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Delignification and Swelling of Eucalpytus Wood Ahead of Enzymatic Hydrolysis of the Cellulosic Fraction
Process Biochemistry, Vol. 30, No. 6, pp. 537-545. 1995 Copyright B 1995 Elsevier Science Ltd Printed in Great Britain. AU rights reserved 0032.9592/95 $9.50 + 0 00 0032-9592(94)00063-S
ELSEVIER
Delignification and Swelling of Eucalpytus Wood Ahead of Enzymatic Hydrolysis of the Cellulosic Fraction J. C. Paraj6,” J. L. Alonso & V. Santos Department (Received
of Chemical Engineering,
University of Vigo (Campus Orense), Las Lagunas, 32004 Orense, Spain
17 July 1994; revised manuscript
received 15 September
1994; accepted 2 October
1994)
An eflective process for the chemical-biotechnological utilization of Eucalyptus globulus wood is reported. In a first stage, a treatment with catalysed acetic acid solutions allowed the solubilization of both lignin and hemicelluloses, leaving cellulose as solid residue. A subsequent swelling treatment with NH,OH solutions caused structural modifications to the cellulosicfraction, giving a substrate highly susceptible to the enzymic hydro1y.si.s. A centered, incomplete, ,factorial design of experiments was carried out in order to establish the inter-relationships between three selected operational variables (NH,OH concentration used in the chemicul treatment and both liquid/solid and enzymelsubstrate ratios employed in the hydrolysis step) on the kinetics and yields of enzymatic hydrolysis. From experimental data, generalized kinetic models giving the injluence ofthe operational van’ables on the hydrolysis stage were developed. Further discussion on relevunt uspects involved in the process is provided.
INTRODUCTION
Fermentative processes are an attractive possibility for the utilization of the carbohydrate fracof LCM. tion Before fermentation, the polysaccharides (cellulose or hemicelluloses) have to be converted into sugars, the authentic carbon source used by micro-organisms. In order to improve the economic features of the processes, the polysaccharide hydrolysis must reach high yield with minimum byproduct generation. The saccharification step can be performed in media catalysed by acids or enzymes. The acid hydrolysis of LCM proceeds with fast kinetics, but the presence of reaction byproducts (such as furfural, acetic acid or hydroxymethylfurfural) can render difficult the utilization of the reaction liquors as fermentation media, leading to the necessity of further ‘refining’ treatments.2-4 On the contrary, the enzymic hydrolysis proceeds with
Lignocellulosic materials (LCM) are interesting raw materials for the chemical industry, owing to several factors such as low price, renewable nature and large availability. Chemicals and fuels can be obtained from LCM by means of a variety of chemical or biotechnological processes. An efficient utilization of LCM can be obtained if their polymeric constituents (lignin, hemicelluloses and cellulose) are separated in a first chemical treatment (‘fractionation’ treatment). In subsequent steps, the main fractions cited above (untouched or as degradation products) can be used in different product applications.’
*To whom correspondence
should
be addressed.
537
538
J. C. Parajb, J. L. Alonso, V Santos
slow kinetics, but shows several advantages, including: (i) high selectivity with low byproduct generation; (ii) the reaction is carried out under mild operational conditions; (iii) the utilization of toxic or corrosive compounds is avoided, and (iv) high sugar yields can be reached with susceptible substrates.“-’ Both lignin and hemicelluloses of native LCM hinder the access of enzymes to cellulose. A chemical pretreatment of substrates (causing lignin removal and hemicellulose degradation) increases the cellulose content of solid residues, allowing significant improvements in both kinetics and yields of reaction.‘-’ This delignification step can also be conceived as a fractionation treatment, leading to separate streams that contain the polymeric constituents of LCM or their degradation products. HCl-catalysed acetic acid solutions are very interesting as chemical agents for performing the fractionation of the raw materials: under selected experimental conditions, extensive delignification and hemicellulose removal can be reached in a single treatment with almost quantitative cellulose recovery. The lignin fractions show little chemical alteration, whereas pentoses and furfural are generated from hemicelluloses. Studies on both kinetics and yields of Eucalyptus globulus wood fractionation by HCl-catalysed acetic acid solutions have been previously reported by the authors.*q9 After the delignification stage, a subsequent ‘swelling’ treatment is often necessary to decrease the cellulose crystallinity. For this purpose, a variety of chemical agents have been used, including KOH, NaOH, NH,OH, mineral acids, n-butylamine and n-propylamine.‘0-12 Ammonia (pure or as aqueous solutions) has been employed as swelling agent in reported studies.‘2,‘3 Compared to other chemicals employed for swelling, ammonia shows several advantages such as high volatilityI and the possibility of further utilization as nitrogen source in subsequent processes of biotransformation.‘2 This work deals with the development of efficient strategies for utilization of Eucalyptus globuZus wood. In a first stage, a treatment with catalysed acetic acid solutions (performed under selected operational conditions) allowed the solubilization of both lignin and hemicelluloses, leaving cellulose as a solid residue. In a second step, structural modification was provoked on the cellulosic fraction by means of treatments with NH,OH, in order to obtain a substrate highly
susceptible to enzymic hydrolysis. A centered, incomplete, factorial design of experiments was carried out in order to establish the inter-relationships between three selected operational variables (NH40H concentration used in the swelling treatments and both liquid/solid and enzyme/substrate ratios employed in the hydrolysis step) on the kinetics and yields of enzymic hydrolysis. MATERIALS
AND METHODS
Raw material Samples of Eucalyptus glob&s wood, collected in a local pulp mill, were milled, screened to select the fraction of particles with a size between 0.25 and 1 mm, homogenized to ensure identical composition among the different aliquots taken from the wood lot, air-dried and stored. Analysis of samples Wood samples were analysed for moisture and submitted to quantitative saccharification with 72% H2S04 under standard conditions.14 I&son lignin was calculated from the oven-dry weight of the solid residue from hydrolysis. Polysaccharide content was calculated from the reducing sugar content of hydrolysates (determined by the Somogyi-Nelson method). The glucan content was calculated from the glucose concentration of hydrolysates (determined by the glucose-oxidase Sigma K- 5 10 method). Delignification Milled and screened wood samples were delignified with acetic acid-HCl-water solutions under conditions selected on the basis of a previous work.Y The operational conditions used for delignification were: normal boiling temperature; 95% acetic acid and 0.2% HCl concentration; liquid/solid ratio, 10 g/g; reaction time, 1 h. The reaction was performed in a batch reactor, and the solid residue was separated by filtration, washed with acetone and air-dried. Aliquots from the solid residue were analysed for lignin, polysaccharide and glucan content using the same procedure above described for untreated wood. Swelling treatments Delignified samples were treated with NH,OH solutions at 60°C during 3 h using a liquid/solid ratio = 10 g/g. The NH,OH concentrations used in experiments were 4, 12 or 20 weight per cent of
Delignification
and swellingof Eucalyptus wood
solutions. At the end of treatments, the solid residues were separated by filtration, washed with water and air-dried. Aliquots of the samples obtained were subjected to chemical analysis (using the same procedures above cited). Enzymatic hydrolysis The enzymic hydrolyses of processed wood samples were performed using Trichoderma reesei cellulases (Celluclast, Novo, Denmark) and AspergiZ1u.s niger B-glucosidase (Novozym, Nova, Denmark). Enzymic activities of commerical solutions were determined using standard methods. The /I-glucosidase/substrate ratio was fixed in 80 IU/g. Both liquid/substrate and enzyme/substrate ratios were considered as operational variables. Other conditions used for hydrolysis were: temperature, 48.5”C; pH 4.85 (citric acid-sodium citrate buffer 0.05 N); reaction time, 48 h. Thymol was used as microbial preservative. Samples were withdrawn from the hydrolysis media at appropriate reaction times, centrifuged, and the supernatant was filtered through 0.45 pm membranes and analysed for reducing sugars by the Somogyi-Nelson method. Fitting of data The sugar concentration-time series of data obtained in the hydrolysis experiments were fitted to a hyperbolic modelI using commercial software (TableCurve from Jandel Scientific, Corta Madera, CA, USA).
RESULTS
AND DISCUSSION
Chemical composition of untreated wood The wood samples used for experimentation were taken from the same wood lot used in a previous study.9 The moisture of air dried-samples accounted for 11.0 weight per cent, whereas the Table 1. Residue yield of treatments and composition
contents in lignin, hemicelluloses and cellulose were 23.3, 17.3 and 47.8 weight per cent (ovendry basis), respectively. Delignification Table 1 shows the yield in solid residue of delignification experiments performed under the conditions selected, as well as the chemical composition of delignified samples. The chemical treatment achieved 90% lignin removal and 87% hemicellulose removal. The cellulosic fraction, measured as glucan, was quantitatively recovered. Taking into account the extent of both lignin and hemicelluloses removal and the high cellulose content of residues, it can be expected that the accessibility of enzymes to the treated substrate was greatly improved. However, limited improvements were observed when the delignified samples were used as substrates for hydrolysis in comparison with experiments performed with untreated wood (see Fig. 1). It was thought that the structural features of cellulose were responsible for the behaviour observed, and that significant improvements could be reached by performing a subsequent swelling treatment with NH,OH. Swelling and enzymatic hydrolysis of delignified wood Delignified wood samples were subjected to treatment with ammonium hydroxide under the conditions specified above. Table 1 shows the residue yield of extractions, as well as the composition of the solid phase obtained. It can be observed that little changes in chemical composition were caused by treatments: the lignin content of samples was not significantly altered, whereas a part of the hemicellulosic fraction was degraded causing a corresponding increase in the per cent of cellulose. Reported results on NH,OH treatments also showed little chemical alterations of substrates,12v13 even if strong operational condi-
of the solid phases obtained
Content (weight per cent, o.d. bask)
Residue yield
Sample
Lignin Delignified Delignified Delignified Delignified
wood + 4% NH,OH + 12% NH,OH + 20% NH,OH
“Residue yield expressed as g residue/100 *Residue yield expressed as g residue/100
54.9” 88.9h 91.0h 89.gh
539
4.4 4.3 4.3 4.2
g untreated wood, o.d. basis. g delignified wood, o.d. basis.
Hemicelluloses 2:; 7.0 6.8
CeIlulose 87-O 89.9 91.5 91.5
540
J. C. Parajb,
J. L. Alonso,
20
I/ Santos
25 REACTION
30 TIME. h
Fig. 1. Dependence of the sugar concentration on the reaction time for untreated wood, delignified samples and substrates subjected to delignification and swelling (operational conditions used for hydrolysis: liquid/solid ratio = 20 g/g; enzyme/ substrate ratio = 8 W/g).
Table 2. Definition and variation ranges of the independent variables and operational conditions assayed (a) Definition and variation ranges of the variables Variable
Nomenclature
NH,OH concentration Liquid/solid ratio employed for hydrolysis Enzyme/substrate ratio (b) Experimental Experiment
Units
Variafion range
AHC LSR
g/l00 g gig
4-20 12-28
ESR
lU/g
3-13
conditions assayed AHC
LSR
ESR
1 2 3 4 2
4 4 4 4 12
12 20 20 28 12 12
8 3 13 8 133
7 ;
12 12
20 20
8 8
10 11
12 12
;: 28
: 13
12 13 14
;: 20
;; 20
: 13
15 16
20 12
::
:
tions were used. However, the structural modifications caused by the swelling stage resulted in marked increases in the susceptibility of substrates, as it can be seen from Fig. 1. In order to achieve further understanding
about the dependence of the enzymic hydrolysis on the operational variables, a second-order, incomplete, centered, factorial design of experiments was carried out. Table 2a shows the definition and variation ranges of the three independent variables selected (NH,OH concentration used in the swelling step and both liquid/solid and enzyme/substrate ratios employed in hydrolysis). The operational conditions assayed (see Table 2b) were chosen as Poirier et a1.,16 following an experimental plan with the structure above cited. For each experiment, sugar concentration-time series of data were determined, and the experimental results were fitted to the hyperbolic model’” c=y,
.‘___, t +t1/2
where y, is the sugar concentration that should be reached at infinite reaction time, and t,/, is the reaction time necessary to achieve the 50% of y, . It can be observed from Fig. 2 the close interrelationship observed between experimental and calculated results in some representative experiences. From the hyperbolic model it follows that y2At1/2
where yZ is the initial reaction rate. For each of the experiments listed in Table 2b, y1 and y, were calculated and considered as dependent variables
Delignificationand swellingof Eucalyptus wood
i.
s I-
25
L p
20
8 2
15
$j
10
.
Exp. Calc. 9Exp.
12
--t
l
Exp. 14 Calc. Exp. 9
-.--- l
Calc. 10 Exp. Erp.
REACTION
14
TIME,
h.
Some representative results achieved in the enzymatic hydrolysis of processed Fig. 2. to the operational conditions of Table 2). Table 3.
541
substrates (experiments
corresponding
Independent dimensionless variables used in this study Nomenclature
Variable
Dimensionless ammonium hydroxide concentration Dimensionless liquid/solid ratio Dimensionless enzyme/substrate ratio
Kzriation range
Dej&zition
XI
(AHC-12)/8
(-1,
x:
(LSR-20)/8
(-1,l)
X3
(ESR-8)/5
I)
(- 1, 1)
AHC, LSR and ESR as in Table 2.
Table 4. Operational conditions assayed (expressed in terms of dimensionless independent variables) and expcrimental results achicvcd Exper.
6 7 8 9 10 11 12 13 14 15 16
Dimensionless, independent wriables
Experimental
x1
x2
xi
Yl (g/l)
Y, (s/Uh)
-1 -1 -1 -1
-1
0 -1 1 0 -I 1 0 0 0 -1
53.5 24.8 38.0 23.0 47.0 54.4 33.3 34.0 34.4 17.9 30.1 61.8 25.4 33.0 26.1 23.9
2.82 1.03 2.70 1.40 1.16 4.1 2.19 1.58 1.81 0.68 1.97 2.79 1.34 3.00 1.37 1-53
0 0 0 0 0 0 0 0 0 1 1 1 I 0
-1 -1 -1 0 0 0
I -:
:, 0 0 1 1
-1 1 0 0
results
in our study. For calculation purposes,” dimensionless and normalized independent variables (X,7 x2 and xX) were defined to measure respectively the ammonium hydroxide concentration, the liquid/solid ratio and the enzyme/substrate ratio. Table 3 lists the inter-relationships between the dimensional independent variables and the dimensionless ones. Table 4 shows the set of operational conditions assayed (expressed in terms of dimensionless independent variables) and the experimental results determined. The dependent and independent variables were correlated by empirical models having the following generalized expression
I
I
k
where y, (j: l-2) are the dependent variables, xi and xk (i or k: 1-3, k 2 i) are the dimensionless
J. C. Para@, J. L. Alamo,
542
VI Sanros
Table 5. Regression coefftcients and statistical parameters (a) Regression
coefficients Variable
Coejicien t b,,j 6, ;2! b:, 13, : 23, b II/
b
b:::
YI
Y2
33.64 0.88 - 15.14 5.05 - 1.30 - 1.40 1.20 0.03 6.97 - 3.45
1.890 0.069 -0.659 0.945 0.000 -0-003 -0.413 0.11 I 0.116 - 0.007
(b) Significance of coefficients - Significant coefficients at the 95% confidence level
LIWIDISDLID
28
Fig. 3. Calculated dependence of the maximum sugar concentration (y,, g/litre) on the liquid/solid ratio and on the enzyme/substrate ratio (data corresponding to samples treated with 12% NH,OH).
b,,, b,,. b,z, bz. b,,, b,,, -
RATID. g,g
Significant coefficients at the 90% confidence level
b 331 (c) Statistical parameters significance of models Variable Yl Y2
measuring
the correlation
R’
Correrted R2
0.9773
0.9432
28.65
0.967 3
0.9183
19.72
and
Fexpu Prob IFexp> F,,P CO.01 co.01
“F,, : defined as the ratio between the mean squares of mo ael and error. ‘Fv,: defined as the statistical value of F for the degrees of freedom of model and error.
to increases
in the liquid/solid
ratio from
12 to
20 g/g. In order to provide further information on the extent of the enzymic hydrolysis, the polysaccharide conversion achieved at infinite reaction time (PC,) was calculated according to the following expression: PC,=WOO, cfh
independent variables and b,. . . b, are the regression coefficients calculated by the least-squares method. Table 5 shows the regression coefficients calculated and their statistical significance, as well as mathematical parameters measuring the correlation and significance of the empirical models. The experimental values determined for the maximum sugar concentration y1 varied in a broad range (17.9-6143 g/litre). The highest values of y, (535-614 g/litre) were obtained at the lowest liquid/solid ratio assayed (12 g/g) in experiments using 8-13 IV/g (experiments 1,5,6 and 12). On the contrary, limited y, values were found in trials carried out with the least enzyme/ substrate ratio assayed at the highest liquid/solid ratio (experiment 10). Figure 3 shows the calculated dependence of y, on the most influential independent variables (liquid/solid ratio and enzyme/substrate ratio) for samples treated with 12% NH,OH. The major effects were associated
where C,, corresponds to the theoretical sugar concentration, which can be calculated from material balances assuming a quantitative transformation of polysaccharides into sugars. It can be conversion noted that the polysaccharide achieved at a given reaction time can be calculated from the above equation considering the sugar concentration achieved at this moment instead y,. The sugar concentration achieved at the desired reaction time is related with y, through the hyperbolic model. The strong influence of LSR on PC, can be assessed by comparing the results calculated for experiments 6 and 11 (both performed with ESR=13 IU/g and AHC= 12 g/100 g). PC, increased from 71.3% (experiment 6) to 92.1% (experiment 11) as ESR increased from 12 to 28 g/g. The replicate experiments (7, 8 and 9) led to PC, in the range 72.8-75-0, which is reasonable considering the complexity of the experimental procedure.
Delignijicationand swellingof Eucalyptus wood
Figure 4 shows the dependence of PC, on liquid/solid ratio and enzyme/substrate ratio (corresponding to samples treated with 12% NH,OH) calculated on the basis of the empirical model developed for y,. For low liquid/solid ratios, improvements in PC, were predicted when the enzyme/substrate ratio increased from 3 to 8 III/g. Further increases up to 13 IU/g did not enhance the polysaccharide conversion. This behaviour agrees with reported studies: several authors’3~‘5~‘8 found that little effects in polysaccharide conversion were caused when the enzyme/substrate ratio exceeded a limit value, which was near 10 III/g. The combined effects of liquid/solid ratio and enzyme/substrate ratio on PC, can be explained on the basis of substrate inhibition: little influence of PC,,, on the operational conditions was observed in the zone of the response surface corresponding to high enzyme/ substrate ratios and low liquid/solid ratios, where significant inhibition effects are expected. On the contrary, a marked dependence of PC, on enzyme/substrate ratio was predicted for experiments carried out with the least liquid/solid ratio assayed. Little improvements in PC, were associated with increases in the ammonium hydroxide concentration in the range considered. The experimental values obtained for the initial hydrolysis rate (y2) varied in a broad range (0.68-4-l g/litre/h). The extreme values were obtained in experiments 10 and 6, performed with the minimum and the maximum enzyme concentration, respectively.
Figure 5 shows the predicted dependence of y, on the most influential independent variables (liquid/solid ratio and enzyme/substrate ratio) for samples treated with 12% NH,OH. It can be observed a linear increase of yZ when either enzyme/substrate ratio was increased or the liquid/solid ratio was decreased. In order to provide further information on the effect caused by the liquid/solid ratio on the hydrolysis rate, an additional variable (roe, initial specific rate, g/g/h) was defined according to the following expression: r”e=$) rh
where r, and C,h are as above. Figure 6 shows the dependence calculated for roe on the ammonium hydroxide concentration and liquid/solid ratio when the enzyme/substrate ratio was fiied in 8 IU/g substrate. Little effects were associated with variations in the NH,OH concentration within the variation range considered. However, when the liquid/solid ratio was increased from 12 to 20 g/g, r,, increased by about 20%. Increases in the solid/liquid ratio above 20 g/g did not signifihas been cantly affect r,,. Similar behaviour reported for the enzymatic hydrolysis of other LCM.‘3*‘5 It can be seen from the empirical models that r,, increased linearly with the enzyme/substrate ratio within the range tested for this variable. The
4MMONI”M
AMMONIUM
HYDROXIDE
CONCENTRATION
9-P ;-A ENZYMEISUSSTR.RATIO. Ill/g
”
543
HYDROXIDE
CONCENTMTIDN
= 12 %
= 12%
LIOUIDISOUD
mno, P/P
3
Calculated dependence of the polysaccharide Fig. 4. conversion PC, on the liquid/solid ratio and on the enzyme/ substrate ratio (data corresponding to samples treated with 12% NH,OH).
LIOUID/SOLIDRATIO. g/g
28
Fig. 5. Calculated dependence of the initial reaction rate (y?, g/litrc/h) on the liquid/solid ratio and on the enzyme/ substrate ratio (data calculated for samples treated with 12% NH,OH).
544
J. C. Parajb, J. L. Alonso, V. Santos ENZYME,S”BSTRATE
f
RATIO
= 8 W/g
0.044
a
.
0.042
2 F: r
0.038
r d
0.04
D-036
y
0.034
E %
0.032 0.03
NH40”
CONCENTKATION,
20
%
Fig. 6. Calculated dependence of the initial specific rate (roe, g/g/h) on the liquid/solid ratio and on the NH,OH concentration (data calculated for experiments performed with an enzyme/substrate ratio of 8 W/g).
AMMONIUM
HYDROXIOE CONCENTRATION
= 12%
__ 3
ENZYMEISUBSTFIATE W/p
RATIO,
Fig. 7. Calculated dependence of the sugar concentration (reached after 48 h reaction time) on the liquid/solid ratio and on the enzyme/substrate ratio (data calculated for samples treated with I 2% NH,OH).
equations predicted a broad variation range for r,, (0.0 l-0.06 g/g/h for samples treated with 12% NH,OH). From the kinetic models developed, the sugar concentration reached at a given reaction time under a fixed set of operational conditions can be calculated. As an example, Fig. 7 shows the calculated dependence of the sugar concentration reached after 48 h reaction time on the liquid/ solid ratio and on the enzyme/substrate ratio for samples treated with 12% NH,OH. It can be
remarked that a sugar concentration of 45 g/litre (or 60% polysaccharide conversion) can be obtained in experiments performed with 12 g liquid/g solid and 13 IU/g substrate. In conclusion, the proposed process provides an efficient scheme for the chemical-biotechnological processing of Eucalyptus wood. The delignification step allowed an extensive separation of lignin and hemicelluloses from the solid residue, which showed an enhanced cellulose content. The solid phase from delignification fits the chemical requirements exiged for good hydrolysis substrates, but its structural features are not adequate for this purpose. A subsequent treatment with NH,OH solutions provoked little changes in chemical composition, but altered the structure of the solids leading to a marked increase in their susceptibility to the enzymic hydrolysis. From experimental data, the kinetics and yields of the enzymatic hydrolysis were modelled by empirical, second-order equations giving a quantitative interpretation of the process. The NH,OH concentration, used as operational variable, did not show a significant influence on the overall process within the limits tested in this study. Low liquor/solid ratios and high enzyme substrate/ratios led to sugar concentrations up to 45 g/litre after 48 h, with polysaccharide conversions in vicinity of 60%. The kinetic models provide the necessary information for performing preliminary evaluations on the feasibility of the hydrolysis stage of the overall process studied.
REFERENCES 1. Myerly, R. S., Nicholson, M. D., Katzen, R. & Taylor, .I. M., The forest refinery. Cherntech, 11 (1981) 186-92. 2. Beck, M. J. & Strickland, R. C., Production of ethanol by bioconversion of wood sugars derived from two-stage dilute acid hydrolysis of hardwood. Biomass, 6 (1984) 101-10. 3. Clark, T. A. & Mackie, K. L., Fermentation inhibitors in wood hydrolysates derived from the softwood Pinus radiata. J. Chem. Tech. Biotechnol., 34 (1984) 101-10. 4. Frazer, I-‘. R. & McCaskey, T. A., Wood hydrolyzate treatments for improved fermentation of wood sugars to 2,3-butanediol. Biomass, 18 (1989) 31-42. 5. Fan, L. T., Lee, Y. H. & Gharpuray, M. M., The nature of lignocellulosics and their pretreatments for enzymic hydrolysis. Adv. Biochem. Eng., 23 (1982) 157-87. 6. Blanch, H. W! & Wilke, C. R., Sugars and chemicals from cellulose. Rev. Chem. Eng., 1 (1983) 71-118. 7. Gharpuray, M. M., Lee, Y. H. & Fan, L. T.; Structural modification of lignocellulosics by pretreatments to enhance enzymatic-hydrolysis. Bioiechnol. Bioeng., 25 (1983) 157-72.
Delignification
and swelling of Eucalyptus
8. Wzquez, D., Lage, M. A., Parajo, J. C. & Vazquez, G., Fractionation of Eucalyptus wood in acetic acid media. Biores. TechnoL, 40 (1992) 13 1-6. 9. Parajo, J. C., Alonso, J. L. & Vaquez, D., On the behaviour of lignin and hemicelluloses during the acetosob processing of wood. Biores. Technol., 46 (1993)
233-40. 10. Kitsos, H. M., Roberts, R. S. & Muzzv, J. D., n-Proovlamine pretreatment of ‘buffered solvent’ pulp to enh&&e enzymatic hvdrolvsis. Biores. Technol.. 39 (1992) 24i-7. _ _ 11. Tanaka, M., Song, G. J., Matsuno, R. & Kamikubo, T., Optimal conditions for pretreatment of rice straw with n-butylamine for enzymatic solubilization. Appl. Microb.
Biotechnol., 22 (1985) 13-18. M., Dale, B. E. & Craig, W. K., Compari12. Mes-Hartree, son of steam and ammonia pretreatment for enzymatic hydrolysis of cellulose. Appl. Microb. Biotechnol., 29
(1988) 462-8. ammonia pretreatment ot 13. Yu Chia, T. C., Supercritical lignocellulosic materials. Biotechnol. Rioeng. Symp, 17
wood
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(1986) 19-32. 14. Browning, B. L. In Methods of Wood Chemistry. John Wiley & Sons, New York, 1967. 15. Holtzapple, M. T. & Humphrey, A. E., The effect of organosolv pretreatment on the enzymatic hydrolysis of poplar. Biozechnol. Bioeng., 26 (1984) 670-6. 16. Poirer, M. G., Ahmed, A., Grandma&on, J. L. & Kaliaguine, S. C. F., Supercritical extraction of wood with ethanol in a tubular reactor. Ind. Eng. Chem. Res., 26 (1987) 1738-43. 17. Box, G. E. P., Hunter, W. G. Kc Hunter,J. S. In Estadtitica Para Investigadores. Ed. Revert&, Barcelona, I988. 18. Hamilton, T. J., Dale, B. E., Ladisch, M. R. & Tsao, G. T., Effect of ferric tartrate/sodium hydroxide solvent pretreatment on enzyme hydrolysis of cellulose in corn residue. Biotechnol. Bioeng., 16 (1984) 781-7. 19. Sattler, W., Esterbauer, H., Gtatter, 0. Kc Steiner, W., The effect of enzyme concentration on the rate of the hydrolysis of cellulose. Biotechnol. Bioeng., 33 (1989)
1221-34.
ELSEVIER
Delignification and Swelling of Eucalpytus Wood Ahead of Enzymatic Hydrolysis of the Cellulosic Fraction J. C. Paraj6,” J. L. Alonso & V. Santos Department (Received
of Chemical Engineering,
University of Vigo (Campus Orense), Las Lagunas, 32004 Orense, Spain
17 July 1994; revised manuscript
received 15 September
1994; accepted 2 October
1994)
An eflective process for the chemical-biotechnological utilization of Eucalyptus globulus wood is reported. In a first stage, a treatment with catalysed acetic acid solutions allowed the solubilization of both lignin and hemicelluloses, leaving cellulose as solid residue. A subsequent swelling treatment with NH,OH solutions caused structural modifications to the cellulosicfraction, giving a substrate highly susceptible to the enzymic hydro1y.si.s. A centered, incomplete, ,factorial design of experiments was carried out in order to establish the inter-relationships between three selected operational variables (NH,OH concentration used in the chemicul treatment and both liquid/solid and enzymelsubstrate ratios employed in the hydrolysis step) on the kinetics and yields of enzymatic hydrolysis. From experimental data, generalized kinetic models giving the injluence ofthe operational van’ables on the hydrolysis stage were developed. Further discussion on relevunt uspects involved in the process is provided.
INTRODUCTION
Fermentative processes are an attractive possibility for the utilization of the carbohydrate fracof LCM. tion Before fermentation, the polysaccharides (cellulose or hemicelluloses) have to be converted into sugars, the authentic carbon source used by micro-organisms. In order to improve the economic features of the processes, the polysaccharide hydrolysis must reach high yield with minimum byproduct generation. The saccharification step can be performed in media catalysed by acids or enzymes. The acid hydrolysis of LCM proceeds with fast kinetics, but the presence of reaction byproducts (such as furfural, acetic acid or hydroxymethylfurfural) can render difficult the utilization of the reaction liquors as fermentation media, leading to the necessity of further ‘refining’ treatments.2-4 On the contrary, the enzymic hydrolysis proceeds with
Lignocellulosic materials (LCM) are interesting raw materials for the chemical industry, owing to several factors such as low price, renewable nature and large availability. Chemicals and fuels can be obtained from LCM by means of a variety of chemical or biotechnological processes. An efficient utilization of LCM can be obtained if their polymeric constituents (lignin, hemicelluloses and cellulose) are separated in a first chemical treatment (‘fractionation’ treatment). In subsequent steps, the main fractions cited above (untouched or as degradation products) can be used in different product applications.’
*To whom correspondence
should
be addressed.
537
538
J. C. Parajb, J. L. Alonso, V Santos
slow kinetics, but shows several advantages, including: (i) high selectivity with low byproduct generation; (ii) the reaction is carried out under mild operational conditions; (iii) the utilization of toxic or corrosive compounds is avoided, and (iv) high sugar yields can be reached with susceptible substrates.“-’ Both lignin and hemicelluloses of native LCM hinder the access of enzymes to cellulose. A chemical pretreatment of substrates (causing lignin removal and hemicellulose degradation) increases the cellulose content of solid residues, allowing significant improvements in both kinetics and yields of reaction.‘-’ This delignification step can also be conceived as a fractionation treatment, leading to separate streams that contain the polymeric constituents of LCM or their degradation products. HCl-catalysed acetic acid solutions are very interesting as chemical agents for performing the fractionation of the raw materials: under selected experimental conditions, extensive delignification and hemicellulose removal can be reached in a single treatment with almost quantitative cellulose recovery. The lignin fractions show little chemical alteration, whereas pentoses and furfural are generated from hemicelluloses. Studies on both kinetics and yields of Eucalyptus globulus wood fractionation by HCl-catalysed acetic acid solutions have been previously reported by the authors.*q9 After the delignification stage, a subsequent ‘swelling’ treatment is often necessary to decrease the cellulose crystallinity. For this purpose, a variety of chemical agents have been used, including KOH, NaOH, NH,OH, mineral acids, n-butylamine and n-propylamine.‘0-12 Ammonia (pure or as aqueous solutions) has been employed as swelling agent in reported studies.‘2,‘3 Compared to other chemicals employed for swelling, ammonia shows several advantages such as high volatilityI and the possibility of further utilization as nitrogen source in subsequent processes of biotransformation.‘2 This work deals with the development of efficient strategies for utilization of Eucalyptus globuZus wood. In a first stage, a treatment with catalysed acetic acid solutions (performed under selected operational conditions) allowed the solubilization of both lignin and hemicelluloses, leaving cellulose as a solid residue. In a second step, structural modification was provoked on the cellulosic fraction by means of treatments with NH,OH, in order to obtain a substrate highly
susceptible to enzymic hydrolysis. A centered, incomplete, factorial design of experiments was carried out in order to establish the inter-relationships between three selected operational variables (NH40H concentration used in the swelling treatments and both liquid/solid and enzyme/substrate ratios employed in the hydrolysis step) on the kinetics and yields of enzymic hydrolysis. MATERIALS
AND METHODS
Raw material Samples of Eucalyptus glob&s wood, collected in a local pulp mill, were milled, screened to select the fraction of particles with a size between 0.25 and 1 mm, homogenized to ensure identical composition among the different aliquots taken from the wood lot, air-dried and stored. Analysis of samples Wood samples were analysed for moisture and submitted to quantitative saccharification with 72% H2S04 under standard conditions.14 I&son lignin was calculated from the oven-dry weight of the solid residue from hydrolysis. Polysaccharide content was calculated from the reducing sugar content of hydrolysates (determined by the Somogyi-Nelson method). The glucan content was calculated from the glucose concentration of hydrolysates (determined by the glucose-oxidase Sigma K- 5 10 method). Delignification Milled and screened wood samples were delignified with acetic acid-HCl-water solutions under conditions selected on the basis of a previous work.Y The operational conditions used for delignification were: normal boiling temperature; 95% acetic acid and 0.2% HCl concentration; liquid/solid ratio, 10 g/g; reaction time, 1 h. The reaction was performed in a batch reactor, and the solid residue was separated by filtration, washed with acetone and air-dried. Aliquots from the solid residue were analysed for lignin, polysaccharide and glucan content using the same procedure above described for untreated wood. Swelling treatments Delignified samples were treated with NH,OH solutions at 60°C during 3 h using a liquid/solid ratio = 10 g/g. The NH,OH concentrations used in experiments were 4, 12 or 20 weight per cent of
Delignification
and swellingof Eucalyptus wood
solutions. At the end of treatments, the solid residues were separated by filtration, washed with water and air-dried. Aliquots of the samples obtained were subjected to chemical analysis (using the same procedures above cited). Enzymatic hydrolysis The enzymic hydrolyses of processed wood samples were performed using Trichoderma reesei cellulases (Celluclast, Novo, Denmark) and AspergiZ1u.s niger B-glucosidase (Novozym, Nova, Denmark). Enzymic activities of commerical solutions were determined using standard methods. The /I-glucosidase/substrate ratio was fixed in 80 IU/g. Both liquid/substrate and enzyme/substrate ratios were considered as operational variables. Other conditions used for hydrolysis were: temperature, 48.5”C; pH 4.85 (citric acid-sodium citrate buffer 0.05 N); reaction time, 48 h. Thymol was used as microbial preservative. Samples were withdrawn from the hydrolysis media at appropriate reaction times, centrifuged, and the supernatant was filtered through 0.45 pm membranes and analysed for reducing sugars by the Somogyi-Nelson method. Fitting of data The sugar concentration-time series of data obtained in the hydrolysis experiments were fitted to a hyperbolic modelI using commercial software (TableCurve from Jandel Scientific, Corta Madera, CA, USA).
RESULTS
AND DISCUSSION
Chemical composition of untreated wood The wood samples used for experimentation were taken from the same wood lot used in a previous study.9 The moisture of air dried-samples accounted for 11.0 weight per cent, whereas the Table 1. Residue yield of treatments and composition
contents in lignin, hemicelluloses and cellulose were 23.3, 17.3 and 47.8 weight per cent (ovendry basis), respectively. Delignification Table 1 shows the yield in solid residue of delignification experiments performed under the conditions selected, as well as the chemical composition of delignified samples. The chemical treatment achieved 90% lignin removal and 87% hemicellulose removal. The cellulosic fraction, measured as glucan, was quantitatively recovered. Taking into account the extent of both lignin and hemicelluloses removal and the high cellulose content of residues, it can be expected that the accessibility of enzymes to the treated substrate was greatly improved. However, limited improvements were observed when the delignified samples were used as substrates for hydrolysis in comparison with experiments performed with untreated wood (see Fig. 1). It was thought that the structural features of cellulose were responsible for the behaviour observed, and that significant improvements could be reached by performing a subsequent swelling treatment with NH,OH. Swelling and enzymatic hydrolysis of delignified wood Delignified wood samples were subjected to treatment with ammonium hydroxide under the conditions specified above. Table 1 shows the residue yield of extractions, as well as the composition of the solid phase obtained. It can be observed that little changes in chemical composition were caused by treatments: the lignin content of samples was not significantly altered, whereas a part of the hemicellulosic fraction was degraded causing a corresponding increase in the per cent of cellulose. Reported results on NH,OH treatments also showed little chemical alterations of substrates,12v13 even if strong operational condi-
of the solid phases obtained
Content (weight per cent, o.d. bask)
Residue yield
Sample
Lignin Delignified Delignified Delignified Delignified
wood + 4% NH,OH + 12% NH,OH + 20% NH,OH
“Residue yield expressed as g residue/100 *Residue yield expressed as g residue/100
54.9” 88.9h 91.0h 89.gh
539
4.4 4.3 4.3 4.2
g untreated wood, o.d. basis. g delignified wood, o.d. basis.
Hemicelluloses 2:; 7.0 6.8
CeIlulose 87-O 89.9 91.5 91.5
540
J. C. Parajb,
J. L. Alonso,
20
I/ Santos
25 REACTION
30 TIME. h
Fig. 1. Dependence of the sugar concentration on the reaction time for untreated wood, delignified samples and substrates subjected to delignification and swelling (operational conditions used for hydrolysis: liquid/solid ratio = 20 g/g; enzyme/ substrate ratio = 8 W/g).
Table 2. Definition and variation ranges of the independent variables and operational conditions assayed (a) Definition and variation ranges of the variables Variable
Nomenclature
NH,OH concentration Liquid/solid ratio employed for hydrolysis Enzyme/substrate ratio (b) Experimental Experiment
Units
Variafion range
AHC LSR
g/l00 g gig
4-20 12-28
ESR
lU/g
3-13
conditions assayed AHC
LSR
ESR
1 2 3 4 2
4 4 4 4 12
12 20 20 28 12 12
8 3 13 8 133
7 ;
12 12
20 20
8 8
10 11
12 12
;: 28
: 13
12 13 14
;: 20
;; 20
: 13
15 16
20 12
::
:
tions were used. However, the structural modifications caused by the swelling stage resulted in marked increases in the susceptibility of substrates, as it can be seen from Fig. 1. In order to achieve further understanding
about the dependence of the enzymic hydrolysis on the operational variables, a second-order, incomplete, centered, factorial design of experiments was carried out. Table 2a shows the definition and variation ranges of the three independent variables selected (NH,OH concentration used in the swelling step and both liquid/solid and enzyme/substrate ratios employed in hydrolysis). The operational conditions assayed (see Table 2b) were chosen as Poirier et a1.,16 following an experimental plan with the structure above cited. For each experiment, sugar concentration-time series of data were determined, and the experimental results were fitted to the hyperbolic model’” c=y,
.‘___, t +t1/2
where y, is the sugar concentration that should be reached at infinite reaction time, and t,/, is the reaction time necessary to achieve the 50% of y, . It can be observed from Fig. 2 the close interrelationship observed between experimental and calculated results in some representative experiences. From the hyperbolic model it follows that y2At1/2
where yZ is the initial reaction rate. For each of the experiments listed in Table 2b, y1 and y, were calculated and considered as dependent variables
Delignificationand swellingof Eucalyptus wood
i.
s I-
25
L p
20
8 2
15
$j
10
.
Exp. Calc. 9Exp.
12
--t
l
Exp. 14 Calc. Exp. 9
-.--- l
Calc. 10 Exp. Erp.
REACTION
14
TIME,
h.
Some representative results achieved in the enzymatic hydrolysis of processed Fig. 2. to the operational conditions of Table 2). Table 3.
541
substrates (experiments
corresponding
Independent dimensionless variables used in this study Nomenclature
Variable
Dimensionless ammonium hydroxide concentration Dimensionless liquid/solid ratio Dimensionless enzyme/substrate ratio
Kzriation range
Dej&zition
XI
(AHC-12)/8
(-1,
x:
(LSR-20)/8
(-1,l)
X3
(ESR-8)/5
I)
(- 1, 1)
AHC, LSR and ESR as in Table 2.
Table 4. Operational conditions assayed (expressed in terms of dimensionless independent variables) and expcrimental results achicvcd Exper.
6 7 8 9 10 11 12 13 14 15 16
Dimensionless, independent wriables
Experimental
x1
x2
xi
Yl (g/l)
Y, (s/Uh)
-1 -1 -1 -1
-1
0 -1 1 0 -I 1 0 0 0 -1
53.5 24.8 38.0 23.0 47.0 54.4 33.3 34.0 34.4 17.9 30.1 61.8 25.4 33.0 26.1 23.9
2.82 1.03 2.70 1.40 1.16 4.1 2.19 1.58 1.81 0.68 1.97 2.79 1.34 3.00 1.37 1-53
0 0 0 0 0 0 0 0 0 1 1 1 I 0
-1 -1 -1 0 0 0
I -:
:, 0 0 1 1
-1 1 0 0
results
in our study. For calculation purposes,” dimensionless and normalized independent variables (X,7 x2 and xX) were defined to measure respectively the ammonium hydroxide concentration, the liquid/solid ratio and the enzyme/substrate ratio. Table 3 lists the inter-relationships between the dimensional independent variables and the dimensionless ones. Table 4 shows the set of operational conditions assayed (expressed in terms of dimensionless independent variables) and the experimental results determined. The dependent and independent variables were correlated by empirical models having the following generalized expression
I
I
k
where y, (j: l-2) are the dependent variables, xi and xk (i or k: 1-3, k 2 i) are the dimensionless
J. C. Para@, J. L. Alamo,
542
VI Sanros
Table 5. Regression coefftcients and statistical parameters (a) Regression
coefficients Variable
Coejicien t b,,j 6, ;2! b:, 13, : 23, b II/
b
b:::
YI
Y2
33.64 0.88 - 15.14 5.05 - 1.30 - 1.40 1.20 0.03 6.97 - 3.45
1.890 0.069 -0.659 0.945 0.000 -0-003 -0.413 0.11 I 0.116 - 0.007
(b) Significance of coefficients - Significant coefficients at the 95% confidence level
LIWIDISDLID
28
Fig. 3. Calculated dependence of the maximum sugar concentration (y,, g/litre) on the liquid/solid ratio and on the enzyme/substrate ratio (data corresponding to samples treated with 12% NH,OH).
b,,, b,,. b,z, bz. b,,, b,,, -
RATID. g,g
Significant coefficients at the 90% confidence level
b 331 (c) Statistical parameters significance of models Variable Yl Y2
measuring
the correlation
R’
Correrted R2
0.9773
0.9432
28.65
0.967 3
0.9183
19.72
and
Fexpu Prob IFexp> F,,P CO.01 co.01
“F,, : defined as the ratio between the mean squares of mo ael and error. ‘Fv,: defined as the statistical value of F for the degrees of freedom of model and error.
to increases
in the liquid/solid
ratio from
12 to
20 g/g. In order to provide further information on the extent of the enzymic hydrolysis, the polysaccharide conversion achieved at infinite reaction time (PC,) was calculated according to the following expression: PC,=WOO, cfh
independent variables and b,. . . b, are the regression coefficients calculated by the least-squares method. Table 5 shows the regression coefficients calculated and their statistical significance, as well as mathematical parameters measuring the correlation and significance of the empirical models. The experimental values determined for the maximum sugar concentration y1 varied in a broad range (17.9-6143 g/litre). The highest values of y, (535-614 g/litre) were obtained at the lowest liquid/solid ratio assayed (12 g/g) in experiments using 8-13 IV/g (experiments 1,5,6 and 12). On the contrary, limited y, values were found in trials carried out with the least enzyme/ substrate ratio assayed at the highest liquid/solid ratio (experiment 10). Figure 3 shows the calculated dependence of y, on the most influential independent variables (liquid/solid ratio and enzyme/substrate ratio) for samples treated with 12% NH,OH. The major effects were associated
where C,, corresponds to the theoretical sugar concentration, which can be calculated from material balances assuming a quantitative transformation of polysaccharides into sugars. It can be conversion noted that the polysaccharide achieved at a given reaction time can be calculated from the above equation considering the sugar concentration achieved at this moment instead y,. The sugar concentration achieved at the desired reaction time is related with y, through the hyperbolic model. The strong influence of LSR on PC, can be assessed by comparing the results calculated for experiments 6 and 11 (both performed with ESR=13 IU/g and AHC= 12 g/100 g). PC, increased from 71.3% (experiment 6) to 92.1% (experiment 11) as ESR increased from 12 to 28 g/g. The replicate experiments (7, 8 and 9) led to PC, in the range 72.8-75-0, which is reasonable considering the complexity of the experimental procedure.
Delignijicationand swellingof Eucalyptus wood
Figure 4 shows the dependence of PC, on liquid/solid ratio and enzyme/substrate ratio (corresponding to samples treated with 12% NH,OH) calculated on the basis of the empirical model developed for y,. For low liquid/solid ratios, improvements in PC, were predicted when the enzyme/substrate ratio increased from 3 to 8 III/g. Further increases up to 13 IU/g did not enhance the polysaccharide conversion. This behaviour agrees with reported studies: several authors’3~‘5~‘8 found that little effects in polysaccharide conversion were caused when the enzyme/substrate ratio exceeded a limit value, which was near 10 III/g. The combined effects of liquid/solid ratio and enzyme/substrate ratio on PC, can be explained on the basis of substrate inhibition: little influence of PC,,, on the operational conditions was observed in the zone of the response surface corresponding to high enzyme/ substrate ratios and low liquid/solid ratios, where significant inhibition effects are expected. On the contrary, a marked dependence of PC, on enzyme/substrate ratio was predicted for experiments carried out with the least liquid/solid ratio assayed. Little improvements in PC, were associated with increases in the ammonium hydroxide concentration in the range considered. The experimental values obtained for the initial hydrolysis rate (y2) varied in a broad range (0.68-4-l g/litre/h). The extreme values were obtained in experiments 10 and 6, performed with the minimum and the maximum enzyme concentration, respectively.
Figure 5 shows the predicted dependence of y, on the most influential independent variables (liquid/solid ratio and enzyme/substrate ratio) for samples treated with 12% NH,OH. It can be observed a linear increase of yZ when either enzyme/substrate ratio was increased or the liquid/solid ratio was decreased. In order to provide further information on the effect caused by the liquid/solid ratio on the hydrolysis rate, an additional variable (roe, initial specific rate, g/g/h) was defined according to the following expression: r”e=$) rh
where r, and C,h are as above. Figure 6 shows the dependence calculated for roe on the ammonium hydroxide concentration and liquid/solid ratio when the enzyme/substrate ratio was fiied in 8 IU/g substrate. Little effects were associated with variations in the NH,OH concentration within the variation range considered. However, when the liquid/solid ratio was increased from 12 to 20 g/g, r,, increased by about 20%. Increases in the solid/liquid ratio above 20 g/g did not signifihas been cantly affect r,,. Similar behaviour reported for the enzymatic hydrolysis of other LCM.‘3*‘5 It can be seen from the empirical models that r,, increased linearly with the enzyme/substrate ratio within the range tested for this variable. The
4MMONI”M
AMMONIUM
HYDROXIDE
CONCENTRATION
9-P ;-A ENZYMEISUSSTR.RATIO. Ill/g
”
543
HYDROXIDE
CONCENTMTIDN
= 12 %
= 12%
LIOUIDISOUD
mno, P/P
3
Calculated dependence of the polysaccharide Fig. 4. conversion PC, on the liquid/solid ratio and on the enzyme/ substrate ratio (data corresponding to samples treated with 12% NH,OH).
LIOUID/SOLIDRATIO. g/g
28
Fig. 5. Calculated dependence of the initial reaction rate (y?, g/litrc/h) on the liquid/solid ratio and on the enzyme/ substrate ratio (data calculated for samples treated with 12% NH,OH).
544
J. C. Parajb, J. L. Alonso, V. Santos ENZYME,S”BSTRATE
f
RATIO
= 8 W/g
0.044
a
.
0.042
2 F: r
0.038
r d
0.04
D-036
y
0.034
E %
0.032 0.03
NH40”
CONCENTKATION,
20
%
Fig. 6. Calculated dependence of the initial specific rate (roe, g/g/h) on the liquid/solid ratio and on the NH,OH concentration (data calculated for experiments performed with an enzyme/substrate ratio of 8 W/g).
AMMONIUM
HYDROXIOE CONCENTRATION
= 12%
__ 3
ENZYMEISUBSTFIATE W/p
RATIO,
Fig. 7. Calculated dependence of the sugar concentration (reached after 48 h reaction time) on the liquid/solid ratio and on the enzyme/substrate ratio (data calculated for samples treated with I 2% NH,OH).
equations predicted a broad variation range for r,, (0.0 l-0.06 g/g/h for samples treated with 12% NH,OH). From the kinetic models developed, the sugar concentration reached at a given reaction time under a fixed set of operational conditions can be calculated. As an example, Fig. 7 shows the calculated dependence of the sugar concentration reached after 48 h reaction time on the liquid/ solid ratio and on the enzyme/substrate ratio for samples treated with 12% NH,OH. It can be
remarked that a sugar concentration of 45 g/litre (or 60% polysaccharide conversion) can be obtained in experiments performed with 12 g liquid/g solid and 13 IU/g substrate. In conclusion, the proposed process provides an efficient scheme for the chemical-biotechnological processing of Eucalyptus wood. The delignification step allowed an extensive separation of lignin and hemicelluloses from the solid residue, which showed an enhanced cellulose content. The solid phase from delignification fits the chemical requirements exiged for good hydrolysis substrates, but its structural features are not adequate for this purpose. A subsequent treatment with NH,OH solutions provoked little changes in chemical composition, but altered the structure of the solids leading to a marked increase in their susceptibility to the enzymic hydrolysis. From experimental data, the kinetics and yields of the enzymatic hydrolysis were modelled by empirical, second-order equations giving a quantitative interpretation of the process. The NH,OH concentration, used as operational variable, did not show a significant influence on the overall process within the limits tested in this study. Low liquor/solid ratios and high enzyme substrate/ratios led to sugar concentrations up to 45 g/litre after 48 h, with polysaccharide conversions in vicinity of 60%. The kinetic models provide the necessary information for performing preliminary evaluations on the feasibility of the hydrolysis stage of the overall process studied.
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Delignification
and swelling of Eucalyptus
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233-40. 10. Kitsos, H. M., Roberts, R. S. & Muzzv, J. D., n-Proovlamine pretreatment of ‘buffered solvent’ pulp to enh&&e enzymatic hvdrolvsis. Biores. Technol.. 39 (1992) 24i-7. _ _ 11. Tanaka, M., Song, G. J., Matsuno, R. & Kamikubo, T., Optimal conditions for pretreatment of rice straw with n-butylamine for enzymatic solubilization. Appl. Microb.
Biotechnol., 22 (1985) 13-18. M., Dale, B. E. & Craig, W. K., Compari12. Mes-Hartree, son of steam and ammonia pretreatment for enzymatic hydrolysis of cellulose. Appl. Microb. Biotechnol., 29
(1988) 462-8. ammonia pretreatment ot 13. Yu Chia, T. C., Supercritical lignocellulosic materials. Biotechnol. Rioeng. Symp, 17
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