Catalyzed steaming as pre-treatment for the enzymatic hydrolysis of bamboo grass culms

Bioresource Technology 64 (1998) 241-243 © 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0960-8524/98 $19.00 ELSEVIER

PII:S0960-8524(97)O0189-2

Short Communication water soluble, and cellulose in the steamed substrate is highly susceptible to attack by cellulase. Recently, the impregnation of wood chips with low levels of acid catalysts before steam treatment has been shown to improve the enzymatic hydrolysis of cellulose in the substrate as well as increase the recovery of soluble hemicellulose sugars (Mackie et al., 1985; Brownell et al., 1986; Sudo et al., 1986; Clark and Mackie, 1987; Rughani et al., 1990, 1992; Ramos et al., 1992). In this study, the culms of Sasa senanensis Rehd., a representative species of bamboo grass, were treated with saturated steam in the presence or absence of acid catalysts. The enzymatic hydrolysis of the steamed substrates was examined. The catalysts were Lewis acids, inorganic salts, acetic acid and organic acid anhydrides.

Catalyzed Steaming as Pre-treatment for the Enzymatic Hydrolysis of Bamboo Grass Culms Abstract

Culms of bamboo grass (Sasa senanensis Rehd.) were treated with saturated steam at 197°C for lO min in the presence of various acid catalysts in order to improve enzymatic susceptibility. Catalyst levels were varied at 5, 10, 20 and 3 0 m m o l k g -1 original material. The catalysts were Lewis acids, NH4CI, (NH4)2S04, acetic acid and organic acid anhydrides. The extent of enzymatic hydrolysis of steamed substrate was improved by the addition of catalyst. The acid catalysts strongly modified the steam treatment of bamboo grass culms. Overall recovery of the steamed solids decreased with increase in concentration of the catalyst. Conversion yields from 60 to 70%, based on polysaccharides present in the original material, were achieved by catalyzed steaming followed by enzymatic hydrolysis. © 1998 Elsevier Science Ltd. All rights reserved

METHODS

The air-dried culms of bamboo grass were crushed in a hammer mill, then screened to eliminate particles smaller than 1 mm. The crushed culms were extracted with hot water in order to enhance the hydrolysis rate of xylan (Aoyama et al., 1995). Before steam treatment, the extracted culms were sprayed with the desired amounts of acid catalyst solution (mmol per dry kg of original material) or water (control), and refrigerated for a few days. The reactor reached to the desired temperature within 1 min. The extracted culms were steamed at 197°C for 10min in the presence or absence of acid catalyst. The catalysts were A1CI3, A12(504)3, FeC13, NH4CI, (NH4)2504, acetic acid, maleic anhydride, succinic anhydride, and phthalic anhydride. Although aqueous solutions of NH4CI and ( N H 4 ) 2 S O 4 a r e neutral, the solutions show strong acidic properties by evaporation of NH3 at elevated temperatures (Sudo et al., 1986). The resulting steamed solid was air-dried and ground in a Wiley mill. Some portions of the steamed and ground solid were extracted with hot water to separate it into soluble xylan fragments and extracted residue using the Japanese Industrial Standard method (JIS P8005-1959). They were subsequently air-dried. Before enzymatic hydrolysis, steamed and hot-water extracted substrates were screened and

Key words: Catalyzed steam treatment, Acid catalysts, Enzymatic hydrolysis, Polysaccharides, Bamboo grass, Sasa senanensis Rehd. INTRODUCTION Bamboo grasses (Bambusoideae), which are perennial grasses with woody culms, are widely distributed in Japan, Korea, Saghalien and the southern part of the Kuril islands. They are an important forest biomass because of the large quantities of growing stock and their rapid growth rate. The chemical composition of bamboo grass culm is similar to that of hardwoods, hence it is a potential source for chemical feedstocks or ruminant feed (Ishii and Tanaka, 1984; Aoyama et al., 1995; Aoyama, 1996). This vast resource could be exploited as feedstock for chemical processes, or for livestock, if an economically viable process for the separation of polysaccharides could be established. Steaming at elevated temperatures is an effective pretreatment for the separation of lignocellulosic materials into their major components. By this treatment, the major part of the hemicellulose becomes 241

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Short communication

the fractions passing through 32 mesh were used. Enzymatic hydrolysis was carried out according to the method of Sudo et al. (1976). The steamed and hot-water extracted substrates (200 mg) was hydrolyzed in a 0.1 M sodium acetate buffer solution (pH 4.8, 10 ml) containing 50 mg commercial cellulase preparation (Meicelase-P, Meiji Seika Co Ltd.) at

40°C for 48 h. The activities of the enzyme preparation have been assayed by Sudo et al. (1976). After incubation the amount of reducing sugars formed was determined by the methods of Somogyi (1952). The hot-water extracts of steamed solids were also hydrolyzed with the cellulase preparation. The Klason lignin content was determined by the method

Table 1. Effect of acid catalysts on the recovery yield and enzymatic hydrolysis of steamed bamboo grass (Sasa senanensis Rehd.) culms

Catalyst

Uncatalyzed

A1CI3 5/ 10 20 30

Yield (%)"

Lignin

Steamed solid

Water extract

85.4

27.5

74.0 69.0 66.1 57.3

(%)~

Yield of reducing sugars

Enzymatic hydrolysis (%)

(%)c

Extracted residue d

Water extracff

33.2

78.1

60.7

56.7

21.6 19.0 17.9 11.6

28.1 29.9 29.6 28.7

80.9 76.7 66.2 59.2

86.7 92.2 100.0 99.3

61.9 71.3 53.8 56.6

74.1 66.5 64.6 59.8

19.5 14.3 13.1 11.9

26.8 27.9 26.4 28.1

80.0 69.9 57.8 44.3

85.9 97.5 69.9 75.3

63.1 63.4 48.7 60.6

76.3 70.9 67.7 66.3

20.3 19.0 20.4 15.1

27.4 28.0 29.6 29.3

71.3 72.0 73.6 71.4

100.0 93.9 94.2 71.6

70.3 63.9 62.6 74.5

75.0 74.3 74.6 73.2

23.7 24.5 23.4 22.7

30.8 31.0 30.2 30.7

76.1 78.9 80.9 85.4

88.0 89.0 99.8 91.8

71.8 73.2 72.6 73.1

73.8 73.4 73.0 72.3

21.4 22.4 21.9 23.1

29.3 28.9 28.0 29.5

77.8 80.2 78.6 79.6

97.9 100.0 95.3 92.9

70.5 74.3 80.6 83.7

76.0 75.3 75.5 74.0

25.9 25.4 25.7 24.8

29.6 29.6 31.1 30.6

79.6 75.3 70.1 73.0

76.7 78.1 86.8 89.9

68.7 60.3 68.0 71.3

79.6 78.4 79.1 76.8

24.2 23.2 23.0 22.0

29.9 29.2 29.9 28.3

86.0 76.5 74.1 72.1

66.2 83.5 72.9 93.2

57.3 62.8 71.5 81.7

77.6 76.0 76.2 74.6

23.6 23.6 24.0 23.0

30.4 29.3 28.8 28.2

75.1 75.3 71.2 75.9

64.7 74.3 67.8 65.9

62.7 56.8 72.5 57.2

78.9 76.6 75.8 75.2

23.4 25.4 25.1 24.2

29.0 31.0 31.2 29.8

79.1 79.5 71.6 74.4

87.4 85.9 92.3 97.0

65.1 68.8 78.4 78.5

A12(504)3 5 10 20 30 FeCl3 5 10 20 30 NH4CI 5 10 20 30

(NH4)2804

5 10 20 30 Acetic acid 5 10 20 30 Maleic Anhydride 5 10 20 30 Succinic anhydride 5 10 20 30 Phthalic anhydride 5 10 20 30

~Based on oven-dried original material. bBased on steamed, extracted residue. CBased on water extract. dBased on polysaccharides in steamed, extracted residue. eBased on polysaccharides in water extract. IAdded amount (mmol).

Short communication of Effland (1977). Each figure in Table 1 is the mean of two replicates.

RESULTS AND DISCUSSION Table 1 shows the effect of catalysts on the recovery yield of steamed solid and its enzymatic hydrolysis. These catalysts strongly modified the steam treatment of bamboo grass culm. When the culms were steamed without acid catalyst, the recovery of steamed solid was 85.4% based on the dry original raw material. About a quarter of the steamed solids was recovered as a mixture of xylose and soluble xylose oligomers. The use of acid catalysts resulted in a reduced recovery of steamed solids, as shown in Table 1. When the culms were steamed in the presence of Lewis acids, AIC13, ml2(SO4) 3 and FeCI3, only glucose could be detected in the complete acid hydrolyzates of the steamed and extracted residues (results not shown). The reduced recovery thus was mainly due to an extensive thermal decomposition of solubilized xylan fragments formed during the catalyzed steam treatments. Although 21-5% of hemicellulose sugars were recovered as a mixture of xylose and soluble xylose oligomers by uncatalyzed steam treatment and subsequent hot water extraction, 40% of total available sugars still remained in the residual fiber after enzymatic hydrolysis. When the steam treatment was carried out in the presence of catalysts, the extent of enzymatic hydrolysis of the steamed substrate was significantly improved. With a few exceptions (acetic acid, maleic anhydride and succinic anhydride), enzymatic hydrolysis yields well above 85% were achieved by the addition of the catalysts even at a low level of 5 mmol. Almost complete hydrolysis of the polysaccharides present in the steamed and extracted residues was attained by the addition of FeC13 and (NH4)2SO4 at a concentration of 5 mmol. The water extracts were less susceptible to the enzyme compared to the extracted residues. Mes-Hartree and Saddler (1983) reported that wheat straw and aspen wood chips pretreated by steam explosion contained substances which reduced /~-glucosidase activity but could be removed by simple water extraction. The relatively low susceptibility of the water extract could be due to the thermal degradation products of sugars and/or of the formation of low molecular weight phenolics during the steam treatment (Buchert et al., 1989). Conversion yields from 60 to 70% based on polysaccharides present in the dry original raw material were achieved by catalyzed steaming followed by enzymatic hydrolysis. In conclusion, the modified steam treatment is a promising approach for the utilization of polysaccharides present in bamboo grass culms.

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REFERENCES Aoyama, M. (1996). Steaming treatment of bamboo grass. II. Characterization of solubilized hemicellulose and enzymatic digestibility of water-extracted residue. Cellulose Chem. Technol., 30, 385-393. Aoyama, M., Seki, K. & Saito, N. (1995). Solubilization of bamboo grass xylan by steaming treatment. Holzforschung, 49, 193-196. Buchert, J., Puls, J. & Poutanen, K. (1989). The use of steamed hemicellulose as substrate in microbial conversions Appl. Biochem. Biotechnol., 20, 309-318. Brownell, H. H., Yu, E. K. C. & Saddler, J. N. (1986). Steam-explosion pretreatment of wood: Effect of chip size, acid, moisture content and pressure drop. Biotechnol. Bioeng., 28, 792-801. Clark, T. A. & Mackie, K. L. (1987). Steam explosion of the Pinus radiata with sulphur dioxide addition. I. Process optimisation. J. Wood Chem. Technol., 7, 373-403. Effland, M. J. (1977). Modified procedure to determine acid-insoluble lignin in wood and pulp. Tappi, 60, 143-144. Ishii, T. & Tanaka, J. (1984). Enzymatic hydrolysis of woods. Part VIII. Chemical composition and enzymatic hydrolysis of bamboo grass Mokuzai Gakkaishi, 30, 230-236. Mackie, K., Brownell, H. H., West, K. L. & Saddler, J. N. (1985). Effect of sulphur dioxide and sulphuric acid on steam explosion of aspenwood. J. Wood Chem. Technol., 5, 405-425. Mes-Hartree, M. & Saddler, J. M. (1983). The nature of inhibitory materials present in pretreated lignocellulosic substrates which inhibit the enzymatic hydrolysis of cellulose. Biotechnol. Lett., 5, 531-536. Ramos, L. P., Breuil, C., Kushner, D. J. & Saddler, J. N. (1992). Steam pretreatment conditions for effective enzymatic hydrolysis and recovery yields of Eucalyptus viminalis wood chips Holzforschung, 46, 149-154. Rughani, J., Wasson, L. & McGinnis, G. (1990). The use of Lewis acids during steam hydrolysis. J. Wood Chem. Technol., 10, 515-530. Rughani, J., Wasson, L., Prewitt, L. & McGinnis, G. (1992). Use of difunctional compounds during Rapid steam hydrolysis (RASH) treatment. J. Wood Chem. Technol., 12, 79-90. Somogyi, M. (1952). Notes on sugar determinatiort J. Biol. Chem., 195, 19-23. Sudo, K., Matsumura, Y. & Shimizu, K. (1976). Enzymatic hydrolysis of woods. I. Effect of delignification on hydrolysis of woods by Trichoderma viride cellulase. Mokuzai Gakkaishi, 22, 670-676. Sudo, K., Shimizu, K., Ishii, T., Fujii, T. & Nagasawa, S. (1986). Enzymatic hydrolysis of woods. Part IX, Catalyzed steam explosion of softwood. Holzforschung, 40, 339-345.

Mayumi Tsuda a*, Masakazu Aoyama a & Nam-Seok Cho b aHokkaido Forest Products Research Institute, Asahikawa, 071-01, Japan hChungbuk National University, Cheongju, 360-763, Republic of Korea (Revised version received 8 November 1997; accepted 17 November 1997) *Author to whom correspondence should be addressed.