 34 74 15

Lignin recovered (%)

m

46 26 73 51;

*Mole oxidant..kg substrate. 4"Calculated assuming negligible loss of cellulose.

oxidants was then undertaken. These oxidants (NaCIO 2. NaC10~, NaCIO4, KCIO 3, KBrO~, N a B r O and KBrO) used at pH 8 with an initial concentration o f 0.5 M and a ratio o f oxidant to substrate o f 15 mol:kg, do not p r o m o t e the rate and yield o f enzymatic hydrolysis o f the pretreated substrate. The yield o f glucose after 4 days was lower or equal to that obtained for untreated substrate in all cases. Very promising results were however obtained for Ca(CIO), which gives better results than NaC10 at the same ratio o f moles o f C10 /kg o f pretreated substratc (Table 4).

Pretreatment c~[ harley H('IO NaCIO at p l l 8

straw

and

bagasse

by

The results are given in Table 4. High yields o f glucose (68 and 74%) arc obtained for b o t h substratcs when 5 m o l oxidant is c o n s u m e d per kg substratc. At Iowcr oxidant c o n s u m p t i o n (2 mol/kg), the limiting yield is lower (40 and 34%) but in any case much higher than that obtained for the untreated substrate. CONCI,USION These results show that the net effect o f a given pretreatment is generally different when applied to pure cellulose or to ligno-cellulosic materials. A sequence o f efficiences for the pretreatments o f pure ccllulose can be deduced from the present work and a previous publication. It is N a O H 18% > untreated > NaCIO 0.5 M > H 2 0 , / F e 2' > 7(50 Mr). T r a n s f o r m a t i o n o f cellulose I into cellulose II increases the reactivity while oxidation decreases it owing to the formation o f new functional groups. The results obtained for wheat straw and spruce wood arc very different. The sequences o f efficiencies for wood and straw are respectively: C a d o x e n "NaC10 pH 8 > N a O H 18% > dil. N a O H > H~O2/ Fe-" > 7 > untreated. Cadoxen -~ N a O H 18% and 5% > NaCIO pH 8 > H,O2/Fe 2" = 7 > untreated. The reactivity o f ligno-cellulosic substrates results from competiti,,e d e g r a d a t i o n o f lignin and cellulose and from t r a n s f o r m a t i o n o f cellulose 1 into cellulose II. Prctreatment with HC10 NaCIO pH 8 c o m p a r e s very favourably with the more expensive cadoxen and N a O H 18% p r e t r e a t m e n t s in the case o f wood. It must be emphasized that the limiting yields reported

here for spruce w o o d pretreated with H C 1 0 NaCIO can be efficiently improved by modifying enzyme and substrate concentration as shown by a detailed kinetic study o f the enzymatic hydrolysis [12]. The pretreatment by H C I O - N a C I O is applicable to a wide range o f ligno-cellulosic materials. Previous work has shown that high yields o f glucose can also be obtained for eucalyptus w o o d pretreated with H C I O - N a C I O [3]. This substrate is completely nonreactive in the absence o f pretreatments. The present results show that high yields o f glucose can be obtained with reduced quantities o f oxidant in the case o f bagasse which is available in large quantities. Similar results are obtained with barley straw. O t h e r halogenated oxidants are not cfficient when used at pH 8, with the exception o f calcium hypochlorite which seems better than sodium hypochlorite when applied to spruce wood. This last point needs further fundamental investigation and application to other substrates.

Acknowledgements--We thank the "Minist6re du Budget", the "Ministere de r Emploi et du Travail" and the "European Community Commission" for financial support to the laboratory. REFERENCES

I. C. R. Wilke, B. Maiorella, A. Sciamanna, D. Wiley and H. Wong, Enzymatic ttydrolysis of Cellulose- Theory and Applications. Noyes Data Corporation, New Jersey (1983). 2. C. David and Ph. Thiry, Eur. Polym. J. 17, 957 (1981). 3. C. David, R. Fornasier, C. Greindl-Fallon and N. Vanlautem, Biotech. Bioengng. 27, 1531 (1985). 4. Unpublished results. 5. C. David and Ph. Thiry, J. appl. Poh'm. Sci. 27, 2395 (1982). 6. M. R. Ladish, C. Ladish and G. T. Tsao, Science 201, 743 (1978). 7. Yuan Zong Lai, A. R. Czerkies and Ing-Luen Shiau, J. appl. Polym. Sci. Appl. Syrup. 37, 943 (1983). 8. L. F. McBurney, Cellulose and Cellulose Deriratives. Vol. V, Part I. lnterscience, New York (1954). 9. M. Lewin and J. A. Epstein, J. Polym. Sci. 58, 1023 (1962). 10. K. V. Sarkanen and C. H. Ludwig, Lignins. Wiley. New York (1971). 11. C. David, Ph. Thiry and R. Fornasier, Appl. Biochern. Biotech. In press. 12. C. David and R. Fornasier, Macromolecules. In press.