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Reutilization of enzymes for saccharification of lignocellulosic materials
Reutilization of enzymes for saccharification of lignocellulosic materials M. V . D e s h p a n d e t a n d K.-E. Eriksson* Swedish Forest Products Research Laboratory, Box 5604, S-114 86 Stockholm, Sweden
Enzymic hydrolysis of steam-exploded wheat straw is initially a fast process which gradually slows down. Since the cellulolytic enzymes account for 60% of the processing costs incurred during saccharification of lignoceUulosics, recirculation of these enzymes is clearly necessary. It is demonstrated that the eellulolytic enzymes have a high affinity for the remaining lignin. Only 50% of the added enzymes are free in solution after almost complete hydrolysis of the straw polysaccharides. Elution of the enzymes from the lignin can result in a total enzyme recovery of up to 90%. However, it is questionable whether elution of enzymes from the lignin is economically feasible as a technical process.
Keywords: CeUulose;lignocellulose; steam explosion; saccharification; cellulases; reutilization
Introduction
Enzymic hydrolysis of lignocellulosic materials involves several stages, including mechanical or chemical pretreatment of the substrate, enzyme production, saccharification and enzyme recovery. Enzyme production costs account for as much as 60% of the total processing costs associated with enzymic hydrolysis of cellulose to glucose] Thus, it was shown recently that 90 95% of the enzymes must be recirculated in order to obtain a saccharification process economically competitive with acid hydrolysis. 2 Since the cellulase system hydrolysing crystalline cellulose is a balanced complex of three different types of enzymes, the recovery of each enzyme component should be the same in relative terms, s'4 The individual enzyme components, exo-l,4-~- and endo-l,4-/3-glucanases and 1,4@ glucosidase in a mixture can now be estimated by reliable methods, s, 6 This paper describes a study of the rates of saccharification of wheat straw, pretreated using a steam explosion technique, during both the initial and later phases of the process. Particular attention has been given to the amount of enzymes adsorbed onto the solid material at various stages of hydrolysis in order to evaluate the extent of enzyme recirculation. Materials and methods
Wheat straw was pretreated in the following way: straw was heated by direct steam to 160°C and then by electric heating to the explosion temperature, usually between 200 and 250°C. The straw material was then exploded by a sudden *To whom correspondence should be addressed. -~Division of Biochemistry, National Chemical Laboratory, Poona 411008, India.
338
Enzyme Microb. Technol., 1984, vol. 6, August
release of the pressure. This pretreatment was carried out by Alfa Laval AB, Tumba, Sweden. To obtain 'a lignin-rich residue' of steam-exploded wheat straw, a 6% (dry weight) suspension of the exploded material was treated with the enzyme mixture, 6 FPU/ml, at 50°C for three 18 h periods. (FPU = filter paper activity, i.e. the ability of the enzyme mixture to saccharify triter paper.) The final residue was washed repeatedly with distilled water to remove enzymes and reducing sugars. A 6% substrate suspension was used in all experiments. Avicel (a microcrystalline cellulose) was obtained from American Viscose Corporation, Marcus Hook, PA, USA. The Trichoderma reesei cellulases and hemicellulases used in the saccharification experiments were supplied by the Alko Company, Helsinki, Finland. In all saccharification experiments, an enzyme concentration of 6 FPU/ml substrate suspension was used. 7 The different enzyme activities, viz. filter paper activity (FPU), endo-1,4-/3-glucanase, exo-1,4n6-glucanase and 1,4-/3glucosidase were assayed as described previously. 6 Reducing sugars released on hydrolysis were estimated by the dinitrosalicylic acid method. 8 Protein concentration was determined by measuring the absorbance at 280 nm. 9 Results a n d discussion
Rate o f reducing sugar formation The two major obstacles preventing an effective enzymic hydrolysis of lignocellulosic materials are lignin and crystalline celluloseJ ° Using 6% (dry wt) suspensions of exploded wheat straw or Avicel as substrates for enzymic hydrolysis, the rate of reducing sugar formation was estimated at various time intervals over a 24 h period. As shown in Figure 1, reducing sugar formation from exploded wheat straw was initially rapid. During the first hour the rate of release varied between 0.75 and 0.3 mg/min per flask (10 ml suspension) decreasing to 0.05 mg/min per flask after 24 h. The rate of reducing sugar formation was also low initially if the exploded straw had been previously treated with enzymes, washed, resuspended and again subjected to a further enzyme treatment. The obvious interpretation of these sugar release patterns is that easily accessible hemicelluloses and amorphous cellulose are first hydrolysed at a high rate. Once these materials are removed, lignin will form a barrier, thus denying the enzymes access to remaining polysaccharides. Furthermore, residual cellulose will be increasingly more crystalline. The low saccharification rate of Avicel is due to the crystallinity of this material. Therefore, any pretreatment step must both remove the lignin and destroy the crystalline nature of cellulose if a fast and high-yield enzyme saccharification process is to be achieved. 0141 ~)229/84/080338-03 $03.00 © 1984 Butterworth & Co. (Publishers) Ltd
Rapid communication 0.8' O
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Time. h Figure 1 Rate of f o r m a t i o n of reducing sugars b y enzymic saccharification of three d i f f e r e n t substrate suspensions (10 ml, 6% d r y w t ) at 5 0 ° 0 over a 24 h period. Substrates: o, steam-exploded wheat straw; A, previously enzyme-treated steam-exploded wheat straw; B, Avicel
Reutilization o f enzymes Enzyme reutilization is necessary in any economically feasible process for the enzymic saccharification of lignocellulosic materials. This implies that enzymes are released into solution automatically once the substrate has been hydrolysed, or can be desorbed cheaply and efficiently. To investigate enzyme adsorption on, and elution from, various substrates, the following experiments were carried out. Enzyme (6 FPU/ml substrate) was added to 6% suspension of: (a) exploded wheat straw, (b) the lignin-rich residue (see Materials and methods) and (c) Avicel. The percentage of enzymes free in solution in the different substrate suspensions was determined at various time intervals over 24 h and the results are presented in Figures 2a-2d. In Figure 2a values are expressed as the percentage of free FPU:s and in Figures 2b-2d as the percentage of free individual enzymes participating in cellulose hydrolysis. It can be seen that the enzymes were adsorbed less on Avicel than on lignin-containing substrates. Further, both endo- and exoglucanases were adsorbed to the same extent almost on the lignin-rich residue as on the exploded straw. Only the/~-glucosidase exhibited markedly less affinity for the lignin-rich residue than for the exploded straw. Although the lignin-rich residue is obtained by a repeated treatment of exploded straw with cellulolytic enzymes and contains only trace amounts of the polysaccharides, it is still able to adsorb ~50% of the endo- and exo-glucanases. This implies that the cellulolytic enzymes have a high affinity for lignin, which is verified by the use of lignin for precipitation of cellulases in the commercial production of these enzymes. It must be concluded that even where almost complete saccharification of a lignocellulosic
material is achieved, only 50% of the enzymes are free in solution and can be directly reutilized. One possibility for reutilization of enzymes, even when adsorbed onto lignin remaining in the substrate, would involve recovery from the residues using different eluents. Such extraction procedures have already been examined in several laboratories. TM 12 In the present study, acetate and phosphate buffers, urea and glycerol solutions have been used to elute enzymes from steam-exploded wheat straw and from the lignin-rich residue. The enzymes were recovered by suspending the respective residues (3% suspensions) in the different eluents to a final volume of 20 ml. The enzymic hydrolysis was carried out at 40°C for 15 rain and the results are described in Table 1. The most effective elution was obtained with phosphate buffer, which gave a total enzyme recovery (recovery in hydrolysate + recovery after elution) of 85, 78 and 65% from exploded straw and 90, 87 and 91% from the lignin-rich residue of endoglucanase, exoglucanase and /3-glucosidase activity, respectively. Water alone also eluted some enzyme activity but the results obtained were not very reproducible. No economical evaluation of the elution costs has been made but it is doubtful if such an approach can be used in a technical process. Figures 2a-2d show that the amount of enzyme free in solution was maximal during the first hour of hydrolysis. Vallander and Eriksson 13 reported that an intermediate removal of the hydrolysate liquid, containing both sugars and enzymes, followed by addition of fresh enzyme gave a much higher saccharification yield compared with an uninterrupted hydrolysis over a 24 h period. It may be possible to develop this observation in order to obtain both a higher degree of saccharification and higher enzyme
Exo- 1.L-,G- glucanase
Filter paper units {FPU) 100
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50
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b
100
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75
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0.5
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1 4
lt2
t
24
0.5
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12
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Time, h Figure 2 A d s o r p t i o n of c e l l u l o l y t i c enzymes on three d i f f e r e n t substrate suspensions (6% d r y wt) at 50°C over a 24 h period. Substrates: o, steam e x p l o d e d wheat straw; 6, lignin-rich residue (see t e x t f o r details); o, Avicel
Enzyme Microb. Technol., 1984, vol. 6, August
339
Rapid communication Table 1 Elution of the cellulolytic enzymes from steam-exploded wheat straw and 'lignin-rich residue' (see t e x t for details) % Enzyme eluted and recovered from Steam-exploded wheat straw
Lignin-rich residue
Acknowledgement Financial support to M.V.D. from the United Nations Industrial Development Organization (UNIDO) and to K.-E.E. from the Swedish Board of Energy Conservation is gratefully acknowledged.
ExoEndo- Exo1,4-/3- 1,4-~- 1,4-~- 1,4-/3- 1,4-~- 1,4-~glucen- glucan- Gluco- glucan- glucan- GlucoEndo-
Recovered in hydrolysate Recovered after elution with 0.05 M acetate buffer, pH 5.0 0.5 M phosphate buffer, pH 7.0 6 M urea in H20 10% glycerol in H=O
ase
ase
sidase
ase
ase
sidase
45
40
35
50
47
66
Received 2 May 1984
References 1 2 3 4
28
21
18
35
25
20
40
38
30
40
40
25
30
27
17
30
30
22
6
30
21
20
32
25
22
7
5
8 9
recovery. However, this approach requires a cheap elution process. When the steam explosion step is carried out according to the Iotech process, ~4 the lignin is easily extracted with NaOH or ethanol after explosion and prior to enzymic hydrolysis. Such a procedure appears likely to give rise both to a higher yield of sugars and to more efficient recirculation of enzymes. This approach is currently being investigated in our laboratory.
340
E n z y m e M i c r o b . T e c h n o l . , 1 9 8 4 , v o l . 6, A u g u s t
10 11 12 13 14
Wilke, C. R., Yang, R. D. and yon Stocker, U. Biotechnol. Bioeng. Syrup. 1976, 6,155-175 Report number 1760302 Ethanol AL. Swedish Board for Energy Conservation, 1983 Eriksson, K.-E. Biotechnol. Bioeng. 1978, 20, 317-332 Ryu, D. D. Y. and Mandels, M. Enzyme Microb. Technol. 1980, 2, 91-102 Ghose, T. K., Montenecourt, B. S. and Eveleigh, D. E. Measurement of Cellulase Activity, Substrates, Assays, Activities and Recommendations prepared for the Biotechnology Commission, International Union of Pure and Applied Chemistry, July 1980 Deshpande, M. V., Eriksson, K.-E. and Pettersson, L. G. Anal. Biochem. 1984, in press Mandels, M., Andriotti, R. and Roche, C. Biotechnol. Bioeng. Syrup. 1976, 6, 21-33 Fisher, E. H. and Stein, E. A. in Biochemical Preparations (A. Meister, ed.) Wiley, New York, 1961, vol. 8, pp. 27-33 Jagannathan, V., Kartar Singh and Damodaran, M. Biochem. J. 1956, 63, 94 -105 MiUett, M. A., Baker, A. J. and Satter, L. D. Biotechnol. Bioeng. Syrup. i976, 6,125-153 Reese,E. T. Process Biochem. 1981, 17, 2-6 Sinitsyn, A. P., Bungay, M. L., Clesceri, L. S. and Bungay, H. R. Appl. Biochem. Biotechnol. 1983, 8, 25-29 Valiander, L. and Eriksson, K.-E. presented at Biotechnology in the Pulp and Paper Industry Pira, London, 12-14 September 1983 'Optimization of steam explosion pretreatment.' Final report, Iotech Corp., Ottawa, Ontario, Canada, 1980
Enzymic hydrolysis of steam-exploded wheat straw is initially a fast process which gradually slows down. Since the cellulolytic enzymes account for 60% of the processing costs incurred during saccharification of lignoceUulosics, recirculation of these enzymes is clearly necessary. It is demonstrated that the eellulolytic enzymes have a high affinity for the remaining lignin. Only 50% of the added enzymes are free in solution after almost complete hydrolysis of the straw polysaccharides. Elution of the enzymes from the lignin can result in a total enzyme recovery of up to 90%. However, it is questionable whether elution of enzymes from the lignin is economically feasible as a technical process.
Keywords: CeUulose;lignocellulose; steam explosion; saccharification; cellulases; reutilization
Introduction
Enzymic hydrolysis of lignocellulosic materials involves several stages, including mechanical or chemical pretreatment of the substrate, enzyme production, saccharification and enzyme recovery. Enzyme production costs account for as much as 60% of the total processing costs associated with enzymic hydrolysis of cellulose to glucose] Thus, it was shown recently that 90 95% of the enzymes must be recirculated in order to obtain a saccharification process economically competitive with acid hydrolysis. 2 Since the cellulase system hydrolysing crystalline cellulose is a balanced complex of three different types of enzymes, the recovery of each enzyme component should be the same in relative terms, s'4 The individual enzyme components, exo-l,4-~- and endo-l,4-/3-glucanases and 1,4@ glucosidase in a mixture can now be estimated by reliable methods, s, 6 This paper describes a study of the rates of saccharification of wheat straw, pretreated using a steam explosion technique, during both the initial and later phases of the process. Particular attention has been given to the amount of enzymes adsorbed onto the solid material at various stages of hydrolysis in order to evaluate the extent of enzyme recirculation. Materials and methods
Wheat straw was pretreated in the following way: straw was heated by direct steam to 160°C and then by electric heating to the explosion temperature, usually between 200 and 250°C. The straw material was then exploded by a sudden *To whom correspondence should be addressed. -~Division of Biochemistry, National Chemical Laboratory, Poona 411008, India.
338
Enzyme Microb. Technol., 1984, vol. 6, August
release of the pressure. This pretreatment was carried out by Alfa Laval AB, Tumba, Sweden. To obtain 'a lignin-rich residue' of steam-exploded wheat straw, a 6% (dry weight) suspension of the exploded material was treated with the enzyme mixture, 6 FPU/ml, at 50°C for three 18 h periods. (FPU = filter paper activity, i.e. the ability of the enzyme mixture to saccharify triter paper.) The final residue was washed repeatedly with distilled water to remove enzymes and reducing sugars. A 6% substrate suspension was used in all experiments. Avicel (a microcrystalline cellulose) was obtained from American Viscose Corporation, Marcus Hook, PA, USA. The Trichoderma reesei cellulases and hemicellulases used in the saccharification experiments were supplied by the Alko Company, Helsinki, Finland. In all saccharification experiments, an enzyme concentration of 6 FPU/ml substrate suspension was used. 7 The different enzyme activities, viz. filter paper activity (FPU), endo-1,4-/3-glucanase, exo-1,4n6-glucanase and 1,4-/3glucosidase were assayed as described previously. 6 Reducing sugars released on hydrolysis were estimated by the dinitrosalicylic acid method. 8 Protein concentration was determined by measuring the absorbance at 280 nm. 9 Results a n d discussion
Rate o f reducing sugar formation The two major obstacles preventing an effective enzymic hydrolysis of lignocellulosic materials are lignin and crystalline celluloseJ ° Using 6% (dry wt) suspensions of exploded wheat straw or Avicel as substrates for enzymic hydrolysis, the rate of reducing sugar formation was estimated at various time intervals over a 24 h period. As shown in Figure 1, reducing sugar formation from exploded wheat straw was initially rapid. During the first hour the rate of release varied between 0.75 and 0.3 mg/min per flask (10 ml suspension) decreasing to 0.05 mg/min per flask after 24 h. The rate of reducing sugar formation was also low initially if the exploded straw had been previously treated with enzymes, washed, resuspended and again subjected to a further enzyme treatment. The obvious interpretation of these sugar release patterns is that easily accessible hemicelluloses and amorphous cellulose are first hydrolysed at a high rate. Once these materials are removed, lignin will form a barrier, thus denying the enzymes access to remaining polysaccharides. Furthermore, residual cellulose will be increasingly more crystalline. The low saccharification rate of Avicel is due to the crystallinity of this material. Therefore, any pretreatment step must both remove the lignin and destroy the crystalline nature of cellulose if a fast and high-yield enzyme saccharification process is to be achieved. 0141 ~)229/84/080338-03 $03.00 © 1984 Butterworth & Co. (Publishers) Ltd
Rapid communication 0.8' O
.c_
0.6' t~
u
0./*"6
,'//i
0
0'.5
I/*
I'2
2'/*
Time. h Figure 1 Rate of f o r m a t i o n of reducing sugars b y enzymic saccharification of three d i f f e r e n t substrate suspensions (10 ml, 6% d r y w t ) at 5 0 ° 0 over a 24 h period. Substrates: o, steam-exploded wheat straw; A, previously enzyme-treated steam-exploded wheat straw; B, Avicel
Reutilization o f enzymes Enzyme reutilization is necessary in any economically feasible process for the enzymic saccharification of lignocellulosic materials. This implies that enzymes are released into solution automatically once the substrate has been hydrolysed, or can be desorbed cheaply and efficiently. To investigate enzyme adsorption on, and elution from, various substrates, the following experiments were carried out. Enzyme (6 FPU/ml substrate) was added to 6% suspension of: (a) exploded wheat straw, (b) the lignin-rich residue (see Materials and methods) and (c) Avicel. The percentage of enzymes free in solution in the different substrate suspensions was determined at various time intervals over 24 h and the results are presented in Figures 2a-2d. In Figure 2a values are expressed as the percentage of free FPU:s and in Figures 2b-2d as the percentage of free individual enzymes participating in cellulose hydrolysis. It can be seen that the enzymes were adsorbed less on Avicel than on lignin-containing substrates. Further, both endo- and exoglucanases were adsorbed to the same extent almost on the lignin-rich residue as on the exploded straw. Only the/~-glucosidase exhibited markedly less affinity for the lignin-rich residue than for the exploded straw. Although the lignin-rich residue is obtained by a repeated treatment of exploded straw with cellulolytic enzymes and contains only trace amounts of the polysaccharides, it is still able to adsorb ~50% of the endo- and exo-glucanases. This implies that the cellulolytic enzymes have a high affinity for lignin, which is verified by the use of lignin for precipitation of cellulases in the commercial production of these enzymes. It must be concluded that even where almost complete saccharification of a lignocellulosic
material is achieved, only 50% of the enzymes are free in solution and can be directly reutilized. One possibility for reutilization of enzymes, even when adsorbed onto lignin remaining in the substrate, would involve recovery from the residues using different eluents. Such extraction procedures have already been examined in several laboratories. TM 12 In the present study, acetate and phosphate buffers, urea and glycerol solutions have been used to elute enzymes from steam-exploded wheat straw and from the lignin-rich residue. The enzymes were recovered by suspending the respective residues (3% suspensions) in the different eluents to a final volume of 20 ml. The enzymic hydrolysis was carried out at 40°C for 15 rain and the results are described in Table 1. The most effective elution was obtained with phosphate buffer, which gave a total enzyme recovery (recovery in hydrolysate + recovery after elution) of 85, 78 and 65% from exploded straw and 90, 87 and 91% from the lignin-rich residue of endoglucanase, exoglucanase and /3-glucosidase activity, respectively. Water alone also eluted some enzyme activity but the results obtained were not very reproducible. No economical evaluation of the elution costs has been made but it is doubtful if such an approach can be used in a technical process. Figures 2a-2d show that the amount of enzyme free in solution was maximal during the first hour of hydrolysis. Vallander and Eriksson 13 reported that an intermediate removal of the hydrolysate liquid, containing both sugars and enzymes, followed by addition of fresh enzyme gave a much higher saccharification yield compared with an uninterrupted hydrolysis over a 24 h period. It may be possible to develop this observation in order to obtain both a higher degree of saccharification and higher enzyme
Exo- 1.L-,G- glucanase
Filter paper units {FPU) 100
tl
C
75
~:r~_...---~- -~'-~ t-
.o
50
25
.c E
"6 c O-
I
I // t
r
I
i //t
I
1.4- .{3- glucosidase
Endo-1./. - ,~- glucanase
b
100
d
75
50
25
,
0.5
. //,
1 4
lt2
t
24
0.5
L . t
t
1 /*
12
2/*
Time, h Figure 2 A d s o r p t i o n of c e l l u l o l y t i c enzymes on three d i f f e r e n t substrate suspensions (6% d r y wt) at 50°C over a 24 h period. Substrates: o, steam e x p l o d e d wheat straw; 6, lignin-rich residue (see t e x t f o r details); o, Avicel
Enzyme Microb. Technol., 1984, vol. 6, August
339
Rapid communication Table 1 Elution of the cellulolytic enzymes from steam-exploded wheat straw and 'lignin-rich residue' (see t e x t for details) % Enzyme eluted and recovered from Steam-exploded wheat straw
Lignin-rich residue
Acknowledgement Financial support to M.V.D. from the United Nations Industrial Development Organization (UNIDO) and to K.-E.E. from the Swedish Board of Energy Conservation is gratefully acknowledged.
ExoEndo- Exo1,4-/3- 1,4-~- 1,4-~- 1,4-/3- 1,4-~- 1,4-~glucen- glucan- Gluco- glucan- glucan- GlucoEndo-
Recovered in hydrolysate Recovered after elution with 0.05 M acetate buffer, pH 5.0 0.5 M phosphate buffer, pH 7.0 6 M urea in H20 10% glycerol in H=O
ase
ase
sidase
ase
ase
sidase
45
40
35
50
47
66
Received 2 May 1984
References 1 2 3 4
28
21
18
35
25
20
40
38
30
40
40
25
30
27
17
30
30
22
6
30
21
20
32
25
22
7
5
8 9
recovery. However, this approach requires a cheap elution process. When the steam explosion step is carried out according to the Iotech process, ~4 the lignin is easily extracted with NaOH or ethanol after explosion and prior to enzymic hydrolysis. Such a procedure appears likely to give rise both to a higher yield of sugars and to more efficient recirculation of enzymes. This approach is currently being investigated in our laboratory.
340
E n z y m e M i c r o b . T e c h n o l . , 1 9 8 4 , v o l . 6, A u g u s t
10 11 12 13 14
Wilke, C. R., Yang, R. D. and yon Stocker, U. Biotechnol. Bioeng. Syrup. 1976, 6,155-175 Report number 1760302 Ethanol AL. Swedish Board for Energy Conservation, 1983 Eriksson, K.-E. Biotechnol. Bioeng. 1978, 20, 317-332 Ryu, D. D. Y. and Mandels, M. Enzyme Microb. Technol. 1980, 2, 91-102 Ghose, T. K., Montenecourt, B. S. and Eveleigh, D. E. Measurement of Cellulase Activity, Substrates, Assays, Activities and Recommendations prepared for the Biotechnology Commission, International Union of Pure and Applied Chemistry, July 1980 Deshpande, M. V., Eriksson, K.-E. and Pettersson, L. G. Anal. Biochem. 1984, in press Mandels, M., Andriotti, R. and Roche, C. Biotechnol. Bioeng. Syrup. 1976, 6, 21-33 Fisher, E. H. and Stein, E. A. in Biochemical Preparations (A. Meister, ed.) Wiley, New York, 1961, vol. 8, pp. 27-33 Jagannathan, V., Kartar Singh and Damodaran, M. Biochem. J. 1956, 63, 94 -105 MiUett, M. A., Baker, A. J. and Satter, L. D. Biotechnol. Bioeng. Syrup. i976, 6,125-153 Reese,E. T. Process Biochem. 1981, 17, 2-6 Sinitsyn, A. P., Bungay, M. L., Clesceri, L. S. and Bungay, H. R. Appl. Biochem. Biotechnol. 1983, 8, 25-29 Valiander, L. and Eriksson, K.-E. presented at Biotechnology in the Pulp and Paper Industry Pira, London, 12-14 September 1983 'Optimization of steam explosion pretreatment.' Final report, Iotech Corp., Ottawa, Ontario, Canada, 1980